Endocrinology, doi:10.1210/en.2007-0660
Endocrinology Vol. 148, No. 12 5624-5634
Copyright © 2007 by The Endocrine Society
Minireview: The Circadian Clockwork of the Suprachiasmatic Nuclei—Analysis of a Cellular Oscillator that Drives Endocrine Rhythms
Elizabeth S. Maywood,
John S. O’Neill,
Johanna E. Chesham and
Michael H. Hastings
Medical Research Council Laboratory of Molecular Biology, Neurobiology Division, Cambridge CB2 0QH, United Kingdom
Address all correspondence and requests for reprints to: Michael H. Hastings, Medical Research Council, Laboratory of Molecular Biology, Neurobiology Division, Hills Road, Cambridge CB2 0QH, United Kingdom. E-mail: mha{at}mrc-lmb.cam.ac.uk.
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Abstract
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The secretion of hormones is temporally precise and periodic, oscillating over hours, days, and months. The circadian timekeeper within the suprachiasmatic nuclei (SCN) is central to this coordination, modulating the frequency of pulsatile release, maintaining daily cycles of secretion, and defining the time base for longer-term rhythms. This central clock is driven by cell-autonomous, transcriptional/posttranslational feedback loops incorporating Period (Per) and other clock genes. SCN neurons exist, however, within neural circuits, and an unresolved question is how SCN clock cells interact. By monitoring the SCN molecular clockwork using fluorescence and bioluminescence videomicroscopy of organotypic slices from mPer1::GFP and mPer1::luciferase transgenic mice, we show that interneuronal neuropeptidergic signaling via the vasoactive intestinal peptide (VIP)/PACAP2 (VPAC2) receptor for VIP (an abundant SCN neuropeptide) is necessary to maintain both the amplitude and the synchrony of clock cells in the SCN. Acute induction of mPer1 by light is, however, independent of VIP/VPAC2 signaling, demonstrating dissociation between cellular mechanisms mediating circadian control of the clockwork and those mediating its retinally dependent entrainment to the light/dark cycle. The latter likely involves the Ca2+/cAMP response elements of mPer genes, triggered by a MAPK cascade activated by retinal afferents to the SCN. In the absence of VPAC2 signaling, however, this cascade is inappropriately responsive to light during circadian daytime. Hence VPAC2-mediated signaling sustains the SCN cellular clockwork and is necessary both for interneuronal synchronization and appropriate entrainment to the light/dark cycle. In its absence, behavioral and endocrine rhythms are severely compromised.
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Introduction
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THE ACTIVITIES OF endocrine systems are cyclical, waxing and waning with periodicities of a few hours (circhoral pulsatility), a day (circadian), a week or more (estrous and menstrual), and over several months (seasonal and annual) (1). Consequently, to understand hormone action, one needs to consider the dimension of biological time. Central to this is an appreciation of the circadian clock, which both modulates the daily activity of circhoral pacemakers and defines the time base for many of the endocrine cycles with longer periods. The purpose of this article, therefore, is to review recent developments in our understanding of the cellular and genetic bases to circadian timing in mammals.
Circadian rhythms are those daily cycles of physiology, metabolism, and behavior that persist (free-run) when an individual is placed into temporal isolation, the most obvious in humans being the cycle of sleep and wakefulness (2, 3). Their persistence under such conditions reveals that they are driven by internal biological oscillators with intrinsic periods of approximately 24 h (hence circa, approximately, and dian, daily). Under normal conditions of life, the oscillators are synchronized (entrained) to the light/dark cycle (detected by the retina) and thereby become a biological clock predictive of solar time. Consequently, the rhythms that they control can preadapt the individual by anticipating the predictable but changing demands and opportunities of day and night. Moreover, by segregating in time noncompatible biochemical processes, circadian clocks make the individual a more efficient biological machine (4, 5). Because of this, circadian clocks are ubiquitous across eukaryotes, and evolution has hardwired them into almost every aspect of our physiology.
The particular relevance of circadian clocks to endocrinology is evidenced by the pronounced hormonal rhythms that span the sleep/wake cycle (3) and the observation that the physiological and pathological actions of a number of hormones are related to their daily patterns of secretion (1, 6). For example, whereas circadian day is characterized by the secretion of corticosteroids, insulin, and catecholamines, circadian night is dominated by an endocrine cocktail that includes GH, prolactin, TSH, arginine vasopressin (AVP), and melatonin (3, 7, 8) (Fig. 1
). This tightly orchestrated endocrine program is the product of an equally precise syncopation of cellular activities within brain, endocrine, and other tissues that is underpinned by circadian coordination of local transcriptomes (9, 10) and thereby local proteomes (11). The capacities of individual cells, be it hormone synthesis and release, nutrient uptake, initiation of cell division, or detoxification of xenobiotics change markedly and deliberately over the 24-h cycle. This extensive circadianprogramming within endocrine and other physiological pathways underpins the marked circadian prevalence characteristic of several major diseases (respiratory, metabolic, and cardiovascular) (4). Moreover, disruption of this regular endocrine program, for example by poor sleep patterns or persistent rotational shift work, can have a severe long-term impact on metabolic and mental health (4, 12); a primary requisite for health is robust circadian coordination of physiology.
