Eliciting the mechanisms by which circadian rhythms are regulated in mammals has involved key work from several disciplines of neurobiology. Endocrinological, neuroanatomical, molecular biological and genetic studies over the past 40 years have all furthered our understanding of a system which impacts greatly upon mammalian biology and behaviour. The realisation that there must be some 'master-clock' within the animal CNS led to a series of detailed experiments to elicit its location. Once the suprachiasmatic nucleus (SCN) of the hypothalamus had been identified as the area crucial to circadian rhythms it was several years before molecular techniques could be applied to ascertain the core genes and their protein products involved in the 'clock' cycle. Several key studies have found that a complex interplay of negative and positive feedback mechanisms between protein complexes and gene expression is crucial to the timing of this cycle. Genetic mutation studies carried out in both Drosophila melanogaster and rodents, provide further evidence for an individual cellular basis for the biological 'clock'. There is still an incomplete understanding of how the individual cycles within neurons of the SCN are coordinated to bring about a circadian effect on various body systems including temperature, autonomic regulation and aspects of the sleep cycle.
Anatomy of the SCN
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The SCN is a paired nucleus, located on either side of the 3rd ventricle in the hypothalamus, just above the optic chiasm (1). Its proximity to the optic tracts, from which it receives afferent projections, and the hypothalamus and midbrain to which it gives efferent projections reflects its function as a coordinator of environmental signals and bodily functions. Two distinct regions have been identified within the SCN, referred to as the shell and the core. The shell region is located in the dorsal and medial parts of the nucleus and possesses smaller neurons with less dendritic arbors than the core (1). The main neurotransmitter throughout the SCN is GABA although the shell also expresses Arginine Vasopressin (AVP). The shell receives afferents from the hypothalamus, basal forebrain, limbic system and other parts of the SCN.
The core possesses more neurons of a greater size than the shell with more extensive dendritic processes. As well as expressing GABA, the core uses Vasoactive Intestinal Peptide (VIP) and GRP as neurotransmitters. The main afferent connections it receives are from the retinohypothalamic tract (RHT) and intergeniculate leaflet, it also receives input from serotonergic neurons of the Raphé nucleus in the midbrain. There is debate as to the functional differences between these two diverse areas as both core and shell neurons can express a circadian cycle of firing patterns (2). Some authors have concluded that the different afferent inputs these two areas receive reflects different sources of information, for example the core may process information related to visual inputs while the shell receives those non-visual inputs that influence circadian rhythm (3).
Discovering the 'Master-Clock'
It was recognised over 200 years ago that organisms regulate biological processes based on a day-night cycle, when the leaves of plants removed from sunlight continued to move depending on where the sun would be (4). It was some time however, before there was considered to be a central 'clock' in the mammalian CNS and only in the last 40 years has the SCN been recognised as the major centre for circadian control. Light microscopy investigations carried out in 1970, identified retinal projections terminating in various nuclei in the hypothalamus including the SCN (5). The importance of the hypothalamic region to certain aspects of circadian regulation was investigated by Stephan et al. in 1972. They found that lesions in the SCN region disrupted circadian rhythms of drinking and activity in rats (6). These discoveries prompted further detailed experiments of the pathways involved in the circadian system.
A series of experiments creating lesions at various points of visual pathways in rats revealed that the retinohypothalamic tract (RHT) plays a critical role in entrainment of the circadian system. Moore et al. (7) measured circadian regulation of two enzymes involved in melatonin production. They found that lesions to the visual optic tracts (sparing the RHT) did not result in impairment of the enzymes' circadian regulation. Adding to these studies, the same authors contributed further evidence that the RHT is a necessary pathway by which light provides input to the circadian system. They also carried out ablations to parts of the SCN and concluded that proper function of the SCN is necessary for maintaining a circadian rhythm (8).
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A new approach to testing the validity of the role of SCN as 'master' circadian pacemaker in the rat CNS was employed by Inouye et al. It involved separating parts of the SCN from surrounding hypothalamic tissue and recording neural discharge frequency from the SCN 'island' and brain areas outside of it. They found a circadian rhythm of discharge from those SCN neurons inside the island which was absent from all other brain areas (9). Further in vivo experiments involved transplanting foetal hamster SCN tissue into adult hamsters with the SCN removed. One such study showed that circadian rhythmic behaviours could be restored through transplantation and so provided evidence for the pivotal role of the SCN in coordinating circadian rhythms (10). Shortly after this study was released, a mutation of the circadian system was discovered in golden hamsters, this provided a new direction of research to help confirm the autonomous role of the SCN in coordinating circadian rhythm (11). Ralph et al. used this mutant breed of hamsters to carry out another transplantation experiment, taking SCN tissue from one circadian genotype and transplanting to another. They found that circadian rhythm was restored to that of the donor genotype, suggesting that the SCN is an autonomous centre of circadian rhythm generation in the mammalian brain (12).
