Differential responses of circadian Per2 rhythms in cultured slices of discrete brain areas from rats showing internal desynchronisation by methamphetamine

Chronic methamphetamine (MAP) treatment desynchronises the behavior rhythms of rats from light–dark cycles. Our previous study (Masubuchi et al., 2000) demonstrated the phase reversal of circadian rhythms in clock gene expression in several brain areas of rats treated with MAP. However, for technical reasons, it was not clear whether the phase shifts were the consequence of phase‐shifted behavior rhythms or reflected phase shifts of extra‐suprachiasmatic nucleus (SCN) oscillators in these areas. In the present study, circadian gene expression rhythms in discrete brain areas were continuously monitored in slice cultures of MAP‐treated rats. Methamphetamine was given to rats carrying a Period2‐dLuciferase reporter system via the drinking water for more than 2 weeks. When behavior rhythms were completely phase reversed, the brain was sampled for slice cultures and circadian bioluminescence rhythms were measured for 5 days in the SCN and four areas of the dopaminergic system, the olfactory bulb, caudate putamen, parietal cortex and substantia nigra. The circadian rhythms in the SCN and caudate putamen were not significantly phase shifted, whereas those in the parietal cortex and substantia nigra showed significant phase‐delay shifts of 6–8 h and that in the olfactory bulb showed phase‐advance shifts of ca. 8 h. Neither the period nor the amplitude of the circadian rhythm was changed by MAP treatment. These findings indicate that the extra‐SCN oscillators in several brain areas are desynchronised from the SCN circadian pacemaker by MAP treatment in parallel with the desynchronisation of behavior rhythms in rats. As the direction and extent of phase shifts of circadian rhythms were different among the areas examined, the brain extra‐SCN oscillators responded differentially to MAP.     

Differential responses of circadian Per2 expression rhythms in discrete brain areas to daily injection of methamphetamine and restricted feeding in rats

Behavioral rhythms induced by methamphetamine (MAP) and daily restricted feeding (RF) in rats are independent of the circadian pacemaker in the suprachiasmatic nucleus (SCN), and have been regarded to share a common oscillatory mechanism. In the present study, in order to examine the responses of brain oscillatory systems to MAP and RF, circadian rhythms in clock gene, Period2, expression were measured in several brain areas in rats. Transgenic rats carrying a bioluminescence reporter of Period2‐dLuciferase were subjected to either daily injection of MAP or RF of 2 h at a fixed time of day for 14 days. As a result, spontaneous movement and wheel‐running activity were greatly enhanced following MAP injection and prior to daily meal under RF. Circadian Per2 rhythms were measured in the cultured brain tissues containing one of the following structures: the olfactory bulb; caudate‐putamen; parietal cortex; substantia nigra; and SCN. Except for the SCN, the circadian Per2 rhythms in the brain tissues were significantly phase‐delayed by 1.9 h on average in MAP‐injected rats as compared with the saline‐controls. On the other hand, the circadian rhythms outside the SCN were significantly phase‐advanced by 6.3 h on average in rats under RF as compared with those under ad libitum feeding. These findings indicate that the circadian rhythms in specific brain areas of the central dopaminergic system respond differentially to MAP injection and RF, suggesting that different oscillatory mechanisms in the brain underlie the MAP‐induced behavior and pre‐feeding activity under RF.     

Circadian PER2::LUC rhythms in the olfactory bulb of freely moving mice depend on the suprachiasmatic nucleus but not on behaviour rhythms

The temporal order of physiology and behaviour in mammals is regulated by the coordination of the master circadian clock in the suprachiasmatic nucleus (SCN) and peripheral clocks in various tissues outside the SCN. Because the circadian oscillator(s) in the olfactory bulb (OB) is regarded as SCN independent, we examined the relationship between the SCN master clock and the circadian clock in the OB. We also examined the role of vasoactive intestinal peptide receptor 2 in the circadian organization of the OB. We continuously monitored the circadian rhythms of a clock gene product PER2 in the SCN and OB of freely moving mice by means of a bioluminescence reporter and an optical fibre implanted in the brain. Robust circadian rhythms were detected in the OB and SCN for up to 19 days. Bilateral SCN lesions abolished the circadian behaviour rhythms and disorganized the PER2 rhythms in the OB. The PER2 rhythms in the OB showed more than one oscillatory component of a similar circadian period, suggesting internal desynchronization of constituent oscillators. By contrast, significant circadian PER2 rhythms were detected in the vasoactive intestinal peptide receptor 2‐deficient mice, despite the substantial deterioration or abolition of circadian behavioural rhythms. These findings indicate that the circadian clock in the OB of freely moving mice depends on the SCN master clock but not on the circadian behavioural rhythms. The circadian PER2::LUC rhythm in the cultured OB was as robust as that in the cultured SCN but reset by slice preparation, suggesting that culturing of the slice reinforces the circadian rhythm.     

