Hourly (ultradian) cellular rhythms: initial studies and some results

Circahoralian cellular rhythms have been revealed after the amendment of quantitative cytochemical methods initiated at the Department of Histology (Moscow State University) and Laboratory of Cytology (Institute of Animal Morphology, Russian Academy of Sciences). The first findings have been confirmed in many laboratories. Circumhoralian kinetics proved to be specific for various cell types (from bacteria to mammalian cells) and cellular functions. Independent physiological investigations found circahoralian rhythms for various tissue functions. Distribution of the rhythms as well as their possible nature and significance for tissue biology are reviewed.     

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.     

Multiple oscillators in the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of the pacemaker that controls circadian rhythms of a variety of physiological functions. Data strongly indicate the majority of the SCN neurons express self-sustaining oscillations that can be detected as rhythms in the spontaneous firing of individual neurons. The period of single SCN neurons in a dissociated cell culture is dispersed in a wide range (from 20h to 28h in rats), but that of the locomotor rhythm is close to 24h, suggesting individual oscillators are coupled to generate an averaged circadian period in the nucleus. Electrical coupling via gap junctions, glial regulation, calcium spikes, ephaptic interactions, extracellular ion flux, and diffusible substances have been discussed as possible mechanisms that mediate the interneuronal rhythm synchrony. Recently, GABA (γ-aminobutyric acid), a major neurotransmitter in the SCN, was reported to regulate cellular communication and to synchronize rhythms through GABA_A receptors. At present, subsequent intracellular processes that are able to reset the genetic loop of oscillations are unknown. There may be diverse mechanisms for integrating the multiple circadian oscillators in the SCN. This article reviews the knowledge about the various circadian oscillations intrinsic to the SCN, with particular focus on the intercellular signaling of coupled oscillators. (Chronobiology International, 18(3), 371-387, 2001)     

The Ultradian Clocks of Eukaryotic Microbes: Timekeeping Devices Displaying a Homeostasis of the Period

Temperature compensation of their period is one of the canonical characteristics of circadian rhythms, yet it is not restricted to circadian rhythms. This short review summarizes the evidence for ultradian rhythms, with periods from 1 minute to several hours, that likewise display a strict temperature compensation. They have been observed mostly in unicellular organisms in which their constancy of period at different temperatures, as well as under different growth conditions (e.g., medium type, carbon source), indicates a general homeostasis of the period. Up to eight different parameters, including cell division, cell motility, and energy metabolism, were observed to oscillate with the same periodicity and therefore appear to be under the control of the same central pacemaker. This suggests that these ultradian clocks should be considered as cellular timekeeping devices that in fast-growing cells take over temporal control of cellular functions controlled by the circadian clock in slow-growing or nongrowing cells. Being potential relatives of circadian clocks, these ultradian rhythms may serve as model systems in chronobiolog-ical research. Indeed, mutations have been found that affect both circadian and ultradian periods, indicating that the respective oscillators share some mechanistic features. In the haploid yeast Schizosaccharomyces pombe, a number of genes have been identified where mutation, deletion, or overex-pression affect the ultradian clock. Since most of these genes play roles in cellular metabolism and signaling, and mutations have pleiotropic effects, it has to be assumed that the clock is deeply embedded in cellular physiology. It is therefore suggested that mechanisms ensuring temperature compensation and general homeostasis of period are to be sought in a wider context. (Chronobiology International, 14(5), 469–479, 1997)     

Circadian rhythms: from gene expression to behavior

Circadian rhythms regulate the functions of living systems at virtually every level of organization, from molecule to organism. In the past year, our understanding of the cellular and molecular processes involved in the generation and regulation of circadian rhythms has advanced considerably. New in vitro model systems for studying circadian oscillators have been developed, a potential regulatory role for cellular immediate-early genes in circadian behavior has been discovered, critical periods for macromolecular synthesis for progression of the circadian clock through its cycle have been defined, and studies of the Drosophila period gene have offered new insight into the clock mechanism. These findings are of particular interest because independent approaches using vertebrates, mollusks and Drosophila all point to a common theme that involves the expression of 'clock proteins' as the basis of the timing mechanism.     