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FIG. 1. Circadian and sleep-dependent control of daily physiological and endocrine rhythms. Daily cycles of physiology (core body temperature and urine volume) and hormone levels (melatonin, cortisol, GH, and prolactin) in human subjects with regular sleep/wake cycles and entrained to a light/dark cycle are shown on the left. Note progressive changes in physiological and endocrine status across day and night, synchronized to the sleep/wake cycle (shaded). When exposed to a constant routine, devoid of time cues and in continuing wakefulness (right), subjects reveal their unmasked circadian cycles. Note maintained physiological rhythms, with slightly reduced amplitudes. Endocrine cycles show differential effects of constant routine; whereas melatonin and cortisol rhythms are strongly circadian, rhythms of GH and prolactin are lost because of their strict dependence on sleep. [Data are redrawn with permission from C. A. Czeisler and E. B. Klerman: Recent Prog Horm Res 54:97–130, 1999 (3 ). © Academic Press.]
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The Suprachiasmatic Nuclei (SCN) as the Brain’s Circadian Pacemaker
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The normal daily profiles of circadian hormones are ultimately dependent upon a master pacemaker in the brain, the SCN (2). The SCN control endocrine rhythms in two ways: first, by directly up- and down-regulating neuroendocrine functions, and second, by controlling cycles of sleep and wakefulness that indirectly influence neuroendocrine activity (3). The relative importance of these routes varies. For example, cortisol and melatonin rhythms are under direct SCN control and little influenced by sleep, whereas nocturnal rhythms of GH and prolactin secretion are almost exclusively dependent on sleep (Fig. 1
). Yet other circadian hormones exhibit intermediate dependence on these two mechanisms.
The SCN lie above the optic chiasm, flanking the third ventricle at the base of the hypothalamus. Each consists of 10,000 or so 
-aminobutyric acid (GABA)-ergic neurons, segregated into a core region characterized by cells that also express vasoactive intestinal polypeptide (VIP) and gastrin-releasing peptide (GRP) and a shell containing AVP-positive cells (13). The core is entrained to light/dark cycles by a direct retinal innervation, the retinohypothalamic tract. This arises from intrinsically photoreceptive retinal ganglion cells that use melanopsin, a novel photopigment with closer homology to invertebrate pigments than conventional rod and cone opsins (14). Light-dependent glutamatergic activation of VIP and other retinorecipient neurons by the retinohypothalamic tract initiates intracellular and intercellular cascades of gene expression, first in the SCN core and then spreading into the shell via GABA, VIP, and GRP signaling pathways (13). In this way, brief exposures to light at dawn and/or dusk are sufficient to entrain the SCN clockwork to solar time, correcting the approximate oscillator to a precise 24-h cycle (15).
Retinal innervation is not necessary, however, for the clock to keep time. In the absence of synchronizing cues, both in vivo and in vitro, the SCN can sustain very precise circadian rhythms of gene expression, electrical firing, and neurotransmitter release (2) and in totally blind human subjects can thereby maintain robust endocrine rhythms (16). Indeed, the timing ability of individual SCN neurons is remarkable; not only can an organotypical SCN slice oscillate for many months in culture without loss of precision, but robust circadian timekeeping is also observed in individual SCN neurons dispersed into primary culture, thereby confirming that individual SCN neurons are self-sustained biological clocks (17). Ablation of the SCN, on the other hand, compromises circadian control of behavioral, metabolic, and endocrine rhythms (2) and disorganizes circadian gene expression rhythms in the periphery (18).
In the intact brain, projections from the SCN are well placed to drive endocrine and other circadian cycles, albeit via indirect, polysynaptic connections (19). The principal circadian relay of the SCN is a crescent-shaped continuum of the medial hypothalamus, running from the sub-paraventricular zone adjacent to the SCN, dorsally and caudally into the dorsomedial hypothalamus (20). Links from there feed into arousal and sleep-regulatory centers of the orexinergic system and ventrolateral preoptic area, respectively, thereby mediating indirect control of sleep-dependent hormones. In addition, extensive efferent pathways run to a diversity of autonomic centers. Consequently, most viscera receive SCN-dependent circadian time cues via their parasympathetic and/or sympathetic innervation (21). In the adrenal, for example, light and circadian signals from the SCN can drive glucocorticoid synthesis and release without accompanying hypothalamo-adenohypophysial activation (22). Similarly, nocturnal secretion of melatonin, an important regulator of seasonal rhythms in many species (1) and of sleep efficiency in humans (23), is driven by a multisynaptic pathway extending from the medial hypothalamus to the sympathetic afferents of the pineal gland. Within the hypothalamus, projections from the SCN to the paraventricular nucleus, the preoptic area, and the mediobasal nuclei are able to regulate daily rhythms of ACTH, gonadotropins, and metabolic hormones, respectively, by controlling the synthesis and release of relevant releasing factors (21).