Using Per gene expression to search for photoperiodic oscillators in the hamster suprachiasmatic nucleus

The circadian pacemaker in the suprachiasmatic nucleus (SCN) is also believed to underlie photoperiodic (seasonal) timekeeping in mammals. This clock has been modeled as a complex pacemaker composed of two coupled circadian oscillators; variability in their mutual phase relationship could account for the ability to measure daylength, with putative morning and evening oscillators synchronized to dawn and dusk, respectively. Recently, several genes have been identified that are believed to be part of the clock's core oscillatory mechanism. Here, we investigate how such molecular oscillations are altered as a function of photoperiod by analyzing Period (Per1, Per2, and Per3) gene expression at the mRNA level using SCN tissue sections and in situ hybridization. Golden hamsters were entrained to complete 24-h light-dark (LD) cycles with either a long (16 h) or a short (8 h) photophase, or they were entrained to the long complete photoperiod and then allowed to free-run in constant darkness. The results show large photoperiod-dependent changes in the duration of high daytime SCN Per1 and Per2 mRNA levels and small changes in the phase difference between their rhythms.     

Immortalized cell lines for real-time analysis of circadian pacemaker and peripheral oscillator properties

In the mammalian circadian system, cell‐autonomous clocks in the suprachiasmatic nuclei (SCN) are distinguished from those in other brain regions and peripheral tissues by the capacity to generate coordinated rhythms and drive oscillations in other cells. To further establish in vitro models for distinguishing the functional properties of SCN and peripheral oscillators, we developed immortalized cell lines derived from fibroblasts and the SCN anlage of mPer2 ^Luc knockin mice. Circadian rhythms in luminescence driven by the mPER2::LUC fusion protein were observed in cultures of mPer2 ^Luc SCN cells and in serum‐shocked or SCN2.2‐co‐cultured mPer2 ^Luc fibroblasts. SCN mPer2 ^Luc cells generated self‐sustained circadian oscillations that persisted for at least four cycles with periodicities of ≈24 h. Immortalized fibroblasts only showed circadian rhythms of mPER2::LUC expression in response to serum shock or when co‐cultured with SCN2.2 cells. Circadian oscillations of luminescence in mPer2 ^Luc fibroblasts decayed after 3–4 cycles in serum‐shocked cultures but robustly persisted for 6–7 cycles in the presence of SCN2.2 cells. In the co‐culture model, the circadian behavior of mPer2 ^Luc fibroblasts was dependent on the integrity of the molecular clockworks in co‐cultured SCN cells as persistent rhythmicity was not observed in the presence of immortalized SCN cells derived from mice with targeted disruption of Per1 and Per2 (Per1^ldc/Per2 ^ldc). Because immortalized mPer2 ^Luc SCN cells and fibroblasts retain their indigenous circadian properties, these in vitro models will be valuable for real‐time comparisons of clock gene rhythms in SCN and peripheral oscillators and identifying the diffusible signals that mediate the distinctive pacemaking function of the SCN.     