The circadian clock system in the mammalian retina

Daily rhythms are a ubiquitous feature of living systems. Generally, these rhythms are not just passive consequences of cyclic fluctuations in the environment, but instead originate within the organism. In mammals, including humans, the master pacemaker controlling 24-hour rhythms is localized in the suprachiasmatic nuclei of the hypothalamus. This circadian clock is responsible for the temporal organization of a wide variety of functions, ranging from sleep and food intake, to physiological measures such as body temperature, heart rate and hormone release. The retinal circadian clock was the first extra-SCN circadian oscillator to be discovered in mammals and several studies have now demonstrated that many of the physiological, cellular and molecular rhythms that are present within the retina are under the control of a retinal circadian clock, or more likely a network of hierarchically organized circadian clocks that are present within this tissue. BioEssays 30:624-633, 2008. (c) 2008 Wiley Periodicals, Inc.     

Circahoralian (Ultradian) metabolic rhythms

This review presents data concerning metabolic rhythms with periods close to one hour (20 to 120 min): their occurrence, biochemical organization, nature, and significance for adaptations and age-related changes of cells and organs. Circahoralian (ultradian) rhythms have been detected for cell mass and size, protein synthesis, enzyme activities, concentration of ATP and hormones, cell respiration, and cytoplasm pH. Rhythms have been observed in bacteria, yeasts, and protozoa, as well as in many cells of metazoans, including mammals, in vivo and in cell cultures. In cell populations, the rhythms are organized by direct cell-cell communication. The biochemical mechanism involves membrane signal factors and cytoplasmic processes resulting in synchronization of individual oscillations to a common rhythm. Phosphorylation of proteins is the key process of coordination of protein synthesis and enzyme activity kinetics. The fractal nature of circahoralian rhythms is discussed as well as the involvement of these rhythms in adaptations of the cells and organs. Senescent decrease in rhythm amplitudes and correspondingly in cell-cell communication has been observed. The possibility of remodeling these changes through the intercellular medium has been predicted and experimentally shown. Perspectives for studies of the organizers and disorganizers of cell-cell communication in the intercellular medium along with appropriate receptors are discussed with special emphasis on aging and pathology. One perspective can be more precise definition of the range of normal biochemical and physiological state with the goal of correction of cellular functions.     

Ultradian, circahoral and circadian structures in endothermic vertebrates and humans

1. For more than 30 years many studies have been carried out concerning rhythms with periods approaching 24 hr (circadian rhythms). 2. The latter have been demonstrated as resulting from environmental 24 hr synchronizers (zeitgebers), but they usually persist in the absence of a 24 hr synchronization, which proves their endogenous nature. 3. Biological rhythms with periods less than 20 hr (ultradian rhythms) and particularly those approaching 1 hr (circahoral rhythms) have been determined: for motility, rest-activity, sleep phases, endocrine secretions and other physiological functions. 4. These ultradian and circahoral rhythms have been found in rodents, birds, monkeys and humans. 5. Existing at all stages of ontogeny, they have been proved to be endogenous and species and strain specific. 6. As these ultradian rhythms can be influenced by environmental factors and sometimes by circadian rhythms they are not truly periodic, so therefore cannot be computed by the usual processes of mathematical time analysis.     

MULTIPLE OSCILLATORS IN THE SUPRACHIASMATIC NUCLEUS

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of the pacemaker that controls circadian rhythms of a variety of physiological functions. Data strongly indicate the majority of the SCN neurons express self-sustaining oscillations that can be detected as rhythms in the spontaneous firing of individual neurons. The period of single SCN neurons in a dissociated cell culture is dispersed in a wide range (from 20h to 28h in rats), but that of the locomotor rhythm is close to 24h, suggesting individual oscillators are coupled to generate an averaged circadian period in the nucleus. Electrical coupling via gap junctions, glial regulation, calcium spikes, ephaptic interactions, extracellular ion flux, and diffusible substances have been discussed as possible mechanisms that mediate the interneuronal rhythm synchrony. Recently, GABA (γ-aminobutyric acid), a major neurotransmitter in the SCN, was reported to regulate cellular communication and to synchronize rhythms through GABA_A receptors. At present, subsequent intracellular processes that are able to reset the genetic loop of oscillations are unknown. There may be diverse mechanisms for integrating the multiple circadian oscillators in the SCN. This article reviews the knowledge about the various circadian oscillations intrinsic to the SCN, with particular focus on the intercellular signaling of coupled oscillators. (Chronobiology International, 18(3), 371–387, 2001)     

