Circadian organization is governed by extra-SCN pacemakers

In mammals, a pacemaker in the suprachiasmatic nucleus (SCN) is thought to be required for behavioral, physiological, and molecular circadian rhythms. However, there is considerable evidence that temporal food restriction (restricted feedisng [RF]) and chronic methamphetamine (MA) can drive circadian rhythms of locomotor activity, body temperature, and endocrine function in the absence of SCN. This indicates the existence of extra-SCN pacemakers: the Food Entrainable Oscillator (FEO) and Methamphetamine Sensitive Circadian Oscillator (MASCO). Here, we show that these extra-SCN pacemakers control the phases of peripheral oscillators in intact as well as in SCN-ablated PER2::LUC mice. MA administration shifted the phases of SCN, cornea, pineal, pituitary, kidney, and salivary glands in intact animals. When the SCN was ablated, disrupted phase relationships among peripheral oscillators were reinstated by MA treatment. When intact animals were subjected to restricted feeding, the phases of cornea, pineal, kidney, salivary gland, lung, and liver were shifted. In SCN-lesioned restricted-fed mice, phases of all of the tissues shifted such that they aligned with the time of the meal. Taken together, these data show that FEO and MASCO are strong circadian pacemakers able to regulate the phases of peripheral oscillators.     

Daily rhythms in olfactory discrimination depend on clock genes but not the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) regulates a wide range of daily behaviors and has been described as the master circadian pacemaker. The role of daily rhythmicity in other tissues, however, is unknown. We hypothesized that circadian changes in olfactory discrimination depend on a genetic circadian oscillator outside the SCN. We developed an automated assay to monitor olfactory discrimination in individual mice throughout the day. We found olfactory sensitivity increased approximately 6-fold from a minimum during the day to a peak in the early night. This circadian rhythm was maintained in SCN-lesioned mice and mice deficient for the Npas2 gene but was lost in mice lacking Bmal1 or both Per1 and Per2 genes. We conclude that daily rhythms in olfactory sensitivity depend on the expression of canonical clock genes. Olfaction is, thus, the first circadian behavior that is not based on locomotor activity and does not require the SCN.     

Circadian rhythms of gastrointestinal function are regulated by both central and peripheral oscillators

Circadian clocks are responsible for daily rhythms in a wide array of processes, including gastrointestinal (GI) function. These are vital for normal digestive rhythms and overall health. Previous studies demonstrated circadian clocks within the cells of GI tissue. The present study examines the roles played by the suprachiasmatic nuclei (SCN), master circadian pacemaker for overt circadian rhythms, and the sympathetic nervous system in regulation of circadian GI rhythms in the mouse Mus musculus. Surgical ablation of the SCN abolishes circadian locomotor, feeding, and stool output rhythms when animals are presented with food ad libitum, while restricted feeding reestablishes these rhythms temporarily. In intact mice, chemical sympathectomy with 6-hydroxydopamine has no effect on feeding and locomotor rhythmicity in light-dark cycles or constant darkness but attenuates stool weight and stool number rhythms. Again, however, restricted feeding reestablishes rhythms in locomotor activity, feeding, and stool output rhythms. Ex vivo, intestinal tissue from PER2::LUC transgenic mice expresses circadian rhythms of luciferase bioluminescence. Chemical sympathectomy has little effect on these rhythms, but timed administration of the β-adrenergic agonist isoproterenol causes a phase-dependent shift in PERIOD2 expression rhythms. Collectively, the data suggest that the SCN are required to maintain feeding, locomotor, and stool output rhythms during ad libitum conditions, acting at least in part through daily activation of sympathetic activity. Even so, this input is not necessary for entrainment to timed feeding, which may be the province of oscillators within the intestines themselves or other components of the GI system.     

In vivo monitoring of peripheral circadian clocks in the mouse

The mammalian circadian system is comprised of a central clock in the suprachiasmatic nucleus (SCN) and a network of peripheral oscillators located in all of the major organ systems. The SCN is traditionally thought to be positioned at the top of the hierarchy, with SCN lesions resulting in an arrhythmic organism. However, recent work has demonstrated that the SCN and peripheral tissues generate independent circadian oscillations in Per1 clock gene expression in vitro. In the present study, we sought to clarify the role of the SCN in the intact system by recording rhythms in clock gene expression in vivo. A practical imaging protocol was developed that enables us to measure circadian rhythms easily, noninvasively, and longitudinally in individual mice. Circadian oscillations were detected in the kidney, liver, and submandibular gland studied in about half of the SCN-lesioned, behaviorally arrhythmic mice. However, their amplitude was decreased in these organs. Free-running periods of peripheral clocks were identical to those of activity rhythms recorded before the SCN lesion. Thus, we can report for the first time that many of the fundamental properties of circadian oscillations in peripheral clocks in vivo are maintained in the absence of SCN control.     