The neurochemical nature of SCN efferent signals is of appreciable current interest, not least because neuronal transplantation studies have indicated that diffusible paracrine factors may play a central role. A screen for secreted factors of the SCN (24) has identified TGF-
and the neuropeptide cardiotropin-like cytokine (25) as likely mediators of SCN output. More recently, prokineticin 2 (PROK2) has been identified as a necessary SCN output. Its expression in the SCN is tightly regulated by the core clock mechanism, and its receptor (PROKR2) is widely expressed in SCN target areas, including the medial thalamus, arcuate nucleus, and dorsomedial hypothalamus (26). Moreover, loss of the genes encoding either the peptide (27) or its cognate receptor PROK2R (28) compromises circadian endocrine, behavioral, and thermoregulatory rhythms, with particular effects in early night. Homeostatic regulation of sleep is also compromised in mice lacking the Prok2 gene (29). Loss of the receptor or ligand had no effect, however, upon cellular timekeeping in the SCN itself, even though both are expressed at high levels in the nucleus. It seems likely, therefore, that the SCN secretes PROK2 alongside other time-stamped factors and thereby defines serial phases of the circadian cycle within target sites. So, how do the SCN keep time?
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Circadian Clock Genes and Timekeeping within the SCN
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The circadian cycles of SCN neuronal firing rate and neurotransmitter release that drive endocrine rhythms are underpinned by a molecular oscillator. This 24-h clockwork consists of interlocked feedback loops encoded by a series of clock genes. The three Period (Per) and two Cryptochrome (Cry) clock genes of mammals were identified through their homology with components of the Drosophila clockwork (2, 4). At the start of circadian day, heteromeric transcriptional regulatory complexes containing the proteins CLOCK and BMAL1 (brain and muscle ARNT-like protein 1) activate these genes by acting through E-box regulatory sequences that carry the motif CACGTG, of which five are present in the mPer1 promoter (30). The cardinal feature of the loops is that, after a delay, the translated period (PER) and cryptochrome (CRY) proteins form complexes in the nucleus and from the beginning of circadian night turn off the activation of their cognate genes by interacting with CLOCK and BMAL1. In the absence of further gene expression, the existing PER and CRY complexes are progressively degraded, and by the end of circadian night, they have disappeared, and the entire cycle is free to start again. The delayed negative feedback loop thus established has a spontaneous intrinsic oscillation of approximately 1 d (2, 4). Mutations that slow the proteasomal degradation of CRY prolong the phase of negative feedback and thereby lengthen circadian period (31, 32), whereas mutations that destabilize PER accelerate the clock and its dependent endocrine rhythms (33, 34, 35). This core loop is stabilized by ancillary feedback pathways involving additional proteins such as REV-ERB (reverse erythroblastic leukemia viral oncogene homolog), RORA (retinoic acid-related orphan receptor), and DEC1 (Drosophila hairy, enhancer of split, cAMP regulated) (36, 37, 38). For example, expression of Bma1 is induced after clearance of its repressor REV-ERB in later circadian night (36), thereby facilitating reinitiation of the cycle just as negative PER and CRY complexes disappear. As well as sustaining a core oscillation, these interlocked loops drive daily waves of gene expression that form the output of the clock, including diverse metabolic proteins, transcription factors, neuropeptide transmitters, and ion channels that determine and support the circadian properties of SCN neurons (10). In this way, the core transcriptional/posttranslational feedback loop regulates events at the neuronal membrane and thereby determines both the pattern of neural and paracrine outputs from the SCN and its responsiveness to afferent signals.
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Real-Time Imaging of Cellular Circadian Gene Expression in the SCN Pacemaker
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By definition, circadian timekeeping is a dynamic process, and so to understand better its cellular and neural bases, we have applied real-time fluorescence and bioluminescence imaging to monitor cellular circadian gene expression in organotypic SCN slices (39). Mice bearing a transgene in which a 3-kb mPer1 genomic sequence drives a short half-life green fluorescent protein (GFP) (40) have been used previously to monitor circadian and light-regulated gene expression in acutely prepared tissue cultures, both SCN (41, 42) and retina (43). For bioluminescence studies, we use SCN from mice carrying the full 7.2-kb mPer1 genomic sequence to drive firefly luciferase (44). Slices are made from early neonatal (fluorescence or bioluminescence) or adult (bioluminescence) mice, and mPer1 activity across the SCN, as reported by either transgene, expresses a very precise and robust oscillation of about 24 h (Fig. 2a) that persists over weeks. Circadian cycling of the cells is synchronized across the nucleus, most having a common peak time in the middle of circadian day (Fig. 2b). In some slices, depending upon their precise orientation, a degree of differential phasing is reflected in a topographical gradient whereby dorsomedial regions of the SCN peak in advance of the lateral parts of the nucleus. Importantly, over the course of successive circadian cycles, there is no evidence for loss of synchrony between the oscillatory cells; their respective phase relationships are retained within and between the paired SCN. This phase clustering in long-term culture contrasts with the situation in acute slices where rhythmic cells are segregated into several phase-specific clusters determined by lighting history of the animal (41, 45). Photoperiodically regulated circadian waveforms are also observed in electrical firing rhythms of acute slices (46). It is evident, therefore, that cellular oscillators in vivo are subject to marked external influences, principally retinally mediated, that establish a complex program of gene expression reflective of photoperiod (47, 48). This modulation of circadian waveform is the origin of seasonal reproductive and metabolic rhythms (1). In its fully relaxed state, however, under long-term culture and devoid of retinal innervation, intrinsic mechanisms draw these diverse cellular components into a simplified circadian program, with only limited and topographically consistent phase differences across the SCN. The nature of these interneuronal signaling pathways is a topic of current interest: what holds the SCN cells together?