Daily rhythms are retained both in spontaneously developed sarcomas and in xenografts grown in immunocompromised SCID mice

The circadian clock generates and regulates many daily physiological, metabolic and behavioral rhythms as well as acute responses to various types of stresses including those induced by anticancer treatment. It has been proposed that modulatory function of the clock may be used for improving the therapeutic efficacy of established anti-cancer treatments. In order to rationally exploit this mechanism, more information is needed to fully characterize the functional status of the molecular clock in tumors of different cellular origin; however, the data describing tumor clocks are still inconsistent. Here we tested the status of clock in two models of tumors derived from connective tissue: sarcomas spontaneously developed in p53-deficient mice and human fibrosarcoma cells grown as xenografts in immunocompromised severe combined immunodeficient (SCID) mice. We show that both types of tumors retain a functional clock, which is synchronized in phase with normal tissues. We also show that spontaneously developed tumors are not only oscillating in the context of an organism where they receive hormonal and metabolic signals but continue oscillating ex vivo in tissue explants demonstrating that tumors have functional clocks capable of timing all their functions. We also provide evidence that similar to liver, tumors can be synchronized by food availability independent of the central pacemaker in the suprachiasmatic nuclei (SCN). These data provide the basis for the design of anticancer therapies that take into account the circadian metabolic and physiological patterns of both the tumor and normal tissues.     

Circadian control of eclosion: interaction between a central and peripheral clock in Drosophila melanogaster

Drosophila melanogaster display overt circadian rhythms in rest:activity behavior and eclosion. These rhythms have an endogenous period of approximately 24 hr and can adjust or "entrain" to environmental inputs such as light. Circadian rhythms depend upon a functioning molecular clock that includes the core clock genes period and timeless (reviewed in and ). Although we know that a clock in the lateral neurons (LNs) of the brain controls rest:activity rhythms, the cellular basis of eclosion rhythms is less well understood. We show that the LN clock is insufficient to drive eclosion rhythms. We establish that the prothoracic gland (PG), a tissue required for fly development, contains a functional clock at the time of eclosion. This clock is required for normal eclosion rhythms. However, both the PG clock function and eclosion rhythms require the presence of LNs. In addition, we demonstrate that pigment-dispersing factor (PDF), a neuropeptide secreted from LNs, is necessary for the PG clock and eclosion rhythms. Unlike other clocks in the fly periphery, the PG is similar to mammalian peripheral oscillators because it depends upon input, including PDF, from central pacemaker cells. This is the first report of a peripheral clock necessary for a circadian event.     

A dual-color luciferase assay system reveals circadian resetting of cultured fibroblasts by co-cultured adrenal glands

