Beyond intuitive modeling: combining biophysical models with innovative experiments to move the circadian clock field forward

Two major approaches have been used to model circadian clocks. Qualitative modeling, used prior to the recent wealth of detailed molecular knowledge, makes general predictions but cannot provide detailed mechanistic insights. The more recent biophysical approach, on the other hand, incorporates the biochemical events that drive the clock and can make detailed and testable molecular predictions. These predictions are being tested using new experimental techniques that measure reaction kinetics and the behavior of individual cells. A joint modeling and experimental approach has recently been used to understand how mutations affecting phosphorylation can lead to a short circadian period in tau mutant hamsters and in humans with familial advanced sleep phase syndrome (FASPS). Another recent study has revealed novel single-cell phenotypes of clock gene mutations, demanding revision of current biophysical models yet validating certain model predictions that were previously overlooked. A new paradigm for clock research is emerging in which modeling inspires new experimental efforts, experimental data inspire new modeling efforts, and joint modeling/experimental studies lead to a deeper understanding of mammalian circadian rhythms.     

Development of a two-dimension manifold to represent high dimension mathematical models of the intracellular Mammalian circadian clock

A new focus for mathematical models of the circadian pacemaker involves the encapsulation within the models of detailed biological processes responsible for generating those circadian rhythms. Representing greater biological detail requires more mathematical equations, which pose a greater challenge for the analysis of such systems. Development of a method that retains the predominant dynamics while still providing biologically detailed information is advantageous. Two high-dimension mathematical models of intracellular mammalian circadian pacemakers, Leloup-Goldbeter and Forger-Peskin, with 19 and 73 differential equations, respectively, have been published. The authors projected each of these high-dimension models onto their respective manifold using proper orthogonal functions (POFs) obtained from the empirical decomposition of the model's phase space to obtain a 2-dimension model. The resulting 2-dimension model, represented by 2 differential equations, predicts most of the salient characteristics of a biological clock including approximately 24-h oscillations, entrainment to an LD cycle, phase response curves, and the amplitude recovery dynamics that emerge following amplitude suppression. The manifold representation simplifies the mathematical analysis, since only 2 variables need to be observed and analyzed to understand the behavior of the biological clock. This reduced model derived from a model based on biological variables can be used for the development and analysis of mathematical models of the coupled mammalian oscillators to understand the dynamics of the integrated circadian pacemaker.     

Chlamydomonas reinhardtii as a unicellular model for circadian rhythm analysis

Chlamydomonas reinhardtii has been used as an experimental model organism for circadian rhythm research for more than 30 yr. Some of the physiological rhythms of this alga are well established, and several clock mutants have been isolated. The cloning of clock genes from these mutant strains by positional cloning is under way and should give new insights into the mechanism of the circadian clock. In a spectacular space experiment, the question of the existence of an endogenous clock vs. an exogenous mechanism has been studied in this organism. With the emergence of molecular analysis of circadian rhythms in plants in 1985, a circadian gene expression pattern of several nuclear and chloroplast genes was detected. Evidence is now accumulating that shows circadian control at the translational level. In addition, the gating of the cell cycle by the circadian clock has been analyzed. This review focuses on the different aspects of circadian rhythm research in C. reinhardtii over the past 30 yr. The suitability of Chlamydomonas as a model system in chronobiology research and the adaptive significance of the observed rhythms will be discussed.     

Chlamydomonas reinhardtii as a unicellular model for circadian rhythm analysis

Chlamydomonas reinhardtii has been used as an experimental model organism for circadian rhythm research for more than 30 yr. Some of the physiological rhythms of this alga are well established, and several clock mutants have been isolated. The cloning of clock genes from these mutant strains by positional cloning is under way and should give new insights into the mechanism of the circadian clock. In a spectacular space experiment, the question of the existence of an endogenous clock vs. an exogenous mechanism has been studied in this organism. With the emergence of molecular analysis of circadian rhythms in plants in 1985, a circadian gene expression pattern of several nuclear and chloroplast genes was detected. Evidence is now accumulating that shows circadian control at the translational level. In addition, the gating of the cell cycle by the circadian clock has been analyzed. This review focuses on the different aspects of circadian rhythm research in C. reinhardtii over the past 30 yr. The suitability of Chlamydomonas as a model system in chronobiology research and the adaptive significance of the observed rhythms will be discussed.     