PPARalpha is a potential therapeutic target of drugs to treat circadian rhythm sleep disorders

Recent progress at the molecular level has revealed that nuclear receptors play an important role in the generation of mammalian circadian rhythms. To examine whether peroxisome proliferator-activated receptor alpha (PPARalpha) is involved in the regulation of circadian behavioral rhythms in mammals, we evaluated the locomotor activity of mice administered with the hypolipidemic PPARalpha ligand, bezafibrate. Circadian locomotor activity was phase-advanced about 3h in mice given bezafibrate under light-dark (LD) conditions. Transfer from LD to constant darkness did not change the onset of activity in these mice, suggesting that bezafibrate advanced the phase of the endogenous clock. Surprisingly, bezafibrate also advanced the phase in mice with lesions of the suprachiasmatic nucleus (SCN; the central clock in mammals). The circadian expression of clock genes such as period2, BMAL1, and Rev-erbalpha was also phase-advanced in various tissues (cortex, liver, and fat) without affecting the SCN. Bezafibrate also phase-advanced the activity phase that is delayed in model mice with delayed sleep phase syndrome (DSPS) due to a Clock gene mutation. Our results indicated that PPARalpha is involved in circadian clock control independently of the SCN and that PPARalpha could be a potent target of drugs to treat circadian rhythm sleep disorders including DSPS.     

Effects of suprachiasmatic nuclei lesions on circadian and ultradian rhythms in body temperature in ocular enucleated rats

Abstract

To test the hypothesis that an oscillator located outside the suprachiasmatic nuclei (SCN) controls the circadian rhythm of body temperature, we conducted a study with 14 blinded rats, 10 of which receiving a SCN lesion. Body temperature was automatically and continuously recorded for about one month by intraperitoneal radio transmitters. Food intake, drinking and locomotor activity were also recorded. Periodograms revealed that 3 rats with histologically verified total bilateral SCN lesions did not exhibit any circadian rhythmicity. The 7 other rats appeared to have partial lesions. They showed shortening of period and severe amplitude reduction in all functions. Thus, no support was found for the hypothesis of a separate circadian ‘temperature oscillator’ located outside the SCN. Nevertheless, after large partial lesions body temperature showed more persistency than some of the other behavioral rhythms.

Ultradian rhythms in temperature persisted after partial and total lesions. Other functions showed parallel ultradian rhythms. In intact rats the ultradian peaks were restricted predominantly to the subjective night. After total lesions these peaks became more or less homogeneously distributed in time but more heterogeneously after partial lesions. So the SCN plays a role in the temporal structure of ultradian rhythms but does not generate them. Non‐24‐hour actograms showed instabilities of period and phase of ultradian rhythms. Intact and lesioned rats were similar with respect to the mean (about 3.5 hrs) and standard deviation (about 1.5 hrs) of ultradian periods in temperature. These features indicate that a mechanism outside the SCN is underlying ultradian rhythmicity, capable of generating short‐term oscillations. Two approaches, homeostatic sleep‐wake relaxation oscillations and multiple circadian oscillators, are discussed.     

Neurotransplantation of the Biological Clock in Mammals, Importance and Perspectives

The tool of neurotransplantation has been successfully introduced in the chronobiology of mammals. Grafting of the foetal suprachiasmatic nucleus (SCN) in the IIIrd ventricle of the brain of SCN-lesioned arhythmic rodents restored free-running circadian activity patterns. This ultimately proves the SCN to be the central circadian pacemaker system. However, recovery is not seen in all animals with a surviving SCN implant and the rhythm is usually not as robust as seen for the intact system. Moreover, the grafted foetal SCN has a partially deviant development, whereas the structure-function relationship after restoration of circadian rhythm was reported to differ in the various studies. This has led to two possible mechanisms of graft action: the one a circadian humoral signal diffusing into the SCN-lesioned host brain, and the other a neuritic afferent outgrowth into the brain. There is, moreover, doubt about the integration of the 'new' SCN in terms of afferent input. Given the fact that the in situ SCN has an extensive efferent and afferent system in the intact brain, the SCN grafting experiments seem to indicate that only limited aspects of the SCN can drive circadian physiological rhythms. However, on the basis of current knowledge on grafting results the present paper recommends performing more sophisticated SCN grafting experiments to contribute to the knowledge on the SCN clock system.     

