In Synch but Not in Step: Circadian Clock Circuits Regulating Plasticity in Daily Rhythms

The suprachiasmatic nucleus (SCN) is a network of neural oscillators that program daily rhythms in mammalian behavior and physiology. Over the last decade much has been learned about how SCN clock neurons coordinate together in time and space to form a cohesive population. Despite this insight, much remains unknown about how SCN neurons communicate with one another to produce emergent properties of the network. Here we review the current understanding of communication among SCN clock cells and highlight a collection of formal assays where changes in SCN interactions provide for plasticity in the waveform of circadian rhythms in behavior. Future studies that pair analytical behavioral assays with modern neuroscience techniques have the potential to provide deeper insight into SCN circuit mechanisms

Discovering circadian clocks in microbes

We humans experience the influence of our circadian clock every day. This clock mechanism causes, for example, a jet lag during transatlantic air travel. We now believe that almost all organisms have developed a circadian clock mechanism.In this thesis I describe the analysis techniques we developed and the newly discovered molecular components of a circadian mechanism in Saccharomyces cerevisiae and Bacillus subtilis. To identify these molecular components, I applied structured zeitgebers, i.e. light and temperature cycling, to yeast and bacillus cultures. All this in conjunction with bioinformatic in-silico approachesIn Bacillus biofilm populations, we found a free-running rhythm of ytvA and KinC activity of nearly 24 hours after entrainment and release to constant dark and temperature conditions. The free-running oscillations are temperature compensated. This is one of the most important features of a circadian clock mechanism, making it very likely that such a system exists in B. subtilis.We found in yeasts that temperature appears to mainly regulate metabolic processes. Light appears to act more indirectly via photo-oxidation of mitochondrial cytochromes.Finally, I present a hypothetical model for an integrated circadian clock mechanism in unicellular microbes with an emphasis on S. cerevisiae. This mechanism involves several metabolic pathways and the main regulator is the stress sensitive transcriptional activator Msn2p. The model shows that in the circadian clock mechanism in yeast, energy metabolism appears to be an important theme. Other processes that are relevant: metabolic process of nitrogen compounds, oxidation-reduction process and fatty acid metabolism. All could serve as a starting point for further research on the circadian clock in yeast

Investigation of Circadian Clock in Peripheral Tissues and Immune-Circadian Interaction in the Domestic Fowl, Gallus Domesticus

The circadian system provides living organisms a means to adapt their internal physiology to constantly changing environmental conditions that exists on our rotating planet, Earth. Clocks in peripheral tissues are referred to as peripheral which may participate in tissue-specific functions. The first step to investigating the circadian regulation in the peripheral tissues of avians was to examine for the presence of avian orthologs of core components of the molecular clock using Quantitative real time (qRTPCR) assays. We investigated the avian spleen for daily and circadian control of core clock genes and regulation of the inflammatory response by the spleen clock. The core clock genes, bmal1, bmal2, per2, per3 and clock displayed both daily and circadian rhythms. Proinflammatory cytokines TNFα, IL-1β, IL-6 and IL-18 exhibited daily and circadian rhythmic oscillations. A differential expression of proinflammatory cytokine induction was observed in the spleen undergoing lipopolysaccharide (LPS)-induced acute inflammation. Exogenous melatonin administration during inflammation seems to enhance some and repress a few inflammatory cytokines, implying that melatonin is pleiotropic molecule. To compare and contrast the role of peripheral clocks in regulating energy balance and reproduction in layer vs. broiler chicken, the visceral adipose tissue (VAT), ovary and hypothalamus were examined for the presence of core clock genes were investigated in these two lines of poultry birds. Quantitative RT-PCR was employed to examine daily control of core clock genes in these three peripheral tissues over a 24hr period. The layer hens exhibit rhythmic oscillations in the mRNA abundance of the core clock genes in the VAT, ovary and the hypothalamus. The hypothalamus and VAT of the broiler hens exhibit rhythmic mRNA abundance of the core clock genes. However, the clock genes in the ovary of the broiler pullets exhibit marked reduction in their amplitude and rhythms over a 24hr period. The broiler hens are prone to poor energy balance, obesity and reproductive capacity. In summary, these data provide evidence for a functional link between the circadian clock and the ovary by determining clock gene regulation under conditions of disrupted or eliminated reproductive function vs. normal reproductive output