The current state and problems of circadian clock studies in cyanobacteria

Circadian rhythms have been observed in innumerable physiological processes in most of organisms. Recent molecular and genetic studies on circadian clocks in many organisms have identified and characterized several molecular regulatory factors that contribute to generation of such rhythms. The cyanobacterium is the simplest organism known to harbor circadian clocks, and it has become one of most successful model organisms for circadian biology. In this review, we will briefly summarize physiological observations and consideration of circadian rhythms in cyanobacteria, molecular genetics of the clock using Synechococcus, and current knowledge of the input and output pathways that support the cellular circadian system. Finally, we will document some current problems in the studies on the cyanobacterial circadian clock.     

Circadian rhythms: perturbing a food-entrained clock

When food is scarce, a food-entrainable circadian clock coordinates mammalian activity rhythms with a predictable daily mealtime. Neural and molecular substrates of this circadian function have long eluded localization, but new studies suggest a critical role for a familiar circadian clock gene.     

Modeling the emergence of circadian rhythms in a clock neuron network

Circadian rhythms in pacemaker cells persist for weeks in constant darkness, while in other types of cells the molecular oscillations that underlie circadian rhythms damp rapidly under the same conditions. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms leading to damped or self-sustained oscillations remain largely unknown. There exist many mathematical models that reproduce the circadian rhythms in the case of a single cell of the Drosophila fly. However, not much is known about the mechanisms leading to coherent circadian oscillation in clock neuron networks. In this work we have implemented a model for a network of interacting clock neurons to describe the emergence (or damping) of circadian rhythms in Drosophila fly, in the absence of zeitgebers. Our model consists of an array of pacemakers that interact through the modulation of some parameters by a network feedback. The individual pacemakers are described by a well-known biochemical model for circadian oscillation, to which we have added degradation of PER protein by light and multiplicative noise. The network feedback is the PER protein level averaged over the whole network. In particular, we have investigated the effect of modulation of the parameters associated with (i) the control of net entrance of PER into the nucleus and (ii) the non-photic degradation of PER. Our results indicate that the modulation of PER entrance into the nucleus allows the synchronization of clock neurons, leading to coherent circadian oscillations under constant dark condition. On the other hand, the modulation of non-photic degradation cannot reset the phases of individual clocks subjected to intrinsic biochemical noise.     

Modeling the influence of circadian rhythms on the acute inflammatory response

A wide variety of modeling techniques have been applied towards understanding inflammation. These models have broad potential applications, from optimizing clinical trials to improving clinical care. Models have been developed to study specific systems and diseases, but the effect of circadian rhythms on the inflammatory response has not been modeled. Circadian rhythms are normal biological variations obeying the 24-h light/dark cycle and have been shown to play a critical role in the treatment and progression of many diseases. Several of the key components of the inflammatory response, including cytokines and hormones, have been observed to undergo significant diurnal variations in plasma concentration. It is hypothesized that these diurnal rhythms are entrained by the cyclic production of the hormones cortisol and melatonin, as stimulated by the central clock in the suprachiasmatic nucleus. Based on this hypothesis, a mathematical model of the interplay between inflammation and circadian rhythms is developed. The model is validated by its ability to reproduce diverse sets of experimental data and clinical observations concerning the temporal sensitivity of the inflammatory response.     

