Socially synchronized circadian oscillators

Daily rhythms of physiology and behaviour are governed by an endogenous timekeeping mechanism (a circadian ‘clock’). The alternation of environmental light and darkness synchronizes (entrains) these rhythms to the natural day–night cycle, and underlying mechanisms have been investigated using singly housed animals in the laboratory. But, most species ordinarily would not live out their lives in such seclusion; in their natural habitats, they interact with other individuals, and some live in colonies with highly developed social structures requiring temporal synchronization. Social cues may thus be critical to the adaptive function of the circadian system, but elucidating their role and the responsible mechanisms has proven elusive. Here, we highlight three model systems that are now being applied to understanding the biology of socially synchronized circadian oscillators: the fruitfly, with its powerful array of molecular genetic tools; the honeybee, with its complex natural society and clear division of labour; and, at a different level of biological organization, the rodent suprachiasmatic nucleus, site of the brain''s circadian clock, with its network of mutually coupled single-cell oscillators. Analyses at the ‘group’ level of circadian organization will likely generate a more complex, but ultimately more comprehensive, view of clocks and rhythms and their contribution to fitness in nature.     

A GABAergic mechanism is necessary for coupling dissociable ventral and dorsal regional oscillators within the circadian clock

BACKGROUND: Circadian rhythms in mammalian behavior, physiology, and biochemistry are controlled by the central clock of the suprachiasmatic nucleus (SCN). The clock is synchronized to environmental light-dark cycles via the retino-hypothalamic tract, which terminates predominantly in the ventral SCN of the rat. In order to understand synchronization of the clock to the external light-dark cycle, we performed ex vivo recordings of spontaneous impulse activity in SCN slices of the rat. RESULTS: We observed bimodal patterns of spontaneous impulse activity in the dorsal and ventral SCN after a 6 hr delay of the light schedule. Bisection of the SCN slice revealed a separate fast-resetting oscillator in the ventral SCN and a distinct slow-resetting oscillator in the dorsal SCN. Continuous application of the GABA(A) antagonist bicuculline yielded similar results as cut slices. Short application of bicuculline at different phases of the circadian cycle increased the electrical discharge rate in the ventral SCN but, unexpectedly, decreased activity in the dorsal SCN. CONCLUSIONS: GABA transmits phase information between the ventral and dorsal SCN oscillators. GABA can act excitatory in the dorsal SCN and inhibits neurons in the ventral SCN. We hypothesize that this difference results in asymmetrical interregional coupling within the SCN, with a stronger phase-shifting effect of the ventral on the dorsal SCN than vice versa. A model is proposed that focuses on this asymmetry and on the role of GABA in phase regulation.     

Intercellular coupling confers robustness against mutations in the SCN circadian clock network

Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor Per2 gene expression in tissues and cells from clock mutant mice. We discovered that Per1 and Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN). Per2 is also required for sustained rhythms, whereas Cry2 and Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for Per1, Per2, and Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.     

Phase angle difference alters coupling relations of functionally distinct circadian oscillators revealed by rhythm splitting

The interactions (i.e., coupling) between multiple oscillators of a circadian system determine basic properties of the integrated pacemaker. Unfortunately, there are few experimental models to investigate the putative interactions of functionally defined oscillators comprising the mammalian circadian pacemaker. Here the authors induce in hamsters a novel circadian entrainment pattern that is characterized by the daily expression of robust wheel-running activity in each scotophase of a 24-h light:dark:light:dark cycle. The daily activity bouts are mediated by 2 circadian oscillators, here designated "daytime" and "nighttime," that have been temporally dissociated under this light regime. To assess the phase dependence of interactions between oscillatory components, the phase relationship of the 2 daily scotophases was manipulated over a 4-h range, and the timing of activity of the daytime and nighttime components under entrained and probe conditions was examined. The average phase angle of entrainment and the day-to-day variability of activity onset of each activity component depended on the phase relationship of the respective scotophases and not on whether the component occurred in the daytime or the nighttime. Short-term denial of wheel access subsequently influenced amount and duration of wheel running but not timing of its onset, suggesting that only the former measures depend on a homeostatic mechanism sensitive to the time elapsed since prior intense running. Replacement of individual photophases with darkness revealed phase attraction between oscillators that was not dependent on the phase relationship of component oscillators but differed for daytime versus nighttime activity components. Entrainment patterns shown here cannot be accounted for by only nonparametric actions of light. Instead, the phase-dependent interactions of oscillators strongly influence entrainment properties, whereas intrinsic functional differences in dissociated oscillators apparently influence their attraction in darkness. This model system may be ideal for identifying genomic and physiological factors that mediate these interactions and thus contribute importantly to system properties of the mammalian circadian clock.     

