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.     

Seasonal molecular timekeeping within the rat circadian clock

In temperate zones duration of daylight, i.e. photoperiod, changes with the seasons. The changing photoperiod affects animal as well as human physiology. All mammals exhibit circadian rhythms and a circadian clock controlling the rhythms is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN consists of two parts differing morphologically and functionally, namely of the ventrolateral (VL) and the dorsomedial (DM). Many aspects of SCN-driven rhythmicity are affected by the photoperiod. The aim of the present overview is to summarize data about the effect of the photoperiod on the molecular timekeeping mechanism in the rat SCN, especially the effect on core clock genes, clock-controlled genes and clock-related genes expression. The summarized data indicate that the photoperiod affects i) clock-driven rhythm in photoinduction of c-fos gene and its protein product within the VL SCN, ii) clock-driven spontaneous rhythms in clock-controlled, i.e. arginine-vasopressin, and in clock-related, i.e. c-fos, gene expression within the DM SCN, and iii) the core clockwork mechanism within the rat SCN. Hence, the whole central timekeeping mechanism within the rat circadian clock measures not only the daytime but also the time of the year, i.e. the actual season.     

Identification of the suprachiasmatic nucleus in birds

Circadian rhythms are generated by an internal biological clock. The suprachiasmatic nucleus (SCN) in the hypothalamus is known to be the dominant biological clock regulating circadian rhythms in mammals. In birds, two nuclei, the so-called medial SCN (mSCN) and the visual SCN (vSCN), have both been proposed to be the avian SCN. However, it remains an unsettled question which nuclei are homologous to the mammalian SCN. We have identified circadian clock genes in Japanese quail and demonstrated that these genes are expressed in known circadian oscillators, the pineal and the retina. Here, we report that these clock genes are expressed in the mSCN but not in the vSCN in Japanese quail, Java sparrow, chicken, and pigeon. In addition, mSCN lesions eliminated or disorganized circadian rhythms of locomotor activity under constant dim light, but did not eliminate entrainment under light-dark (LD) cycles in pigeon. However, the lesioned birds became completely arrhythmic even under LD after the pineal and the eye were removed. These results indicate that the mSCN is a circadian oscillator in birds.     

Signalling entrains the peripheral circadian clock

In mammals, 24-h rhythms of behaviour and physiology are regulated by the circadian clock. The circadian clock is controlled by a central clock in the brain's suprachiasmatic nucleus (SCN) that synchronizes peripheral clocks in peripheral tissues. Clock genes in the SCN are primarily entrained by light. Increasing evidence has shown that peripheral clocks are also regulated by light and hormones independent of the SCN. How the peripheral clocks deal with internal signals is dependent on the relevance of a specific cue to a specific tissue. In different tissues, most genes that are under circadian control are not overlapping, revealing the tissue-specific control of peripheral clocks. We will discuss how different signals control the peripheral clocks in different peripheral tissues, such as the liver, gastrointestinal tract, and pancreas, and discuss the organ-to-organ communication between the peripheral clocks at the molecular level.     

Clock genes, oscillators, and cellular networks in the suprachiasmatic nuclei

The mammalian SCN contains a biological clock that drives remarkably precise circadian rhythms in vivo and in vitro. Recent advances have revealed molecular and cellular mechanisms required for the generation of these daily rhythms and their synchronization between SCN neurons and to the environmental light cycle. This review of the evidence for a cell-autonomous circadian pacemaker within specialized neurons of the SCN focuses on 6 genes implicated within the pace making mechanism, an additional 4 genes implicated in pathways from the pacemaker, and the intercellular and intracellular mechanisms that synchronize SCN neurons to each other and to solar time.     

Postnatal ontogenesis of the circadian clock within the rat liver

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbalpha, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbalpha expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbalpha, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.     

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.     

Establishment of cell lines derived from the rat suprachiasmatic nucleus

Physiological and behavioral circadian rhythms in mammals are orchestrated by a central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Photic input entrains the phase of the central clock, and many peripheral clocks are regulated by neural or hormonal output from the SCN. We established cell lines derived from the rat embryonic SCN to examine the molecular network of the central clock. An established cell line exhibited the stable circadian expression of clock genes. The circadian oscillation was abruptly phase-shifted by forskolin, and abolished by siBmal1. These results are compatible with in vivo studies of the SCN.