Circadian and photic modulation of daily rhythms in diurnal mammals

The temporal niche that an animal occupies includes a coordinated suite of behavioral and physiological processes that set diurnal and nocturnal animals apart. The daily rhythms of the two chronotypes are regulated by both the circadian system and direct responses to light, a process called masking. Here we review the literature on circadian regulations and masking responses in diurnal mammals, focusing on our work using the diurnal Nile grass rat (Arvicanthis niloticus) and comparing our findings with those derived from other diurnal and nocturnal models. There are certainly similarities between the circadian systems of diurnal and nocturnal mammals, especially in the phase and functioning of the principal circadian oscillator within the hypothalamic suprachiasmatic nucleus (SCN). However, the downstream pathways, direct or indirect from the SCN, lead to drastic differences in the phase of extra‐SCN oscillators, with most showing a complete reversal from the phase seen in nocturnal species. This reversal, however, is not universal and in some cases the phases of extra‐SCN oscillators are only a few hours apart between diurnal and nocturnal species. The behavioral masking responses in general are opposite between diurnal and nocturnal species, and are matched by differential responses to light and dark in several retinorecipient sites in their brain. The available anatomical and functional data suggest that diurnal brains are not simply a phase‐reversed version of nocturnal ones, and work with diurnal models contribute significantly to a better understanding of the circadian and photic modulation of daily rhythms in our own diurnal species.     

Daily exposure to cold phase‐shifts the circadian clock of neonatal rats in vivo

Maternal rhythms entrain the prenatal and neonatal circadian clock in the suprachiasmatic nucleus (SCN) before light entrainment is established. However, the responsible time cues for maternal entrainment are not identified. To examine the role of cyclic changes of ambient temperature in maternal entrainment, blind neonatal rats carrying a clock gene (Per2) bioluminescence reporter were exposed to either of three ambient temperatures (10, 20 or 30 °C) during 6‐h maternal separation in the early light phase. Cold exposure was performed from postnatal day 1 (P1) to P5. On P6, the SCN was harvested and cultured for photometric monitoring of the circadian rhythm in Per2 expression. Here we demonstrate that the daily cold exposure phase‐delayed the circadian Per2 expression rhythms at P6 in a temperature‐dependent manner. Exposure to 10 °C produced the largest phase‐shift of 12.7 h, and exposure to 20 and 30 °C yielded moderate shifts of 4.1 and 4.5 h, respectively. There was no significant difference in the phase‐shifts between the latter two temperatures, indicating that ambient temperature is not the sole factor for the phase‐shift. Behavioral rhythms that developed after weaning reflected the phase‐shift of clock gene expression rhythm in the SCN. These findings indicate that a daily exposure to an ambient temperature of 10 °C during the neonatal period is capable of resetting the circadian clock in the SCN, but other factors yet unidentified are also involved in maternal entrainment.     

Depression‐like behaviour in mice is associated with disrupted circadian rhythms in nucleus accumbens and periaqueductal grey

An association between circadian rhythms and mood regulation is well established, and disturbed circadian clocks are believed to contribute to the development of mood disorders, including major depressive disorder. The circadian system is coordinated by the suprachiasmatic nucleus (SCN), the master pacemaker in the hypothalamus that receives light input from the retina and synchronizes circadian oscillators in other brain regions and peripheral tissues. Lacking the tight neuronal network that couples single‐cell oscillators in the SCN, circadian clocks outside the SCN may be less stable and more susceptible to disturbances, for example by clock gene mutations or uncontrollable stress. However, non‐SCN circadian clocks have not been studied extensively in rodent models of mood disorders. In the present study, it was hypothesized that disturbances of local circadian clocks in mood‐regulating brain areas are associated with depression‐like behaviour in mice. Using the learned helplessness procedure, depression‐like behaviour was evoked in mice bearing the PER2::LUC circadian reporter, and then circadian rhythms of PER2 expression were examined in brain slices from these mice using luminometry and bioluminescence imaging. It was found that helplessness is associated with absence of circadian rhythms in the nucleus accumbens and the periaqueductal grey, two of the most critical brain regions within the reward circuit. The current study provides evidence that susceptibility of mice to depression‐like behaviour is associated with disturbed local circadian clocks in a subset of mood‐regulating brain areas, but the direction of causality remains to be determined.     