In mammals, circadian rhythms of various organs and tissues are synchronized by pacemaker neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. Glucocorticoids released from the adrenal glands can synchronize circadian rhythms in other tissues. Many hormones show circadian rhythms in their plasma concentrations; however, whether organs outside the SCN can serve as master synchronizers to entrain circadian rhythms in target tissues is not well understood. To further delineate the function of the adrenal glands and the interactions of circadian rhythms in putative master synchronizing organs and their target tissues, here we report a simple co-culture system using a dual-color luciferase assay to monitor circadian rhythms separately in various explanted tissues and fibroblasts. In this system, circadian rhythms of organs and target cells were simultaneously tracked by the green-emitting beetle luciferase from Pyrearinus termitilluminans (ELuc) and the red-emitting beetle luciferase from Phrixothrix hirtus (SLR), respectively. We obtained tissues from the adrenal glands, thyroid glands, and lungs of transgenic mice that expressed ELuc under control of the promoter from a canonical clock gene, mBmal1. The tissues were co-cultured with Rat-1 fibroblasts as representative target cells expressing SLR under control of the mBmal1 promoter. Amplitudes of the circadian rhythms of Rat-1 fibroblasts were potentiated when the fibroblasts were co-cultured with adrenal gland tissue, but not when co-cultured with thyroid gland or lung tissue. The phases of Rat-1 fibroblasts were reset by application of adrenal gland tissue, whereas the phases of adrenal gland tissue were not influenced by Rat-1 fibroblasts. Furthermore, the effect of the adrenal gland tissue on the fibroblasts was blocked by application of a glucocorticoid receptor (GR) antagonist. These results demonstrate that glucocorticoids are strong circadian synchronizers for fibroblasts and that this co-culture system is a useful tool to analyze humoral communication between different tissues or cell populations.     

Hierarchically coupled ultradian oscillators generating robust circadian rhythms

Ensembles of mutually coupled ultradian cellular oscillators have been proposed by a number of authors to explain the generation of circadian rhythms in mammals. Most mathematical models using many coupled oscillators predict that the output period should vary as the square root of the number of participating units, thus being inconsistent with the well-established experimental result that ablation of substantial parts of the suprachiasmatic nuclei (SCN), the main circadian pacemaker in mammals, does not eliminate the overt circadian functions, which show no changes in the phases or periods of the rhythms. From these observations, we have developed a theoretical model that exhibits the robustness of the circadian clock to changes in the number of cells in the SCN, and that is readily adaptable to include the successful features of other known models of circadian regulation, such as the phase response curves and light resetting of the phase.     

MELATONIN CIRCADIAN RHYTHM IN THE RETINA OF MAMMALS

Melatonin has been traditionally considered to be derived principally from the pineal gland. However, several investigations have now demonstrated that melatonin synthesis occurs also in the retina (and in other organs as well) of several vertebrate classes, including mammals. As in the pineal, melatonin synthesis in the retina is elevated at night and reduced during the day. Since melatonin receptors are present in the retina and retinal melatonin does not contribute to the circulating levels, retinal melatonin probably acts locally as a neuromodulator. Melatonin synthesis in the retinas of mammals is under control of a circadian oscillator located within the retina itself, and circadian rhythms in melatonin synthesis and/or release have been described for several species of rodents. These rhythms are present in vivo, persist in vitro, are entrained by light, and are temperature compensated. The recent cloning of the gene responsible for the synthesis of the enzyme arylalkylamine N-acetyltransferase (the only enzyme unique to the melatonin synthetic pathway) will facilitate localizing the cellular site of melatonin synthesis in the retina and investigating the molecular mechanism responsible for the generation of retinal melatonin rhythmicity. Melatonin has been implicated in many retinal functions, and the levels of melatonin and dopamine appear to regulate several aspects of retinal physiology that relate to light and dark adaptation. In conclusion, it seems that retinal melatonin is involved in several functions, but its precise role is yet to be understood. (Chronobiology International, 17(5), 599–612, 2000)     

Compartmentation of NAD+-dependent signalling

NAD(+) plays central roles in energy metabolism as redox carrier. Recent research has identified important signalling functions of NAD(+) that involve its consumption. Although NAD(+) is synthesized mainly in the cytosol, nucleus and mitochondria, it has been detected also in vesicular and extracellular compartments. Three protein families that consume NAD(+) in signalling reactions have been characterized on a molecular level: ADP-ribosyltransferases (ARTs), Sirtuins (SIRTs), and NAD(+) glycohydrolases (NADases). Members of these families serve important regulatory functions in various cellular compartments, e.g., by linking the cellular energy state to gene expression in the nucleus, by regulating nitrogen metabolism in mitochondria, and by sensing tissue damage in the extracellular compartment. Distinct NAD(+) pools may be crucial for these processes. Here, we review the current knowledge about the compartmentation and biochemistry of NAD(+)-converting enzymes that control NAD(+) signalling.