Transplantation of Foetal SCN into the Brain of non SCN-Lesioned Rats

Transplantation of foetal SCN tissue into the brain of arrhythmic SCN-lesioned rats and hamsters has shown to be effective in restoring circadian rhythms. Transplantation of the SCN into normal untreated rats has not been described so far as function is concerned. In rats certain demands have to be met for successful grafting of the SCN. Location, age and method of transplantation play an important role in the survival and function of the graft. This paper describes a method for SCN transplantation in normal rats. Results show transplant survival in 95% and successful grafting of SCN tissue in 85% of the treated rats as shown by VP and VIP staining. Disturbed circadian eating, drinking and activity rhythms are noted when grafts are located very near the endogenous SCN. Rhythms of wheel running and body temperature were less affected. The method described seems therefore well suited to do further research with circadian rhythms.     

Circadian rhythmicity of vasopressin levels in the cerebrospinal fluid of suprachiasmatic nucleus-lesioned and -grafted rats

Transplantation of the fetal suprachiasmatic nucleus (SCN) in arrhythmic SCN-lesioned rats can reinstate circadian drinking rhythms in 40% to 50% of the cases. In the current article, it was investigated whether the failure in the other rats could be due to the absence of a circadian rhythm in the grafted SCN, using a circadian vasopressin (VP) rhythm in the cerebrospinal fluid (CSF) as the indicator for a rhythmic SCN. CSF was sampled in continuous darkness from-intact control rats and SCN-lesioned and -grafted rats. VP could be detected in all samples, with concentrations of 15 to 30 pg/ml in the control rats and 5 to 15 pg/ml in the grafted rats. A circadian VP rhythm with a two- to threefold difference between peak and nadir values was found in all 7 control rats but in only 4 of 13 experimental rats, despite the presence of a VP-positive SCN in all grafts. A circadian VP rhythm was present in 2 drinking rhythm-recovered rats (6 of 13) and in 2 nonrecovery rats. Apparently, in these latter rats, the failure of the grafted SCN to restore a circadian drinking rhythm cannot be attributed to a lack of rhythmicity in the SCN itself. Thus, the presence of a rhythmic grafted SCN, as is deduced from a circadian CSF VP rhythm, appears not to be sufficient for restoration of a circadian drinking rhythm in SCN-lesioned arrhythmic rats.     

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.     

Electrophysiology of the Circadian Pacemaker in Mammals

The neurons of the mammalian suprachiasmatic nuclei (SCN) control circadian rhythms in molecular, physiological, endocrine, and behavioral functions. In the SCN, circadian rhythms are generated at the level of individual neurons. The last decade has provided a wealth of information on the genetic basis for circadian rhythm generation. In comparison, a modest but growing number of studies have investigated how the molecular rhythm is translated into neuronal function. Neuronal attributes have been measured at the cellular and tissue level with a variety of electrophysiological techniques. We have summarized electrophysiological research on neurons that constitute the SCN in an attempt to provide a comprehensive view on the current state of the art.     

Electrophysiology of the circadian pacemaker in mammals

The neurons of the mammalian suprachiasmatic nuclei (SCN) control circadian rhythms in molecular, physiological, endocrine, and behavioral functions. In the SCN, circadian rhythms are generated at the level of individual neurons. The last decade has provided a wealth of information on the genetic basis for circadian rhythm generation. In comparison, a modest but growing number of studies have investigated how the molecular rhythm is translated into neuronal function. Neuronal attributes have been measured at the cellular and tissue level with a variety of electrophysiological techniques. We have summarized electrophysiological research on neurons that constitute the SCN in an attempt to provide a comprehensive view on the current state of the art.     

A neural clockwork for encoding circadian time.

Many daily biological rhythms are governed by an innate timekeeping mechanism or clock. Endogenous, temperature-compensated circadian clocks have been localized to discrete sites within the nervous systems of a number of organisms. In mammals, the master circadian pacemaker is the bilaterally paired suprachiasmatic nucleus (SCN) in the anterior hypothalamus. The SCN is composed of multiple single cell oscillators that must synchronize to each other and the environmental light schedule. Other tissues, including those outside the nervous system, have also been shown to express autonomous circadian periodicities. This review examines 1) how intracellular regulatory molecules function in the oscillatory mechanism and in its entrainment to environmental cycles; 2) how individual SCN cells interact to create an integrated tissue pacemaker with coherent metabolic, electrical, and secretory rhythms; and 3) how such clock outputs are converted into temporal programs for the whole organism.     

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.     

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.     

Neural mechanisms for entrainment and generation of mammalian circadian rhythms.

The identification of a direct retinohypothalamic tract (RHT) terminating in the supra-chiasmatic nuclei (SCN) has focused attention on the role of these structures in the entrainment and generation of circadian rhythms in mammals. Light effects on circadian rhythms are mediated by both the RHT and portions of the classical visual system. The complex interactions of these systems are reflected both in their direct anatomical connections and in the functional changes in entrainment produced by interruption of either set of projections. Destruction of the RHT/SCN eliminated both normal entrainment and normal free-running circadian rhythms. No circadian rhythms has survived SCN ablation in rodents, but a variety of non-circadian cycles can be generated by lesioned animals. The complex behavioral patterns produced by SCN-lesioned hamsters suggest that circadian oscillators continue to function in these animals, but that their activity is no longer integrated into a single circadian framework. The available evidence indicates that the mammalian pacemaking system comprises a set of independent oscillators normally regulated by the SCN and by light information that is transmitted via several retinofugal pathways.     