Behavioural rhythm splitting in the CS mouse is related to clock gene expression outside the suprachiasmatic nucleus

CS mice exhibit a spontaneous splitting in the circadian rhythm of locomotor activity under constant darkness, suggesting that they contain two weakly coupled oscillators in the circadian clock system regulating locomotor activity rhythm. In order to clarify whether the two oscillators are located in the suprachiasmatic nucleus (SCN), a site of the master circadian pacemaker in mammals, circadian rhythms in mRNA of mouse Period genes (mPer1, mPer2 and mPer3) in the SCN and cerebral cortex were examined during rhythm splitting by in situ hybridization. In the SCN, mPer1 and mPer2 showed a circadian rhythm with a single peak in both split and unsplit mice. The rhythms of mPer1 and mPer2 were slightly phase delayed during rhythm splitting in reference to the activity onset, but the phase relationship between the two rhythms was not changed. In the cerebral cortex, the expression of mPer1 and mPer2 underwent the bimodal fluctuation with peaks temporally corresponding to split activity components. The unsplit mice showed the circadian rhythms with a single peak. There was no difference in the mPer3 rhythms in either the SCN or the cerebral cortex between the split and unsplit mice. These results indicate that the circadian oscillations of mPer1, mPer2 and mPer3 in the SCN are not related to the rhythm splitting of CS mice. The split rhythms of the CS mice are suggested to be caused by uncoupling of oscillators located outside the SCN from the SCN circadian pacemaker.     

Electrophysiological Effects of Melatonin on Mouse Per1 and non‐Per1 Suprachiasmatic Nuclei Neurones In Vitro

The master circadian pacemaker in the suprachiasmatic nuclei (SCN) regulates the nocturnal secretion of the pineal hormone melatonin. Melatonin, in turn, has feedback effects on SCN neuronal activity rhythms via high affinity G protein‐coupled receptors (MT₁ and MT₂). However, the precise effects of melatonin on the electrical properties of individual SCN neurones are unclear. In the present study, we investigated the acute effects of exogenous melatonin on SCN neurones using whole‐cell patch‐clamp recordings in brain slices prepared from Per1::d2EGFP‐expressing transgenic mice. In current‐clamp mode, bath applied melatonin, at near‐physiological concentrations (1 nm ), hyperpolarised the majority (63.7%) of SCN neurones tested at all times of the projected light/dark cycle. In addition, melatonin depolarised a small proportion of cells (11.0%). No differences were observed for the effects of melatonin between Per1::GFP or non‐Per1::GFP SCN neurones. Melatonin‐induced effects were blocked by the MT₁/MT₂ antagonist, luzindole (1 μm ) and the proportion of SCN neurones responsive to melatonin was greatly reduced in the presence of either tetrodotoxin (200 or 500 nm ) or gabazine (20 μm ). In voltage‐clamp recordings, 1 nm melatonin increased the frequency of GABA‐mediated currents. These findings indicate, for the first time, that exogenous melatonin can alter neuronal excitability in the majority of SCN neurones, regardless of whether or not they overtly express the core clock gene Per1. The results also suggest that melatonin acts mainly by modulating inhibitory GABAergic transmission within the SCN. This may explain why exogenous application of melatonin has heterogenous effects on individual SCN neurones.     

The complex relationship between the light‐entrainable and methamphetamine‐sensitive circadian oscillators: evidence from behavioral studies of Period‐mutant mice

The methamphetamine‐sensitive circadian oscillator (MASCO) is an enigmatic circadian clock whose output is observed during continuous consumption of low‐dose methamphetamine. The MASCO rhythm persists when the light‐entrainable pacemaker in the suprachiasmatic nucleus (SCN) is lesioned, but the anatomical location of MASCO is unknown. We recently found that the period of the MASCO rhythm is unusually short (21 h) in mice with disruption of all three paralogs of the canonical clock gene, Period. In this study, we investigated the contribution of each Period paralog to timekeeping in MASCO. We measured wheel‐running activity rhythms in intact and SCN‐lesioned Per1‐, 2‐ and 3‐mutant mice administered methamphetamine, and found that none of the mice displayed a short (21‐h) period, demonstrating that no single Period gene is responsible for the short‐period MASCO rhythm of Per1^?/?/Per2^?/?/Per3^?/? mice. We also found that the periods of activity rhythms in constant darkness were lengthened by methamphetamine treatment in intact wild‐type, Per1^?/? and Per3^?/? mice but not Per2^?/? mice, and Per2^?/? mice had two distinct activity rhythms upon release to constant light. These data suggest that the SCN and MASCO are not coupled in Per2^?/? mice. The MASCO rhythm in Per1^?/?/Per2^?/? mice in constant darkness alternated between a short (22‐h) and a long (27‐h) period. This pattern could result from two coupled oscillators that are not synchronised to each other, or from a single oscillator displaying birhythmicity. Finally, we propose a working model of the in vivo relationship between MASCO and the SCN that poses testable hypotheses for future studies.     