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FIG. 2. Real-time fluorescence recordings of cellular circadian gene expression in long-term organotypic SCN slice cultures of Per1::GFP mice. a, Serial video images captured at 12-h intervals during the first 2 d of recording from a slice that had been in culture for 15 d before analysis. Bar, 500 µm. V, Third ventricle. b, GFP emission from 15 individual neurons from another slice, showing synchronous circadian gene expression across the SCN. Rhythm amplitude varied between units, and peak phases were distributed across approximately 3–4 h in mid-circadian day. [Redrawn with permission from E. S. Maywood et al.: Prog Brain Res 153:253–269, 2006 (68). © Elsevier.] c, Comparison of mPer1-driven GFP expression during 4 d of recording and the distribution of AVP neurons in the slice revealed by post hoc immunostaining. Note close correspondence between circadian gene expression and AVP neurons in dorsomedial shell SCN. Bar, 500 µm. V, Third ventricle.
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Under phase-contrast optics, the shell and core subdivisions of the SCN are readily apparent, and the strongest circadian gene expression appears to originate from the medial region of the shell, immediately adjacent to the third ventricle and extending in a dorsal and laterally projecting arc. Post hoc immunostaining of the slices confirms that the strongest circadian gene expression maps to the dorsomedial shell characterized by AVP neurons (Fig. 2c) rather than the densely packed VIP cells of the ventral SCN (39). These pronounced mPer1 oscillations mark out the AVP-rich dorsomedial shell as a major pacemaker. This is consistent with in vivo experiments in which rodents are subject to a sharp phase shift in the lighting cycle. Whereas rhythmic, photically driven Per expression in the core adjusts rapidly to the new lighting conditions, resynchronization of circadian Per expression in the shell progresses more slowly, reflecting the inertia of its innate oscillatory function. Importantly, the rate of readjustment of gene expression rhythms within the shell correlates directly with the rate at which behavioral and endocrine rhythms readjust to the new lighting regime (49). The intrinsic oscillator of the SCN shell may, therefore, be the behaviorally relevant pacemaker, whereas the core is specialized for entrainment and so tracks ambient lighting (13).
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The Core Molecular Oscillator Is Sustained by Intercellular Signaling: Effects of Tetrodotoxin (TTX)
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The linear, hierarchical view of the neuronal clock described earlier, with an autonomous transcriptional timekeeper that dictates but is not affected by downstream cellular rhythms, has, however, recently been questioned (50). An emerging view is that events at the cell membrane and within the cytosol may contribute to pacemaking. Furthermore, interneuronal communication mediated across the membrane and through cytosolic signals may not only synchronize SCN neurons but may also be necessary to sustain high-amplitude cellular rhythms (51). To examine the role of intercellular signaling in SCN circadian timing, mPer1::GFP slices were treated with TTX (0.5 µM) to block Na+-dependent action potentials. This had a dramatic effect on molecular timekeeping (Fig. 3a). Within the first cycle of TTX addition, the amplitude of oscillation across the SCN fell by over 50% and continued to decline further. Moreover, the precisely organized sinusoidal waveform to gene expression became disrupted. These effects of TTX were reversible upon washout, the oscillation regaining amplitude and temporal definition (Fig. 3, a and b). The reduced amplitude of circadian gene expression across the SCN did not arise from an immediate uncoupling of otherwise highly rhythmic individual units. Rather, the amplitude of cellular gene expression was reduced as indicated by comparison of signal variance over the circadian cycle immediately before TTX and the second cycle after TTX (Fig. 3c). This was confirmed by analyzing the circadian profiles of oscillation within individual units as the amplitude declined (Fig. 3, d and e). This loss of coherent circadian oscillations in the SCN reported by mPer1::GFP is consistent with findings from a previous study that used mPer1::luciferase activity (44), indicating that the attenuation is unlikely to be an artifact of the reporter used to monitor the clockwork. Moreover, TTX very rapidly blocks the circadian peak of transcription of AVP in organotypic SCN slices (52), a major output gene of the clockwork (53). Together, the results show that the intracellular molecular loop is dependent on spontaneous electrical activity within the SCN and, by implication, interneuronal signaling.
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FIG. 3. Treatment with TTX reversibly blocks the molecular clockwork. a, Representative circadian cycle of mPer1-driven fluorescence integrated across a single SCN in organotypic slice culture. Note loss of amplitude and precision to circadian gene expression with exposure to TTX (0.5 µM, down arrow) and restoration upon washout (up arrow). b, Combined data from four slices showing effect of exposure to TTX on the amplitude of circadian fluorescence across the SCN. Data are expressed as mean + SEM relative to rhythm amplitude before TTX treatment. Post TTX indicates after TTX washout. c, Fluorescence variance images from slice depicted in A on the circadian cycle immediately before and the second cycle after addition of TTX. Note massively reduced amplitude and distribution of mPer1 activity, with persistent oscillation most obvious in dorsomedial SCN (arrows). d, Representative images of Per1::GFP activity in six individual neurons (horizontal rows) before and during exposure to TTX. The high-contrast enhancement necessary to show low-amplitude cycles under TTX causes images of peak fluorescence before TTX to be overexposed. e, Graphical plots of circadian gene expression from individual neurons before and during exposure to TTX. Note immediate loss of amplitude to circadian rhythm of fluorescence from individual cells but maintained synchrony over the first few cycles. Data are plotted as deviation from running 24-h mean.