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.     

Forced desynchronization of dual circadian oscillators within the rat suprachiasmatic nucleus

The circadian clock in the suprachiasmatic nucleus of the hypothalamus (SCN) contains multiple autonomous single-cell circadian oscillators and their basic intracellular oscillatory mechanism is beginning to be identified. Less well understood is how individual SCN cells create an integrated tissue pacemaker that produces a coherent read-out to the rest of the organism. Intercellular coupling mechanisms must coordinate individual cellular periods to generate the averaged, genotype-specific circadian period of whole animals. To noninvasively dissociate this circadian oscillatory network in vivo, we (T.C. and A.D.-N.) have developed an experimental paradigm that exposes animals to exotic light-dark (LD) cycles with periods close to the limits of circadian entrainment. If individual oscillators with different periods are loosely coupled within the network, perhaps some of them would be synchronized to the external cycle while others remain unentrained. In fact, rats exposed to an artificially short 22 hr LD cycle express two stable circadian motor activity rhythms with different period lengths in individual animals. Our analysis of SCN gene expression under such conditions suggests that these two motor activity rhythms reflect the separate activities of two oscillators in the anatomically defined ventrolateral and dorsomedial SCN subdivisions. Our "forced desychronization" protocol has allowed the first stable separation of these two regional oscillators in vivo, correlating their activities to distinct behavioral outputs, and providing a powerful approach for understanding SCN tissue organization and signaling mechanisms in behaving animals.     

Leptin-sensitive neurons in the arcuate nuclei contribute to endogenous feeding rhythms

Neural sites that interact with the suprachiasmatic nuclei (SCN) to generate rhythms of unrestricted feeding remain unknown. We used the targeted toxin, leptin conjugated to saporin (Lep-SAP), to examine the importance of leptin receptor-B (LepR-B)-expressing neurons in the arcuate nucleus (Arc) for generation of circadian feeding rhythms. Rats given Arc Lep-SAP injections were initially hyperphagic and rapidly became obese (the "dynamic phase" of weight gain). During this phase, Lep-SAP rats were arrhythmic under 12:12-h light-dark (LD) conditions, consuming 59% of their total daily intake during the daytime, compared with 36% in blank-SAP (B-SAP) controls. Lep-SAP rats were also arrhythmic in continuous dark (DD), while significant circadian feeding rhythms were detected in all B-SAP controls. Approximately 8 wk after injection, Lep-SAP rats remained obese but transitioned into a "static phase" of weight gain marked by attenuation of their hyperphagia and rate of weight gain. In this phase, Arc Lep-SAP rats exhibited circadian feeding rhythms under LD conditions, but were arrhythmic in continuous light (LL) and DD. Lep-SAP injections into the ventromedial hypothalamic nucleus did not cause hyperphagia, obesity, or arrhythmic feeding in either LD or DD. Electrolytic lesion of the SCN produced feeding arrhythmia in DD but not hyperphagia or obesity. Results suggest that both Arc Lep-SAP neurons and SCN are required for generation of feeding rhythms entrained to photic cues, while also revealing an essential role for the Arc in maintaining circadian rhythms of ad libitum feeding independent of light entrainment.     

Circadian regulation of cardiovascular function: a role for vasoactive intestinal peptide

The circadian system, driven by the suprachiasmatic nucleus (SCN), regulates properties of cardiovascular function. The dysfunction of this timing system can result in cardiac pathology. The neuropeptide vasoactive intestinal peptide (VIP) is crucial for circadian rhythms in a number of biological processes including SCN electrical activity and wheel running behavior. Anatomic evidence indicates that SCN neurons expressing VIP are well positioned to drive circadian regulation of cardiac function through interactions with the autonomic centers. In this study, we tested the hypothesis that loss of VIP would result in circadian deficits in heart rate (HR) and clock gene expression in cardiac tissue. We implanted radiotelemetry devices into VIP-deficient mice and wild-type (WT) controls and continuously recorded HR, body temperature, and cage activity in freely moving mice. Under light-dark conditions, VIP-deficient mice displayed weak rhythms in HR, body temperature, and cage activity, with onsets that were advanced in phase compared with WT mice. Similarly, clock gene expression in cardiac tissue was rhythmic but phase advanced in mutant mice. In constant darkness, the normal circadian rhythms in HR were lost in VIP-deficient mice; however, most mutant mice continued to exhibit circadian rhythms of body temperature with shortened free-running period. The loss of VIP altered, but did not abolish, autonomic regulation of HR. Analysis of the echocardiograms did not find any evidence for a loss of cardiac function in VIP-deficient mice, and the size of the hearts did not differ between genotypes. These results demonstrate that VIP is an important regulator of physiological circadian rhythmicity in the heart.     