Entrainment of circadian clocks in mammals by arousal and food

Circadian rhythms in mammals are regulated by a system of endogenous circadian oscillators (clock cells) in the brain and in most peripheral organs and tissues. One group of clock cells in the hypothalamic SCN (suprachiasmatic nuclei) functions as a pacemaker for co-ordinating the timing of oscillators elsewhere in the brain and body. This master clock can be reset and entrained by daily LD (light-dark) cycles and thereby also serves to interface internal with external time, ensuring an appropriate alignment of behavioural and physiological rhythms with the solar day. Two features of the mammalian circadian system provide flexibility in circadian programming to exploit temporal regularities of social stimuli or food availability. One feature is the sensitivity of the SCN pacemaker to behavioural arousal stimulated during the usual sleep period, which can reset its phase and modulate its response to LD stimuli. Neural pathways from the brainstem and thalamus mediate these effects by releasing neurochemicals that inhibit retinal inputs to the SCN clock or that alter clock-gene expression in SCN clock cells. A second feature is the sensitivity of circadian oscillators outside of the SCN to stimuli associated with food intake, which enables animals to uncouple rhythms of behaviour and physiology from LD cycles and align these with predictable daily mealtimes. The location of oscillators necessary for food-entrained behavioural rhythms is not yet certain. Persistence of these rhythms in mice with clock-gene mutations that disable the SCN pacemaker suggests diversity in the molecular basis of light- and food-entrainable clocks.     

When clocks go bad: neurobehavioural consequences of disrupted circadian timing

Progress in unravelling the cellular and molecular basis of mammalian circadian regulation over the past decade has provided us with new avenues through which we can explore central nervous system disease. Deteriorations in measurable circadian output parameters, such as sleep/wake deficits and dysregulation of circulating hormone levels, are common features of most central nervous system disorders. At the core of the mammalian circadian system is a complex of molecular oscillations within the hypothalamic suprachiasmatic nucleus. These oscillations are modifiable by afferent signals from the environment, and integrated signals are subsequently conveyed to remote central neural circuits where specific output rhythms are regulated. Mutations in circadian genes in mice can disturb both molecular oscillations and measurable output rhythms. Moreover, systematic analysis of these mutants indicates that they can express an array of abnormal behavioural phenotypes that are intermediate signatures of central nervous system disorders. Furthermore, the response of these mutants to psychoactive drugs suggests that clock genes can modify a number of the brain’s critical neurotransmitter systems. This evidence has led to promising investigations into clock gene polymorphisms in psychiatric disease. Preliminary indications favour the systematic investigation of the contribution of circadian genes to central nervous system disease.     

Circadian rhythms in the retina of rats with photoreceptor degeneration

Previous studies have demonstrated that the mammalian retina contains a circadian clock system that controls several retinal functions. In mammals the location of the retinal circadian clock is unknown whereas, in non-mammalian vertebrates, earlier work has demonstrated that photoreceptor cells contain the circadian clock. New experimental evidence has suggested that in mammals the retinal circadian clock may be located outside the photoreceptor cells. In this study we report that circadian rhythms in Aa-nat mRNA (in vivo) and melatonin synthesis (in vitro) are still present in the retina of rats lacking photoreceptors. The circadian pacemaker(s) controlling such rhythms is probably located in kainic acid sensitive neurons in the inner retina since kainic acid injections abolished the rhythmicity. These data are the first direct demonstration that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors and the suprachiasmatic nuclei of the hypothalamus.     

Modulation of circadian clocks by nutrients and food factors

Daily activity rhythms that are dominated by internal clocks are called circadian rhythms. A central clock is located in the suprachiasmatic nucleus of the hypothalamus, and peripheral clocks are located in most mammalian peripheral cells. The central clock is entrained by light/dark cycles, whereas peripheral clocks are entrained by feeding cycles. The effects of nutrients on the central and peripheral clocks have been investigated during the past decade and much interaction between them has come to light. For example, a high-fat diet prolongs the period of circadian behavior, a ketogenic diet advances the onset of locomotor activity rhythms, and a high-salt diet advances the phase of peripheral molecular clocks. Moreover, some food factors such as caffeine, nobiletin, and resveratrol, alter molecular and/or behavioral circadian rhythms. Here, we review nutrients and food factors that modulate mammalian circadian clocks from the cellular to the behavioral level.     

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.     

Latitudinal clines: an evolutionary view on biological rhythms

Properties of the circadian and annual timing systems are expected to vary systematically with latitude on the basis of different annual light and temperature patterns at higher latitudes, creating specific selection pressures. We review literature with respect to latitudinal clines in circadian phenotypes as well as in polymorphisms of circadian clock genes and their possible association with annual timing. The use of latitudinal (and altitudinal) clines in identifying selective forces acting on biological rhythms is discussed, and we evaluate how these studies can reveal novel molecular and physiological components of these rhythms.