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Intercellular VIP-ergic Signals Sustain and Synchronize Cellular Molecular Timekeeping in the SCN
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What intrinsic SCN interneuronal signals might be important in maintaining cellular clocks? VIP is an abundant SCN neuropeptide, and its receptor VIP/PACAP2 (VPAC2) (encoded by the Vipr2 gene), is highly expressed in the SCN. Targeted knockout of the genes encoding either VIP or VPAC2 compromises circadian rhythms of electrical firing in individual SCN neurons in acute SCN slices (54) and also in dispersed SCN cultures (55). It is also associated with disorganized circadian rest/activity behavior in mutant mice (56) (Fig. 4a). Furthermore, downstream endocrine cycles, including corticosteroid and estrous rhythms, are disrupted in Vipr2–/– mice (57, 58). Of course, these compromised electrical, endocrine, and behavioral rhythms do not necessarily imply loss of core pacemaker function. They may merely reflect uncoupling between the core clockwork and cellular output signals. In situ hybridization analyses of Per gene expression in the SCN of VPAC2 null mice indicate, however, that the core molecular oscillation is impaired right across the SCN, with mPer1 and mPer2 mRNA levels basal in the mutant, regardless of sample time (56). These snapshot studies failed, therefore, to support the hypothesis that cellular clocks remained active and at high amplitude within the mutant SCN and were simply uncoupled from output pathways. Rather, they suggested that cellular circadian timekeeping within the SCN required neuropeptidergic signals.
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FIG. 4. Compromised circadian behavior and circadian timekeeping in SCN of VPAC2 receptor knockout mice. a, Representative wheel-running actograms of wild-type (WT, left) and Vipr2 null mice (KO, right), carrying the mPer1::luciferase reporter gene. Mice were initially held on a cycle of 12 h light and 12 h dim red light (LD), with dim red light indicated by black bar, and then transferred to continuous dim red light (DD) at the red line. Note loss of coherent behavior in mutant under DD. b, Recording of Per1-driven bioluminescence from organotypic SCN slices of mice depicted in a. Note high-amplitude coherent cycles in wild-type SCN and loss of amplitude and coherence in mutant. Mice were killed for slice culture at points indicated by arrows.
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To characterize the role of peptidergic interneuronal signaling in maintaining the core molecular oscillator, the mPer1::GFP and mPer1::luciferase mice were crossed into Vipr2 mutant mice. Having confirmed that null mutants exhibited disordered circadian behavioral rhythms in vivo (Fig. 4a), the SCN of homozygous mutants carrying the mPer1::luciferase reporter were dissected and placed into organotypic culture and circadian gene expression monitored by a photomultiplier assembly. Loss of the VPAC2 receptor had two effects; first, overall levels of SCN gene expression, as reported by bioluminescence, were reduced by an order of magnitude, and second, the coherence of the circadian timekeeping seen in the wild type was severely compromised in the mutant (Fig. 4b). A comparable effect was also observed in SCN slices carrying the mPer1::GFP reporter, insofar as slices from wild-type and heterozygous (Vipr2+/–) mice exhibited robust circadian gene expression, whereas it was dramatically impaired in homozygous mutants (Fig. 5, a and b). This was not, however, a simple desynchronization of otherwise rhythmic cellular or regional components. Expression of the Per1::GFP transgene was extremely weak across the SCN and barely above the level of detection, such that very few rhythmic cells could be identified (39). With the more sensitive luciferase reporter analyzed with a CCD camera, rhythmic cells were detectable in the Vipr2–/– SCN (Fig. 5c), but they occurred at an appreciably lower density than in heterozygote or wild-type slices (
50% reduction), and the overall level of bioluminescent activity was less than 20% of the wild-type condition (39). These rhythmic cells tended to cluster in the medial shell, and some if not all were AVP-immunoreactive (AVP-ir). Another marked effect of the Vipr2–/– mutation was desynchronization of these remaining rhythmic cells; at any one time across the SCN, weakly rhythmic cells could be seen at all phases of oscillation (Fig. 5d). VIP has been proposed as a synchronizing factor within the SCN (55, 59). In the absence of the VPAC2 receptor, therefore, some or all of the molecular clocks within the SCN might be expected to desynchronize, leading to a variety of phase relationships for cellular mPer1 rhythms. Alternatively, it has been proposed as necessary to sustain cellular rhythms (55, 56), in which case its loss would suspend the molecular clockwork. The current results show that VIP-ergic signaling is necessary for both synchronization and maintenance of molecular timekeeping in SCN cells.