Experiments using tetrodotoxin to block Na+ dependent action potentials in the SCN were used to separate the input and output components of the circadian pathway from the SCN itself. The authors concluded that circadian rhythmicity continued without the conduction of action potentials, providing further evidence that the SCN can function as an autonomous pacemaker (13). The use of immunofluorescence methods to determine the presence of the protein Synapsin I in developing synapses enabled its use in neurodevelopmental studies (14). One such study looked at the timing of synaptogenesis in the SCN of rats, finding that the majority of synapses were formed in the days to weeks following birth (15). When considered alongside previous studies demonstrating circadian glucose utilization in the pre-natal SCN of rats (16), this study strongly suggests the presence of an exclusive circadian mechanism expressed by cells of the SCN. Further evidence for the expression of circadian rhythms by individual cells within the SCN came with the development of microelectrode array techniques (17). This allowed the direct study of neurons cultured from rat SCN which were subsequently found to express individual circadian firing rhythms (2).
The studies referred to above, strongly reliant upon concurrent advances in biological experimental techniques, paved the way for molecular and genetic investigation into the nature of the circadian oscillatory mechanism in individual SCN neurons.
Molecular Mechanism in Drosophila
The molecular mechanisms underlying cellular circadian rhythmicity have been well studied in both Drosophila and mammals. Several core 'clock' genes (genes that are essential for the generation and regulation of circadian rhythms) have been identified. Mutations to the gene period (per) in Drosophila have been discovered that lengthen, shorten or abolish the circadian rhythm (perl, pers and per0 respectively) (18). PER, the protein product of the per gene is expressed rhythmically, peaking during circadian night. This pattern of expression is maintained when the flies are kept in constant darkness, suggesting that per expression is regulated by a cellular circadian mechanism (19). It has also been noted that per mRNA levels vary with circadian time and that PER protein (directly or indirectly) affects per expression, most likely through a negative feedback loop (20).
Another mutant circadian gene timeless (tim) interacts with per to affect expression of PER and both protein products form a complex, PER-TIM that enters the nucleus to inhibit expression of per and tim (21). The formation of another heterodimer, dCLOCK-CYC is essential to circadian behaviour in Drosophila by promoting rhythmical expression of per and tim. It is thought that the PER-TIM complex acts to inhibit the activity of dCLOCK-CYC thereby forming a further negative feedback loop and indirectly inhibiting their own expression (22). These complicated mechanisms of positive and negative feedback are crucial to maintaining circadian rhythms in Drosophila. The importance of light as a source of signal input to the circadian system was in part explained through the discovery that levels of CRY decrease with increased light levels. CRY is the protein product of cry, a cryptochrome gene, the regulation of which is influenced by the other Drosophila clock genes (23). Rapid breakdown of TIM in the presence of light, thereby disrupting the PER-TIM complex and allowing for reaccumulation of per and tim RNA, is another possible mechanism by which light provides regulation for the Drosophila circadian cycle (24), (25).
Molecular Mechanisms in Mammals
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The principles underlying expression and regulation of clock genes and their products in Drosophila can be applied to the molecular level of circadian regulation in mammals. Two genes found to be of particular importance in mammalian circadian rhythm are Clock and Bmal1. Studies to identify Clock used a mutagenic chemical N-ethyl-N-nitrosourea (ENU), resulting in a mutation that lengthened the circadian period in both homozygous and heterozygous genotypes. The chromosomal location of this gene could then be identified using linkage analysis (26). These genes code for proteins in the basic Helix-loop-Helix-PAS family. This enables the proteins, CLOCK and BMAL1 to heterodimerise through the PAS domain forming a complex that can pass into the nucleus and promote transcription of other clock genes, discussed below (27). Levels of Clock mRNA and CLOCK protein have been found to remain constant in mouse SCN (28) as opposed to Bmal1 which shows significant circadian oscillation, with levels peaking around the middle of circadian night (29).