Circadian rhythms in the mouse reproductive axis during the estrous cycle and pregnancy

Molecular and behavioral timekeeping is regulated by the circadian system which includes the brain's suprachiasmatic nucleus (SCN) that translates environmental light information into neuronal and endocrine signals aligning peripheral tissue rhythms to the time of day. Despite the critical role of circadian rhythms in fertility, it remains unexplored how circadian rhythms change within reproductive tissues during pregnancy. To determine how estrous cycle and pregnancy impact phase relationships of reproductive tissues, we used PER2::Luciferase (PER2::LUC) circadian reporter mice and determined the time of day of PER2::LUC peak (phase) in the SCN, pituitary, uterus, and ovary. The relationships between reproductive tissue PER2::LUC phases changed throughout the estrous cycle and late pregnancy and were accompanied by changes to PER2::LUC period in the SCN, uterus, and ovary. To determine if the phase relationship adaptations were driven by sex steroids, we asked if progesterone, a hormone involved in estrous cyclicity and pregnancy, could regulate Per2‐luciferase expression. Using an in vitro transfection assay, we found that progesterone increased Per2‐luciferase expression in immortalized SCN (SCN2.2) and arcuate nucleus (KTAR) cells. In addition, progesterone shortened PER2::LUC period in ex vivo uterine tissue recordings collected during pregnancy. As progesterone dramatically increases during pregnancy, we evaluated wheel‐running patterns in PER2::LUC mice. We confirmed that activity levels decrease during pregnancy and found that activity onset was delayed. Although SCN, but not arcuate nucleus, PER2::LUC period changed during late pregnancy, onset of locomotor activity did not correlate with SCN or arcuate nucleus PER2::LUC period.     

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.     

Estrogen differentially regulates expression of Per1 and Per2 genes between central and peripheral clocks and between reproductive and nonreproductive tissues in female rats

Although it has long been established that estrogen alters circadian rhythms in behavior, physiology, and reproductive functions in mammals, the molecular mechanism for these effects remains unknown. To explore the possibility that estrogen affects circadian rhythms by changing the expression of clock-related genes, we investigated the effects of chronic treatment with 17beta-estradiol (E2) on the expression of Per1 and Per2 genes in the brain (suprachiasmatic nucleus and cerebral cortex) and periphery (liver, kidney, and uterus) of ovariectomized rats by means of in situ hybridization and northern blotting. In the brain, E2 treatment advanced the peak of Per2 mRNA expression in the SCN; however, it failed to affect the rhythm of Per2 mRNA expression in the CX and Per1 mRNA expression in both the SCN and the CX. In nonreproductive peripheral tissues (liver and kidney), E2 delayed the phase and increased the amplitude of Per1 mRNA expression. In the reproductive tissues (uterus), biphasic rhythms in Per1 and Per2 mRNA were observed after E2 treatment. These findings suggest that the effects of estrogen are different between central and peripheral clock in the brain, and between reproductive and nonreproductive tissues in the periphery.     

Regional circadian period difference in the suprachiasmatic nucleus of the mammalian circadian center

The suprachiasmatic nucleus (SCN) is the mammalian circadian rhythm center. Individual oscillating neurons have different endogenous circadian periods, but they are usually synchronized by an intercellular coupling mechanism. The differences in the period of each oscillating neuron have been extensively studied; however, the clustering of oscillators with similar periods has not been reported. In the present study, we artificially disrupted the intercellular coupling among oscillating neurons in the SCN and observed regional differences in the periods of the oscillating small‐latticed regions of the SCN using a transgenic rat carrying a luciferase reporter gene driven by regulatory elements from a per2 clock gene (Per2::dluc rat). The analysis divided the SCN into two regions – a region with periods shorter than 24 h (short‐period region, SPR) and another with periods longer than 24 h (long‐period region, LPR). The SPR was located in the smaller medial region of the dorsal SCN, whereas the LPR occupied the remaining larger region. We also found that slices containing the medial region of the SCN generated shorter circadian periods than slices that contained the lateral region of the SCN. Interestingly, the SPR corresponded well with the region where the SCN phase wave is generated. We numerically simulated the relationship between the SPR and a large LPR. A mathematical model of the SCN based on our findings faithfully reproduced the kinetics of the oscillators in the SCN in synchronized conditions, assuming the existence of clustered short‐period oscillators.     