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FIG. 5. Attenuated and desynchronized cellular circadian oscillation in VPAC2 receptor knockout mice. a, Representative serial video images captured during the first 3 d of recording from an organotypic slice from a heterozygous (Het) mouse and two slices both from a homozygous (KO) mutant mouse. Post hoc immunostaining confirmed the presence of AVP-ir perikarya in the dorsomedial SCN of both the Het and KO slices. b, Plots of relative fluorescence intensity integrated over the right SCN from the Het ( ) and KO ( and  ) slices. c, Integrated bioluminescent signal showing distribution of a limited number of cells exhibiting circadian gene expression in Vipr2–/– slice (left) and phase image of same slice (right), indicating that the active cells lie in the medial SCN shell. oc, Optic chiasm; V, third ventricle. Bar, 500 µm. d, Circadian cycles of gene expression recorded from six representative cells identified in slice in C. Although rhythmic bioluminescence is evident, it is poorly organized within individual neurons and is not synchronized between cells. a–d, [Redrawn with permission from E. S. Maywood et al.: Curr Biol 16:599–605, 2006 (39 ). © Cell Press.]
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Cellular Actions of VIP Underlying Synchronization and Maintenance of the Clockwork
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How might VIP-ergic signals sustain and synchronize SCN clock cells? Electrophysiological studies show that neurons in the Vipr2–/– SCN are hyperpolarized (60). To test whether hyperpolarization might, at least in part, explain the compromised molecular clockwork, Vipr2–/– SCN slices were acutely depolarized, either by application of high extracellular potassium levels or by the SCN neuropeptide GRP. Both treatments rapidly induced mPer1-driven luciferase activity (39), and concomitant with this activation, the asynchronous SCN neurons were forced to a common phase of oscillation, thereby synchronizing across the slice. Synchrony was, however, transient, and with continued recording, the cellular oscillators once more diverged in phase. These results indicate that reduced circadian gene expression in the Vipr2–/– SCN cell may be a consequence of hyperpolarization, implying that at some level the molecular clockwork is dependent upon ionic events at the cell membrane, driven by VIP.
Consistent with the view that a clock-relevant role of VIP signaling is to maintain membrane depolarization is the observation that the spontaneous firing rates of SCN cells in Vipr2–/– slices are low compared with wild-type controls (54). Conversely, hyperpolarization of wild-type SCN slices by low [K+] medium attenuates mPer1 expression rhythms (61). Certainly, membrane polarity/ionic fluxes appear to sustain the SCN molecular clockwork rather than simply be a consequence of its output because buffering of the circadian cycle of [Ca2+] levels abrogates rhythmic expression of both mPer1 and mPER2 (61). Changes in intracellular [Ca2+] likely regulate mPer expression via Ca2+/cAMP response element (CRE) sequences, which are also responsive to cAMP. It may be significant, therefore, that the VPAC2 receptor is positively linked to adenylyl cyclase. This raises the possibility that E-box-dependent activation of mPer1 by CLOCK and BMAL1 cannot sustain circadian expression if CRE-mediated drive to Per is compromised because in the absence of VIP-ergic signals, hyperpolarization reduces intracellular [Ca2+] and/or cAMP levels are insufficient to activate CREs. This suggests that treatments that acutely increase intracellular cAMP levels would temporarily restore circadian function to Vipr2–/– SCN.
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Competent Retinal Signaling in Vipr2–/– SCN but Loss of Its Circadian Modulation
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Between 3 and 5 h after Per1::GFP slices are first prepared from neonatal pups, a very strong GFP signal is elicited from the SCN (Fig. 6, a and b). This acute activation of mPer1 in Vipr2–/– SCN is indistinguishable from that seen in heterozygous and wild-type animals. Acute regulation of the mPer1 gene is therefore retained in the Vipr2–/– mutant slice in vitro, even though circadian control is lost. To test whether this dissociation between circadian and acute regulation of mPer1 activation in vitro reflects the in vivo condition, Vipr2–/– mutant mice and wild-type controls were transferred from a lighting cycle of 12 h light, 12 h dim red light to continuous dim red light (DD) and on the second cycle exposed to DD or a white light pulse for 10 min at either circadian day [circadian time (CT) 08] or circadian night (CT20), phases when the wild-type SCN clock is unresponsive or responsive to light, respectively. In situ hybridization revealed that in wild-type mice under DD, expression of mPer1 mRNA in the SCN was high at CT08 and low at CT20, the typical circadian profile (62) (Fig. 6, c and d). In Vipr2–/– mice, mPer1 mRNA was low at both times, as noted previously (56) and consistent with the absence of strong circadian expression in mutant SCN slices in vitro. As anticipated, the brief light pulse induced mPer1 expression in wild-type mice at CT20 but not at CT08. This is the characteristic circadian gating of the responses to light that underpins the differential behavioral resetting effects of light delivered in circadian night and circadian day (63). Light also acutely induced mPer1 expression in the Vipr2–/– SCN, confirming that acute physiological regulation remained intact in the Vipr2–/– background even though circadian regulation to the gene was compromised. Moreover, photic induction occurred in both subjective day and subjective night, indicating that circadian gating of the response of the core molecular loop to light is lost in the mutant SCN.