Neurotransplantation of the Biological Clock in Mammals,Importance and Perspectives

The tool of neurotransplantation has been successfully introduced in the chronobiology of mammals. Grafting of the foetal suprachiasmatic nucleus (SCN) in the IIIrd ventricle of the brain of SCN-lesioned arhythmic rodents restored free-running circadian activity patterns. This ultimately proves the SCN to be the central circadian pacemaker system. However, recovery is not seen in all animals with a surviving SCN implant and the rhythm is usually not as robust as seen for the intact system. Moreover, the grafted foetal SCN has a partially deviant development, whereas the structure-function relationship after restoration of circadian rhythm was reported to differ in the various studies. This has led to two possible mechanisms of graft action: the one a circadian humoral signal diffusing into the SCN-lesioned host brain, and the other a neuritic afferent outgrowth into the brain. There is, moreover, doubt about the integration of the ‘new’ SCN in terms of afferent input. Given the fact that the in situ SCN has an extensive efferent and afferent system in the intact brain, the SCN grafting experiments seem to indicate that only limited aspects of the SCN can drive circadian physiological rhythms. However, on the basis of current knowledge on grafting results the present paper recommends performing more sophisticated SCN grafting experiments to contribute to the knowledge on the SCN clock system.     

Circadian Regulation of Food-Anticipatory Activity in Molecular Clock–Deficient Mice

In the mammalian brain, the suprachiasmatic nucleus (SCN) of the anterior hypothalamus is considered to be the principal circadian pacemaker, keeping the rhythm of most physiological and behavioral processes on the basis of light/dark cycles. Because restriction of food availability to a certain time of day elicits anticipatory behavior even after ablation of the SCN, such behavior has been assumed to be under the control of another circadian oscillator. According to recent studies, however, mutant mice lacking circadian clock function exhibit normal food-anticipatory activity (FAA), a daily increase in locomotor activity preceding periodic feeding, suggesting that FAA is independent of the known circadian oscillator. To investigate the molecular basis of FAA, we examined oscillatory properties in mice lacking molecular clock components. Mice with SCN lesions or with mutant circadian periods were exposed to restricted feeding schedules at periods within and outside circadian range. Periodic feeding led to the entrainment of FAA rhythms only within a limited circadian range. Cry1^−/− mice, which are known to be a “short-period mutant,” entrained to a shorter period of feeding cycles than did Cry2^−/− mice. This result indicated that the intrinsic periods of FAA rhythms are also affected by Cry deficiency. Bmal1 ^−/− mice, deficient in another essential element of the molecular clock machinery, exhibited a pre-feeding increase of activity far from circadian range, indicating a deficit in circadian oscillation. We propose that mice possess a food-entrainable pacemaker outside the SCN in which canonical clock genes such as Cry1, Cry2 and Bmal1 play essential roles in regulating FAA in a circadian oscillatory manner.     

Restoration of Orcadian Behavior by Anterior Hypothalamic Grafts Containing the Suprachiasmatic Nucleus: Graft/Host Interconnections

Destruction of the hypothalamic suprachiasmatic nucleus (SCN) disrupts circadian behavior. Transplanting SCN tissue from fetal donors into SCN-lesioned recipients can restore circadian behavior to the arhythmic hosts. In the transplantation model employing fetal hamster donors and SCN-lesioned hamsters as hosts, the period of the restored circadian behavior is hamster-typical. However, when fetal rat anterior hypothalamic tissue containing the SCN is implanted into SCN-lesioned rats, the period of the restored circadian rhythm is only rarely typical of that of the intact rat. The use of an anterior hypothalamic heterograft model provides new approaches to donor specificity of restored circadian behavior and with the aid of species-specific markers, provides a means for assessing connectivity between the graft and the host. Using an antibody that stains rat and mouse neuronal tissue but not hamster neurons, it has been demonstrated that rat and mouse anterior hypothalamic heterografts containing the SCN send numerous processes into the host (hamster) neuropil surrounding the graft, consistent with graft efferents reported in other hypothalamic transplantation models in which graft and host tissue can be differentiated (i.e., Brattleboro rat and hypogonadal mouse). Moreover, SCN neurons within anterior hypothalamic grafts send an appropriately restricted set of efferent projections to the host brain which may participate in the functional recovery of circadian locomotor activity.     