Development and validation of computational models for mammalian circadian oscillators

Circadian rhythms are endogenous rhythms with a cycle length of approximately 24 h. Rhythmic production of specific proteins within pacemaker structures is the basis for these physiological and behavioral rhythms. Prior work on mathematical modeling of molecular circadian oscillators has focused on the fruit fly, Drosophila melanogaster. Recently, great advances have been made in our understanding of the molecular basis of circadian rhythms in mammals. Mathematical models of the mammalian circadian oscillator are needed to piece together diverse data, predict experimental results, and help us understand the clock as a whole. Our objectives are to develop mathematical models of the mammalian circadian oscillator, generate and test predictions from these models, gather information on the parameters needed for model development, integrate the molecular model with an existing model of the influence of light and rhythmicity on human performance, and make models available in BioSpice so that they can be easily used by the general community. Two new mammalian models have been developed, and experimental data are summarized. These studies have the potential to lead to new strategies for resetting the circadian clock. Manipulations of the circadian clock can be used to optimize performance by promoting alertness and physiological synchronization.     

Spontaneous synchronization of coupled circadian oscillators

In mammals, the circadian pacemaker, which controls daily rhythms, is located in the suprachiasmatic nucleus (SCN). Circadian oscillations are generated in individual SCN neurons by a molecular regulatory network. Cells oscillate with periods ranging from 20 to 28 h, but at the tissue level, SCN neurons display significant synchrony, suggesting a robust intercellular coupling in which neurotransmitters are assumed to play a crucial role. We present a dynamical model for the coupling of a population of circadian oscillators in the SCN. The cellular oscillator, a three-variable model, describes the core negative feedback loop of the circadian clock. The coupling mechanism is incorporated through the global level of neurotransmitter concentration. Global coupling is efficient to synchronize a population of 10,000 cells. Synchronized cells can be entrained by a 24-h light-dark cycle. Simulations of the interaction between two populations representing two regions of the SCN show that the driven population can be phase-leading. Experimentally testable predictions are: 1), phases of individual cells are governed by their intrinsic periods; and 2), efficient synchronization is achieved when the average neurotransmitter concentration would dampen individual oscillators. However, due to the global neurotransmitter oscillation, cells are effectively synchronized.     

Are circadian oscillators structurally unstable?

The common belief is that all biological oscillations are of limit cycle type. It is shown in this article that the phase response curves simulated on a two-species Lotka-Volterra linear (i.e. non-limit cycle type) oscillator, do look similar to those obtained by experimental methods by different workers. The form of the phase response curves, the existence of singularities and the mirror-image symmetry of opposite perturbations are modelled on the Lotka-Volterra system. The study, which is strongly indicative of the possibility that the underlying oscillator (or oscillators) is (are) not structurally stable, also indicates the necessity of designing critical experiments, capable of distinguishing between limit cycle and non-limit cycle oscillators, since the single-pulse phase resetting does nothing to distinguish between them.     

Intercellular synchronization of diffusively coupled Ca2+ oscillators

We examine the synchrony in the dynamics of localized [Ca^2 +]_i oscillations among a group of cells exhibiting such complex Ca^2 + oscillations, connected in the form of long chain, via diffusing coupling where cytosolic Ca^2 + and inositol 1,4,5-triphosphate are coupling molecules. Based on our numerical results, we could able to identify three regimes, namely desynchronized, transition and synchronized regimes in the (T − k_e) (time period-coupling constant) and (A − k_e) (amplitude-coupling constant) spaces which are supported by phase plots (Δϕ verses time) and recurrence plots, respectively. We further show the increase of synchronization among the cells as the number of coupling molecules increases in the (T − k_e) and (A − k_e) spaces.     