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FIG. 6. Acute expression of mPer1 and activation of retinal signaling pathways is preserved in Vipr2–/– mice despite loss of circadian regulation. a, Representative fluorescence images of Per1::GFP expression in acutely prepared SCN slices from wild-type (WT), heterozygous (Het), and homozygous (Vipr2–/–) knockout (KO) mice. Signal was predominantly in the dorsomedial SCN in all three genotypes. Bar, 500 µm. b, Mean + SEM fluorescence emission from Per1::GFP-positive (white bars) and -negative (gray bars) slices with wild-type (WT; n = 10 and 4), heterozygous (Het; n = 14 and 3), or homozygous (KO; n = 6 and 7) VPAC2 knockout backgrounds. c, Representative mPer1 in situ hybridization images of coronal brain sections from wild-type (WT) and Vipr2–/– (KO) mice held in darkness or exposed to light for 10 min at CT08 or CT20. Arrows indicate SCN. Bar, 1 mm. d, Semiquantitative analysis of in situ hybridization signal intensity for mPer1 mRNA in SCN of WT and KO mice, exposed to darkness (gray bars) or light (white bars) at CT08 or CT20. e, Representative images for pCREB-ir in SCN of WT and VPAC2 KO mice exposed to darkness or light at CT08 or CT20. Bar, 500 µm. f, Abundance of pCREB-ir nuclei (mean + SEM) in SCN of WT and VPAC2 KO mice exposed to darkness (gray bars) or light (white bars) at CT08 or CT20. g, Semiquantitative analysis of in situ hybridization signal intensity for cFos mRNA in SCN of WT and KO mice, exposed to darkness (gray bars) or light (white bars) at CT08 or CT20. h, Abundance of pERK-ir nuclei (mean + SEM) in SCN of WT and VPAC2 KO mice exposed to darkness (gray bars) or light (white bars) at CT08 or CT20. Data in d, g, f, and h are plotted as mean + SEM, n = 4 per time point.
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Photic activation of mPer1 in the SCN is mediated by glutamatergic retinal afferents, acting via N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors (64, 65), which leads to gene expression after phosphorylation of CRE-binding protein (CREB) (66). In the absence of light, levels of phosphorylated CREB (pCREB) in the SCN were low in both wild-type and mutant mice (Fig. 6, e and f). In wild-type mice, light at CT20 induced pCREB-ir in the core SCN, but light at CT08 was ineffective. In contrast, pCREB-ir was acutely induced by light at both CT20 and CT08 in Vipr2–/– mice, again revealing the presence of photic responses and the absence of the circadian gate. The immediate-early gene cFos is a well-characterized target of pCREB, and this too was differentially regulated in wild-type and Vipr2–/– mice. Whereas photic induction of cFos mRNA was gated by the circadian clock in wild-type mice, occurring at CT20 but not CT08, light delivered to Vipr2–/– mice activated cFos mRNA at both phases (Fig. 6g). Finally, the Ca2+-regulated phosphorylation of the transcription factor kinase ERK is a component of the light-dependent glutamatergic signaling cascade and is also under circadian regulation, as evidenced by higher levels of pERK-ir during circadian day than night (Fig. 6h). This cycle was absent in Vipr2–/– mice, levels being basal at both CT08 and CT20. Nevertheless, at both phases, pERK-ir was markedly increased by light in the mutants, as reported previously (67), whereas wild-type SCN responded only at CT20.
These data reveal a loss of circadian patterning to mPer1 and cFos gene expression and ERK activation within the SCN of Vipr2–/– mice in vivo that parallels the compromised circadian mPer1 expression detected by fluorescence and bioluminescence imaging in vitro. Nevertheless, the critical signaling cascades of the SCN that mediate entrainment by light remain active in the mutant, and specifically, mPer1 can be induced by a glutamatergic, Ca2+-dependent cascade that involves pERK and pCREB. These VPAC2-independent signaling pathways may contribute to the maintenance of daily activity patterns and low-amplitude SCN gene expression rhythms of Vipr2–/– mice held on a full lighting schedule (56). They are, however, insufficient to initiate or entrain an autonomous circadian rhythm (57). In addition, because the circadian pacemaking mechanism of the mutant is dysfunctional, these signaling cascades, which are normally responsive to light only during subjective night, are activated by light during both subjective night and day. The loss of circadian gating to photic induction of pCREB, mPer1, and cFos mRNA in the SCN core extends previous reports of consistent photic pERK activation and c-FOS protein induction across circadian time in the Vipr2–/– SCN (67) and indicates that the role of the functional clockwork is to suppress responses of the SCN core to retinal activation during circadian daytime. This rhythm in responsiveness may depend upon intrinsic clock mechanisms of the retinorecipient core neurons and/or regulation of the core by clock cells of the shell. In either case, loss of coherent clock function in the SCN after loss of VIP-ergic signals compromises this circadian modulation. The continued response to light may arise because every cell retains sensitivity or because the retinorecipient cell population in Vipr2–/– mice is desynchronized (as is seen in the shell) such that at any point some cells are responsive, even if others are not. This model would predict responses to light of approximately 50% of those seen in wild-type mice, whereas in the current study, the magnitude of photic responses is equivalent to wild type. Within the limitations of single time-point analyses, therefore, desynchrony among core neurons seems less likely to explain the effect, and it may be that core neurons are permanently in a condition equivalent to circadian night; i.e. they are relatively hyperpolarized and able to respond to retinal activation by glutamatergic depolarization and calcium-dependent gene expression. Importantly, in the absence of effective VIP-ergic signals from core to shell, this wave of clock gene activation within the core is unable to extend into the shell.