Light-Dependent Development of Circadian Gene Expression in Transgenic Zebrafish

The roles of environmental stimuli in initiation and synchronization of circadian oscillation during development appear to vary among different rhythmic processes. In zebrafish, a variety of rhythms emerge in larvae only after exposure to light-dark (LD) cycles, whereas zebrafish period3 (per3) mRNA has been reported to be rhythmic from day 1 of development in constant conditions. We generated transgenic zebrafish in which expression of the firefly luciferase (luc) gene is driven by the zebrafish per3 promoter. Live larvae from these lines are rhythmically bioluminescent, providing the first vertebrate system for high-throughput measurement of circadian gene expression in vivo. Circadian rhythmicity in constant conditions was observed only after 5–6 d of development, and only if the fish were exposed to LD signals after day 4. Regardless of light exposure, a novel developmental profile was observed, with low expression during the first few days and a rapid increase when active swimming begins. Ambient temperature affected the developmental profile and overall levels of per3 and luc mRNA, as well as the critical days in which LD cycles were needed for robust bioluminescence rhythms. In summary, per3-luc zebrafish has revealed complex interactions among developmental events, light, and temperature in the expression of a clock gene

Searching for Order in Body Clocks

Physiological rhythms are central to life. Mammalian behavior and metabolism are organized around the day and night by the regulated action of cell-autonomous clocks that exist throughout our bodies. At the core of this molecular clockwork are multiple coupled feedback loops that generate sustained circadian rhythms in gene expression to ultimately orchestrate mammalian physiology. In this work we provide evidence for the role of metabolism in regulating the core clock. We present genes involved in energetic and redox pathways which we identified to be essential for the robustness of cellular timekeepers to temperature fluctuations. We developed the first computational model for circadian redox oscillations that contributes to the understanding of how cellular redox balance might adjust circadian rates in response to perturbations and convey timing information to the core molecular oscillator. Moreover, we show that our mathematical model can be coupled with prior published models of the transcriptional clockwork resulting in 1:1 entrainment. This experimental-theoretical approach exemplifies the need of a dynamic analysis at the system level to understand complex biological processes and provides insights into how basic timekeeping mechanisms are integrated into cellular physiology. Such knowledge might highlight new ways by which functional consequences of circadian timekeeping can be explored in the context of human health and disease

A spatio-temporal model to reveal oscillator phenotypes in molecular clocks: Parameter estimation elucidates circadian gene transcription dynamics in single-cells.

We propose a stochastic distributed delay model together with a Markov random field prior and a measurement model for bioluminescence-reporting to analyse spatio-temporal gene expression in intact networks of cells. The model describes the oscillating time evolution of molecular mRNA counts through a negative transcriptional-translational feedback loop encoded in a chemical Langevin equation with a probabilistic delay distribution. The model is extended spatially by means of a multiplicative random effects model with a first order Markov random field prior distribution. Our methodology effectively separates intrinsic molecular noise, measurement noise, and extrinsic noise and phenotypic variation driving cell heterogeneity, while being amenable to parameter identification and inference. Based on the single-cell model we propose a novel computational stability analysis that allows us to infer two key characteristics, namely the robustness of the oscillations, i.e. whether the reaction network exhibits sustained or damped oscillations, and the profile of the regulation, i.e. whether the inhibition occurs over time in a more distributed versus a more direct manner, which affects the cells' ability to phase-shift to new schedules. We show how insight into the spatio-temporal characteristics of the circadian feedback loop in the suprachiasmatic nucleus (SCN) can be gained by applying the methodology to bioluminescence-reported expression of the circadian core clock gene Cry1 across mouse SCN tissue. We find that while (almost) all SCN neurons exhibit robust cell-autonomous oscillations, the parameters that are associated with the regulatory transcription profile give rise to a spatial division of the tissue between the central region whose oscillations are resilient to perturbation in the sense that they maintain a high degree of synchronicity, and the dorsal region which appears to phase shift in a more diversified way as a response to large perturbations and thus could be more amenable to entrainment

Evolving a 24-hr oscillator in budding yeast

We asked how a new, complex trait evolves by selecting for diurnal oscillations in the budding yeast, Saccharomyces cerevisiae. We expressed yellow fluorescent protein (YFP) from a yeast promoter and selected for a regular alternation between low and high fluorescence over a 24-hr period. This selection produced changes in cell adhesion rather than YFP expression: clonal populations oscillated between single cells and multicellular clumps. The oscillations are not a response to environmental cues and continue for at least three cycles in a constant environment. We identified eight putative causative mutations in one clone and recreated the evolved phenotype in the ancestral strain. The mutated genes lack obvious relationships to each other, but multiple lineages change from the haploid to the diploid pattern of gene expression. We show that a novel, complex phenotype can evolve by small sets of mutations in genes whose molecular functions appear to be unrelated to each other. DOI: http://dx.doi.org/10.7554/eLife.04875.00