Enhanced Food Anticipatory Activity Associated with Enhanced Activation of Extrahypothalamic Neural Pathways in Serotonin2C Receptor Null Mutant Mice

The ability to entrain circadian rhythms to food availability is important for survival. Food-entrained circadian rhythms are characterized by increased locomotor activity in anticipation of food availability (food anticipatory activity). However, the molecular components and neural circuitry underlying the regulation of food anticipatory activity remain unclear. Here we show that serotonin_2C receptor (5-HT2CR) null mutant mice subjected to a daytime restricted feeding schedule exhibit enhanced food anticipatory activity compared to wild-type littermates, without phenotypic differences in the impact of restricted feeding on food consumption, body weight loss, or blood glucose levels. Moreover, we show that the enhanced food anticipatory activity in 5-HT2CR null mutant mice develops independent of external light cues and persists during two days of total food deprivation, indicating that food anticipatory activity in 5-HT2CR null mutant mice reflects the locomotor output of a food-entrainable oscillator. Whereas restricted feeding induces c-fos expression to a similar extent in hypothalamic nuclei of wild-type and null mutant animals, it produces enhanced expression in the nucleus accumbens and other extrahypothalamic regions of null mutant mice relative to wild-type subjects. These data suggest that 5-HT2CRs gate food anticipatory activity through mechanisms involving extrahypothalamic neural pathways.     

The pineal gland influences rat circadian activity rhythms in constant light.

Mammalian circadian organization is believed to derive primarily from circadian oscillators within the hypothalamic suprachiasmatic nuclei (SCN). The SCN drives circadian rhythms of a wide array of functions (e.g., locomotion, body temperature, and several endocrine processes, including the circadian secretion of the pineal hormone melatonin). In contrast to the situation in several species of reptiles and birds, there is an extensive literature reporting little or no effect of pinealectomy on mammalian circadian rhythms. However, recent research has indicated that the SCN and circadian systems of several mammalian species are highly sensitive to exogenous melatonin, raising the possibility that endogenous pineal hormone may provide feedback in the control of overt circadian rhythms. To determine the role of the pineal gland in rat circadian rhythms, the effects of pinealectomy on locomotor rhythms in constant light (LL) and constant darkness (DD) were studied. The results indicated that the circadian rhythms of pinealectomized rats but not sham-operated controls dissociated into multiple ultradian components in LL and recoupled into circadian patterns only after 12-21 days in DD. The data suggest that pineal feedback may modulate sensitivity to light and/or provide coupling among multiple circadian oscillators within the SCN.     

Identification of the suprachiasmatic nucleus in birds

Circadian rhythms are generated by an internal biological clock. The suprachiasmatic nucleus (SCN) in the hypothalamus is known to be the dominant biological clock regulating circadian rhythms in mammals. In birds, two nuclei, the so-called medial SCN (mSCN) and the visual SCN (vSCN), have both been proposed to be the avian SCN. However, it remains an unsettled question which nuclei are homologous to the mammalian SCN. We have identified circadian clock genes in Japanese quail and demonstrated that these genes are expressed in known circadian oscillators, the pineal and the retina. Here, we report that these clock genes are expressed in the mSCN but not in the vSCN in Japanese quail, Java sparrow, chicken, and pigeon. In addition, mSCN lesions eliminated or disorganized circadian rhythms of locomotor activity under constant dim light, but did not eliminate entrainment under light-dark (LD) cycles in pigeon. However, the lesioned birds became completely arrhythmic even under LD after the pineal and the eye were removed. These results indicate that the mSCN is a circadian oscillator in birds.     