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The SCN Are at the Head of a Circadian Control Hierarchy
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Having considered the cellular basis to circadian timing in the SCN, it is necessary finally to place the SCN into a broader context. A few years ago, the view was that, via its various outputs, the SCN clock drives rhythms of behavior, hormone secretion, and autonomic activity that in turn impose upon target tissues circadian patterns of gene expression and hence physiology. What is now apparent from real-time analyses of circadian gene expression in tissue explants and primary cell cultures is that most, if not all, cells have essentially the same molecular clockwork as that in the SCN. There may be differences in details, for example tissue-specific splice variants of BMAL1 and later relative phasing of CRY expression in peripheral tissues (4). Nevertheless, it is clear that the major organ systems, including heart, lungs, kidney, liver, and pituitary all contain local, autonomously rhythmic circadian clocks. Indeed, individual fibroblasts in culture express robust circadian cycles of gene expression that persist for weeks (17). The principal characteristic difference between the SCN and peripheral oscillators is that whereas SCN neurons can synchronize to each other and thereby sustain circadian cycles indefinitely at the tissue level, peripheral clock cells do not appear able to do so, and as they drift out of phase in culture, tissue-level oscillations are lost. A simple stimulus such as a serum shock can resynchronize the cells and reinitiate tissue-level oscillations. The in vivo role of the SCN, therefore, is to deliver daily time cues across the body that maintain the phase relationships of cellular clocks both within and between tissues, thereby ensuring high-amplitude, stable circadian cycles of physiology in tune with solar time. This internal synchronization is achieved via the diverse outputs of the SCN listed above: behavioral, autonomic, and endocrine. Daily endocrine rhythms should not, therefore, be considered merely as the endpoint of a circadian control cascade. Rather, they are one link in the circadian program, conveying temporal cues from the SCN to subordinate tissue-based oscillators. For example, the corticosteroid rhythm is extremely important in coordinating rhythmic liver metabolism, and a single acute treatment with the receptor agonist dexamethasone is able to activate approximately 60% of the hepatic circadian transcriptome (18). Identification of the relative contribution of different synchronizing cues to local circadian physiology may prove to be the major practical application of circadian endocrinology because it should provide novel avenues though which to control clinically relevant, locally rhythmic processes such as vasoconstriction, detoxification of xenobiotics, cell division, or renal filtration.
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Conclusion
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The circadian patterning intrinsic to endocrine activity is dependent upon the central pacemaker of the SCN. Individual neurons of the SCN are competent circadian clocks, but it is clear that intercellular, neural circuit functions mediated by VIP/VPAC2 signaling sustain as well as synchronize the intracellular molecular timer. This interneuronal synchronization is necessary to drive endocrine and other dependent rhythms in the organism, as evidenced by compromised estrous and corticosteroid cycles in VPAC2-null mice. VIP-ergic signaling is not necessary, however, for acute activation of clock gene expression by retinal innervation, revealing a dichotomy between pathways mediating either the circadian or the acute regulation of the cellular clock. The molecular clockwork of the SCN is also active in peripheral tissues, but these cells do not appear able to communicate effectively and so require regular input from the SCN to maintain intra- and intertissue synchrony. Endocrine rhythms, such as the release of corticosteroids before the start of wakefulness, form an important component of the mechanism of internal synchronization. Future studies will therefore be directed toward understanding the neurochemical and molecular bases of interneuronal signaling within the SCN and its relationship to the maintenance and synchronization of SCN clocks cells. Furthermore, it will be important to define how the SCN pacemaker via its endocrine and other rhythmic outputs is able to maintain adaptive circadian physiology.
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Footnotes
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We are grateful to Drs. Beth Klerman, Steve Lockley, and Chuck Czeisler (Harvard Medical School) for their generous assistance in the design of Fig. 1 and Mr. Paul Margiotta (MRC-LMB) for its production.
This work was supported by the Medical Research Council, the Biotechnology and Biological Research Council (UK), and "EUCLOCK" Framework Program 6 of the European Union.
Disclosure Statement: The authors have nothing to disclose.
First Published Online September 27, 2007
Based on talk given at the "From Molecular Clocks to Human Health" session at the Sixth International Congress of Neuroendocrinology, Pittsburgh, PA, June 2006.
Abbreviations: AVP, Arginine vasopressin; BMAL1, brain and muscle ARNT-like protein; CRE, Ca2+/cAMP response element; CREB, CRE-binding protein; CRY, cryptochrome; CT, circadian time; DD, continuous dim red light; GABA, -aminobutyric acid; GFP, green fluorescent protein; GRP, gastrin-releasing peptide; ir, immunoreactive; PACAP, pituitary adenylyl cyclase-activating peptide; pCREB, phosphorylated CREB; PER, period; PROK2, prokineticin 2; SCN, suprachiasmatic nuclei; TTX, tetrodotoxin; VIP, vasoactive intestinal peptide; VPAC2, VIP/PACAP2.
Received May 17, 2007.
Accepted for publication July 12, 2007.
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Abstract
Introduction