The other core clock genes and proteins that have been identified in mammals include per, a mammalian homologue of the same gene found in Drosophila (30). This gene was initially identified using a modified form of PCR and there are now three forms, per1, per2 and per3 that have been described structurally and in terms of their circadian oscillation (31). The genes cry1 and cry2 are also key components of the mammalian circadian oscillatory mechanism as was shown in knockout studies where mutant mice lost circadian rhythmicity when placed in dark conditions (32). Extensive investigation has been carried out into exactly how these genes and their products interact to bring about a rhythmic cycle of gene transcription and mRNA translation. The finding that levels of per and cry mRNA peaks during mid-late circadian night, a number of hours after the peak of Bmal1 levels suggests that BMAL1 plays a role in promoting expression of per and cry. It is now widely acknowledged that the CLOCK-BMAL1 heterodimer that passes into the nucleus, act as a positive feedback loop on per and cry genes promoting their transcription. Per and cry gene products are also thought to form a complex, PER-CRY which enters the nucleus and acts in a negative feedback manner on Clock and Bmal1, decreasing levels of their transcription. CRY is thought to play the biggest part in this autoregulatory mechanism (33). The effect of decreased levels of CLOCK-BMAL1 is to remove the positive feedback effect on per and cry transcription and consequently lead to lower levels of per and cry mRNA. When PER-CRY levels drop below a threshold their negative influence on Clock and Bmal1 is removed and the cycle of CLOCK-BMAL1 and PER-CRY accumulation begins again (28).
Another component of the circadian oscillatory mechanism was recently identified. REV-ERBα is a nuclear receptor which is thought to act somewhere between the positive and negative feedback loops. CLOCK-BMAL1 is thought to activate REV-ERBα transcription while the PER-CRY complex inhibits it, furthermore REV-ERBα inhibits transcription of BMAL1-CLOCK, indirectly inhibiting its own transcription (34).
Post-translational modification of clock proteins is thought to play an important
role in determining circadian patterns of gene expression. The enzyme casein kinase I epsilon (CKIε ) is implicated in the phosphorylation of PERIOD proteins, promoting their breakdown. Evidence for the importance of CKIε is based on mutation studies from the same tau mutant hamsters discovered by Ralph et al. Positional syntenic cloning of the tau mutation revealed that the CKIε enzyme is coded for by the deficient gene in these hamsters. In vitro affinity kinase assays were then used to show that the CKIε in mutant hamsters is deficient in phosphorylating PER proteins (36). This deficiency of post-translational modification results in a shorter circadian period in tau mutant hamsters, in homozygous mutants the period is as short as 20 hours compared to 24 in normal hamsters. These studies show the importance of post-translational modification of clock proteins in regulating the circadian cycle. There is further relevance to these findings when considering the human genetic condition Familial Advanced Sleep Phase Syndrome (FASPS). Sufferers experience earlier onset and termination of sleep, with no difference in duration to the normal. Linkage analysis identified a gene, hPer2, the product of which, contains a phosphorylation site for CKIε. A mutation at this site in FASPS sufferers reduces the affinity of CKIε binding, pointing to a fault in post-translational phosphorylation as the mechanism behind their altered sleep-wake cycle (37).
There is still a great deal to be learnt about how these complex molecular cycles within cells of the SCN are related to output signals affecting various body systems and so influencing circadian behaviour. Vasopressin and TGF-α are both thought to have roles in coordinating some aspects of circadian behaviour. Prokineticin 2 (PK2) mRNA is found in SCN neurons and its expression is thought the be controlled in a circadian rhythm. PK2 was found to influence locomotor activity in rats so it is possible that this protein provides a link between the molecular oscillation of clock proteins and output from the SCN to influence certain aspects of behaviour (38).
Biological rhythms play a fundamental role in regulating various life-processes in almost all organisms (39). In mammals, the complex interplay between environmental stimuli and neural processes is coordinated by a relatively small area of the CNS. There is substantial evidence to support the role of the SCN in the hypothalamus as the 'master-clock' of circadian rhythms. It is understood how light entrains the circadian system through RHT projections from retinal ganglion cells to the SCN. Individual neurons in the SCN maintain a cycle of transcription of clock genes through complex negative and positive feedback mechanisms over a 24hr period. Significant progress in research methods, along with some serendipitous discoveries such as that of the tau mutant hamster, contributed to the advances in this field. The mechanism by which circadian rhythms within the SCN are translated into behavioural circadian patterns remains obscure and should be an important area for research in the future. Disorders such as FASPS, jet-lag, manic-depressive disorder and seasonal-affective disorder, provide a significant clinical context within which such research can take place.