Cardiovascular control by the suprachiasmatic nucleus: neural and neuroendocrine mechanisms in human and rat

The risk for cardiovascular incidents is highest in the early morning, which seems partially due to endogenous factors. Endogenous circadian rhythms in mammalian physiology and behavior are regulated by the suprachiasmatic nucleus (SCN). Recently, anatomical evidence has been provided that SCN functioning is disturbed in patients with essential hypertension. Here we review neural and neuroendocrine mechanisms by which the SCN regulates the cardiovascular system. First, we discuss evidence for an endogenous circadian rhythm in cardiac activity, both in humans and rats, which is abolished after SCN lesioning in rats. The immediate impact of retinal light exposure at night on SCN-output to the cardiovascular system, which signals 'day' in both diurnal (human) and nocturnal (rat) mammals with opposite effects on physiology, is discussed. Furthermore, we discuss the impact of melatonin treatment on the SCN and its potential medical relevance in patients with essential hypertension. Finally, we argue that regional differentiation of the SCN and autonomous nervous system is required to explain the multitude of circadian rhythms. Insights into the mechanisms by which the SCN affects the cardiovascular system may provide new strategies for the treatment of disease conditions known to coincide with circadian rhythm disturbances, as is presented for essential hypertension.     

Developmental and functional aspects of grafting of the suprachiasmatic nucleus in the Brattleboro and the arrhythmic rat

The development of suprachiasmatic nuclei (SCN) dissected from fetal rats and grafted in adult rat brains has provided additional insights in the normal ontogeny of the SCN. The SCN survives rather easily and develops to its typical adult cytoarchitectonical arrangement of contiguous clusters of vasopressin (VP)-, vasoactive intestinal polypeptide (VIP)- and somatostatin (SOM)- immunoreactive cells. Neither site of implantation, nor the establishment of efferent or afferent connections of the grafted SCN seems to be essential to allow it to develop normally into this distinguishing cytology. This independent maturation does certainly not contradict with its known endogenous and independent potency of circadian pacemaker function in the brain. If the fetal SCN is grafted in such a way that it could merge with the parenchyma of the brain of a VP-deficient Brattleboro rat, the VP neurons of the SCN often establish efferent connections with the genuine target areas of this nucleus as could be shown immunocytochemically. When the fetal SCN is grafted homotopically in the brain of SCN-lesioned rat (or hamster), the surviving SCN neurons are able to reverse the arrhythmicity of these rats. Free-running circadian rhythm of drinking or motor behaviour in constant darkness are induced within weeks after grafting. A correlation between this restorative effect and the immunocytochemical staining pattern of the SCN in the transplant and/or the afferent and efferent connections between graft and host brain, could, however, not be shown conclusively. Transplants with surviving SCN are also seen when arrhythmicity was still present, which made us conclude that there has to be a neural connection between graft and host rather than a neurohumoral control in order to explain the restorative effect of the SCN graft in SCN-lesioned animals.     

Vasopressin: its possible role in circadian time keeping.

In this review article an insight has been made into the strong possibility of the role of vasopressin (VP) in the control of circadian rhythms which has emerged from the results of the recent experiments in this field. A role for VP, which is identified in the suprachiasmatic nuclei (SCN) of mammals, as a neurotransmitter/neuromodulator in the central nervous system has been postulated for some time now. The presence of certain abnormalities in the circadian rhythms in VP deficient Brattleboro rats has suggested that this neuropeptide is a likely candidate in controlling circadian rhythms. The coexistence of VP and corticotropin releasing factor (CRF), their interrelation with reference to their role in the hypothalamus-pituitary-adrenocortical glucocorticoid axis not only in states of stress but also in day-to-day life has also been discussed. The possible role of dynorphin, which is co-synthesized with VP in the hypothalamic neurons, and other opioids in the control of circadian rhythms has been highlighted. The pineal, SCN relation in the process of development of circadian rhythms has also been reviewed briefly.     

How many pieces to build a circadian clock?

Biological rhythms represent a fundamental property of various living organisms. In particular, circadian rhythms, i.e. rhythms with a period close to 24 hours, help organisms to adapt to environmental daily rhythms. Although various factors can entrain or reset rhythms, they persist even in the absence of external timing cue, showing that their generation is endogenous. Indeed, the suprachiasmatic nucleus (SCN) of the hypothalamus is considered to be the main circadian clock in mammals. Isolated SCN neurons have been shown to display circadian rhythms, and in each cell, a set of genes, called "clock genes", are devoted to the generation and regulation of rhythms. Recently, it has become obvious that the clock located in the SCN is not homogenous, but is rather composed of multiple functional components somewhat reminiscent of its neurochemical organization. The significance and implications of these findings are still poorly understood but pave the way for future exciting studies. Here, current knowledge concerning these distinct neuronal populations and the ways through which synchronization could be achieved, as well as the potential role of neuropeptides in both photic and non-photic resetting of the clock, are summarized. Finally, we discuss the role of the SCN within the circadian system, which also includes oscillators located in various tissues and cell types.