Populations of biochemical oscillators as circadian clocks

Various types of populations of interacting oscillators were analyzed and their synchronization states were determined. One of the systems involving biochemical oscillators was simulated on the computer and the occurence of rhythm splitting was observed. A comparison of its attributes with experimental results on circadian ryhythms showed good agreement. This allows us to distinguish between types of mechanisms held responsible for the splitting phenomenon in the past. The present model also offers a new explanation about the differences of light action on diurnal and nocturnal organisms.     

Melatonin: A coupling device between oscillators in the circadian system of the ruin lizard Podarcis sicula

• 1.1. Chronic administration of melatonin (in silastic capsules) lengthened the free-running period of the locomotor rhythm and shortened the circadian activity time in Podarcis sicula held in constant temperature and darkness.
• 2.2. Lizards displaying a bimodal pattern of activity invariably became unimodal after melatonin administration.
• 3.3. The results support the hypothesis that melatonin acts as a coupling device between circadian oscillators driving the locomotor rhythm in Podarcis sicula.

     

Temperature-sensitive and circadian oscillators of Neurospora crassa share components

In Neurospora crassa, the interactions between products of the frequency (frq), frequency-interacting RNA helicase (frh), white collar-1 (wc-1), and white collar-2 (wc-2) genes establish a molecular circadian clockwork, called the FRQ-WC-Oscillator (FWO), which is required for the generation of molecular and overt circadian rhythmicity. In strains carrying nonfunctional frq alleles, circadian rhythms in asexual spore development (conidiation) are abolished in constant conditions, yet conidiation remains rhythmic in temperature cycles. Certain characteristics of these temperature-synchronized rhythms have been attributed to the activity of a FRQ-less oscillator (FLO). The molecular components of this FLO are as yet unknown. To test whether the FLO depends on other circadian clock components, we created a strain that carries deletions in the frq, wc-1, wc-2, and vivid (vvd) genes. Conidiation in this ΔFWO strain was still synchronized to cyclic temperature programs, but temperature-induced rhythmicity was distinct from that seen in single frq knockout strains. These results and other evidence presented indicate that components of the FWO are part of the temperature-induced FLO.     

Peripheral circadian oscillators in mammals: time and food

Peripheral cells from mammalian tissues, while perfectly capable of circadian rhythm generation, are not light sensitive and thus have to be entrained by nonphotic cues. Feeding time is the dominant zeitgeber for peripheral mammalian clocks: Daytime feeding of nocturnal laboratory rodents completely inverts the phase of circadian gene expression in many tissues, including liver, heart, kidney, and pancreas, but it has no effect on the SCN pacemaker. It is thus plausible that in intact animals, the SCN synchronizes peripheral docks primarily through temporal feeding patterns that are imposed through behavioral rest-activity cycles. In addition, body temperature rhythms, which are themselves dependent on both feeding patterns and rest-activity cycles, can sustain circadian, clock gene activity in vivo and in vitro. The SCN may also influence the phase of rhythmic gene expression in peripheral tissues through direct chemical pathways. In fact, many chemical signals induce circadian gene expression in tissue culture cells. Some of these have been shown to elicit phase shifts when injected into intact animals and are thus candidates for physiologically relevant timing cues. While the response of the SCN to light is strictly gated to respond only during the night, peripheral oscillators can be chemically phase shifted throughout the day. For example, injection of dexamethasone, a glucocorticoid receptor agonist, resets the phase of circadian liver gene expression during the entire 24-h day. Given the bewildering array of agents capable of influencing peripheral clocks, the identification of physiologically relevant agents used by the SCN to synchronize peripheral clocks will clearly be an arduous undertaking. Nevertheless, we feel that experimental systems by which this enticing problem can be tackled are now at hand.