Twelve-hour days in the brain and behavior of split hamsters

Hamsters will spontaneously ‘split’ and exhibit two rest–activity cycles each day when housed in constant light (LL). The suprachiasmatic nucleus (SCN) is the locus of a brain clock organizing circadian rhythmicity. In split hamsters, the right and left SCN oscillate 12 h out of phase with each other, and the twice‐daily locomotor bouts alternately correspond to one or the other. This unique configuration of the circadian system is useful for investigation of SCN communication to efferent targets. To track phase and period in the SCN and its targets, we measured wheel‐running and FOS expression in the brains of split and unsplit hamsters housed in LL or light–dark cycles. The amount and duration of activity before splitting were correlated with latency to split, suggesting behavioral feedback to circadian organization. LL induced a robust rhythm in the SCN core, regardless of splitting. The split hamsters’ SCN exhibited 24‐h rhythms of FOS that cycled in antiphase between left and right sides and between core and shell subregions. In contrast, the medial preoptic area, paraventricular nucleus of the hypothalamus, dorsomedial hypothalamus and orexin‐A neurons all exhibited 12‐h rhythms of FOS expression, in‐phase between hemispheres, with some detectable right–left differences in amplitude. Importantly, in all conditions studied, the onset of FOS expression in targets occurred at a common phase reference point of the SCN oscillation, suggesting that each SCN may signal these targets once daily. Finally, the transduction of 24‐h SCN rhythms to 12‐h extra‐SCN rhythms indicates that each SCN signals both ipsilateral and contralateral targets.     

Light-induced phase shifts of circadian activity rhythms and immediate early gene expression in the suprachiasmatic nucleus are attenuated in old C3H/HeN mice

Alterations in the mechanisms of entrainment and/or response of the circadian pacemaker to zeitgebers may contribute to age related changes in sleep/wake rhythms. This study examined the effect of age on light-induced phase shifts of circadian activity rhythms and on the expression of the immediate early genes c-fos and jun-B in the suprachiasmatic nucleus (SCN) of young and old C3H/HeN mice. Mice (4 months or 16 months at the beginning of the experiment) were housed in constant darkness with circadian rhythms assessed by running wheel activity. Mice were exposed to light pulses of 30, 100, 300 or 1000 lux and steady state phase shifts of circadian activity rhythms determined. In young mice exposed to light at circadian time (CT) 14, light pulses of 30, 100, 300 or 1000 lux induced phase delays of circadian activity rhythms of similar magnitude (averaging 2.8 h). Phase delays following photic stimulation were reduced in the old mice at all light levels (averaging 1.1 h, P<0.001). Following behavioral testing, mice were exposed to light (1000 lux) at CT 14 for determination of the light-induced expression of c-fos and jun-B mRNA in the SCN by in situ hybridization histochemistry. Immediate early gene expression following light exposure was reduced by 42% (c-fos) and 48% (jun-B) in the SCN of old mice compared to young controls (P<0.001). Together, these results suggest an age related reduction in responsiveness to light by the circadian pacemaker.     

From clock to functional pacemaker

In mammals, the central pacemaker that coordinates 24‐hr rhythms is located in the suprachiasmatic nucleus (SCN). Individual neurons of the SCN have a molecular basis for rhythm generation and hence, they function as cell autonomous oscillators. Communication and synchronization among these neurons are crucial for obtaining a coherent rhythm at the population level, that can serve as a pace making signal for brain and body. Hence, the ability of single SCN neurons to produce circadian rhythms is equally important as the ability of these neurons to synchronize one another, to obtain a bona fide pacemaker at the SCN tissue level. In this chapter we will discuss the mechanisms underlying synchronization, and plasticity herein, which allows adaptation to changes in day length. Furthermore, we will discuss deterioration in synchronization among SCN neurons in aging, and gain in synchronization by voluntary physical activity or exercise.