Circadian rhythms in the auditory system

Circadian clocks have been found in numerous cell types and tissues of the body, allowing organisms to coordinate their daily biological functions. They generate cycles of behavioral and physiological processes with a period length of ~24h, which are synchronized to the rotation of the Earth and the subsequent daily changes in illumination, temperature and humidity. The circadian system evolved in order to ensure anticipation and adaptation to these predictable environmental changes, thereby optimizing physiological functions. The current view is that all the cellular clocks of the body are organized in a hierarchical system, which consists of a central master clock and a network of peripheral clocks found in different organs. The circadian organization facilitates temporal control of physiological functions and ensures tissue homeostasis. Prior to this thesis, the consequences of circadian regulation in the auditory system had not been deeply explored. The studies presented here are aiming to investigate the role of the circadian rhythmicity in regulating auditory function. We focused on mechanisms underlying noise sensitivity, involving neurotrophic factors and glucocorticoid hormones. Our results demonstrate that circadian rhythms play a significant role in modulating auditory sensitivity to noise trauma. Mice were found to be more vulnerable to night noise exposure, which triggered permanent hearing loss, whereas day-exposed mice recovered to normal hearing thresholds. This diurnal variation was associated with the presence of a circadian cochlear clock and the circadian control of the brain-derived neurotrophic factor (BDNF). We next found a molecular clock machinery in a central auditory structure, the inferior colliculus (IC), which demonstrated a differential response to day or night noise trauma, and was independent from that of the cochlear clock. Focusing on the cochlear clock, we next identified self-sustained single-cell oscillators originating from sensory and neuronal populations. Cellular clocks were tonotopically arranged, suggesting that networks of individual oscillators may organize circadian rhythms along the length of the cochlea. Finally, we examined the interaction between glucocorticoids and the cochlear clock in regulating the diurnal sensitivity to noise. We found that the absence of circadian glucocorticoid rhythms abolished the greater vulnerability to noise trauma at night, as hearing thresholds recovered completely. This response was linked to glucocorticoid-dependent control of inflammatory cochlear genes. Finally, treatment with the synthetic glucocorticoid dexamethasone at day time, but not at night, protected against noise damage, highlighting the importance of endogenous glucocorticoid rhythms on the effects of otoprotective drugs. In summary, sensitivity to noise insults is greater at specific phases of the circadian cycle, both at behavioral and molecular level and is mediated through complex interaction between circadian clocks, BDNF and glucocorticoids. Overall this thesis is describing a novel feature of the auditory system that would likely have major clinical implications

Interaction of external forcing and noise in bio-inspired oscillator systems

Biologische und bioinspirierte Oszillatoren sind faszinierende Systeme, die aus technischer Sicht untersucht werden sollten. Biologische Oszillatoren sind von Natur aus verrauscht, erzeugen aber dennoch stabile Schwingungsrhythmen und passen ihre Periode an die Periode der Eingangssignale an, ein Prozess, der als Entrainment bekannt ist. Oszillatorsysteme kommen nicht nur in der Natur, sondern auch in vielen vom Menschen geschaffenen Systemen vor, zum Beispiel in Bewegungsgeneratoren für Roboter oder in elektrischen Schaltkreisen. Daher ist ein besseres Verständnis der Konstruktionsprinzipien biologischer Oszillatoren und ihrer Strategien, mit Rauschen umzugehen oder es sogar zu nutzen, um ihr Entrainment an die Eingangssignale zu unterstützen, für ein besseres Verständnis der oszillatorischen Systeme und ihrer technischen Anwendungen unerlässlich. Der Hauptbeitrag der vorliegenden Arbeit ist die numerische Untersuchung und Analyse einer Population stochastischer Oszillatoren unter wechselnden Parametern des Eingangssignals und variierender Rauschintensität. Das theoretische Kapitel der Arbeit zeigt, dass Rauschen die Empfindlichkeit gegenüber schwachen externen Signalen erhöhen kann und somit die Anpassung an einen größeren Bereich von Eingangsamplituden und -perioden im Vergleich zu einem äquivalenten deterministischen System ermöglicht. Das Rauschen erhöht auch die Phasenreaktion auf einen stufenförmigen Eingangsimpuls und beschleunigt die Erholung von einer Jet-Lag-artigen Störung. Es wird ferner gezeigt, dass diese Effekte nicht nur auf biologische Oszillatoren beschränkt sind, sondern auch für eine größere Anzahl von generischen Oszillatorsystemen mit einem Grenzzyklus zu gelten scheinen. Im letzten Teil des theoretischen Kapitels wird ein neuartiger schrittweiser Anpassungsalgorithmus vorgestellt, der eine Parameteranpassung von stochastischen Oszillatorpopulationen ermöglicht. Alle im theoretischen Kapitel entwickelten Methoden wurden als Open-Source-Softwarepaket zur Verfügung gestellt. Im letzten Teil der Arbeit wird eine praktische Anwendung für die entwickelten Methoden vorgestellt. Hier wird ein stochastisches Gleichungsmodell entwickelt, um die Tag-Nacht-Rhythmen in Zebrafisch-Zelllinien zu untersuchen. Das Modell wird anschließend verwendet, um festzustellen, wie verschiedene Medikamente die Synchronisation und Stochastizität der biologischen Uhr beeinflussen