Physiological and anatomic evidence for regulation of the heart by suprachiasmatic nucleus in rats

The suprachiasmatic nucleus (SCN) is the mammalian biological clock that generates the daily rhythms in physiology and behavior. Light can phase shift the rhythm of the SCN but can also acutely affect SCN activity and output, e.g., output to the pineal. Recently, multisynaptic SCN connections to other organs were also demonstrated. Moreover, they were shown to affect those organs functionally. The aim of the present study was to investigate the role of the SCN in the regulation of the heart. First, we demonstrated that heart rate (HR) in SCN-intact, but not SCN-lesioned (SCNx), male Wistar rats had a clear circadian rhythm, which was not caused by locomotor activity. Second, we demonstrated that light at night reduces HR in intact but not in SCNx rats. Finally, we demonstrated the presence of a multisynaptic autonomic connection from SCN neurons to the heart with the retrograde pseudorabies virus tracing technique. Together, these results demonstrate that the SCN affects the heart in rats and suggest that this is mediated by a neuronal mechanism.     

High orexin-A neuron activity and RACK1 expression might be involved in the restricted feeding-entrained behaviors in mice

Under normal conditions, circadian rhythms of rodents are derived from the suprachiasmatic nucleus (SCN) and primarily entrained by light. But food-related signals, such as restricted feeding (RF), can also affect circadian rhythms and result in food-entrained locomotor activities in mice, suggesting that an additional oscillating circadian pacemaker besides the SCN is responsible for this regulation. However, little is known about its detailed molecular mechanisms. Our study found that RF during subjective day under continuous darkness augmented mice wheel-running locomotors during the RF period. Additionally, the orexin-A (OXA) neuron activity was increased obviously, and the mRNA and protein levels of RACK1 were significantly elevated. The activation of OXA neurons was prior to the initiation of RF and the elevation of RACK1. These results suggest that OXA and RACK1 may be involved in wheel-running locomotor activities entrained by RF during subjective day in mice.     

Temporal reorganization of the suprachiasmatic nuclei in hamsters with split circadian rhythms.

A dual oscillator basis for mammalian circadian rhythms is suggested by the splitting of activity rhythms into two components in constant light and by the photoperiodic control of pineal melatonin secretion and phase-resetting effects of light. Because splitting and photoperiodism depend on incompatible environmental conditions, however, these literatures have remained distinct. The refinement of a procedure for splitting hamster rhythms in a 24-h light-dark:light-dark cycle has enabled the authors to assess the ability of each of two circadian oscillators to initiate melatonin secretion and to respond to light pulses with behavioral phase shifting and induction of Fos-immunoreactivity in the suprachiasmatic nuclei (SCN). Hamsters exposed to a regimen of afternoon novel wheel running (NWR) split their circadian rhythms into two distinct components, dividing their activity between the latter half of the night and the afternoon dark period previously associated with NWR. Plasma melatonin concentrations were elevated during both activity bouts of split hamsters but were not elevated during the afternoon period in unsplit controls. Light pulses delivered during either the nighttime or afternoon activity bout caused that activity component to phase-delay on subsequent days and induced robust expression of Fos-immunoreactivity in the SCN. Light pulses during intervening periods of locomotor inactivity were ineffective. The authors propose that NWR splits the circadian pacemaker into two distinct oscillatory components separated by approximately 180 degrees, with each expressing a short subjective night.     

Cognitive Performance as a Zeitgeber: Cognitive Oscillators and Cholinergic Modulation of the SCN Entrain Circadian Rhythms

The suprachiasmatic nucleus (SCN) is the primary circadian pacemaker in mammals that can synchronize or entrain to environmental cues. Although light exerts powerful influences on SCN output, other non-photic stimuli can modulate the SCN as well. We recently demonstrated that daily performance of a cognitive task requiring sustained periods of attentional effort that relies upon basal forebrain (BF) cholinergic activity dramatically alters circadian rhythms in rats. In particular, normally nocturnal rats adopt a robust diurnal activity pattern that persists for several days in the absence of cognitive training. Although anatomical and pharmacological data from non-performing animals support a relationship between cholinergic signaling and circadian rhythms, little is known about how endogenous cholinergic signaling influences SCN function in behaving animals. Here we report that BF cholinergic projections to the SCN provide the principal signal allowing for the expression of cognitive entrainment in light-phase trained animals. We also reveal that oscillator(s) outside of the SCN drive cognitive entrainment as daily timed cognitive training robustly entrains SCN-lesioned arrhythmic animals. Ablation of the SCN, however, resulted in significant impairments in task acquisition, indicating that SCN-mediated timekeeping benefits new learning and cognitive performance. Taken together, we conclude that cognition entrains non-photic oscillators, and cholinergic signaling to the SCN serves as a temporal timestamp attenuating SCN photic-driven rhythms, thereby permitting cognitive demands to modulate behavior.