Contributions Made to Chronobiology by Studies of Fiddler Crab Rhythms

One of the classic organisms used in chronobiological research is the fiddler crab (genus Uca), an animal unique in that it displays both circadian and tidal (i.e., circalunidian) rhythms. The pioneering work on this animal helped produce the early evidence for many of the standard properties now recognized for all circadian rhythms: near temperature independence of the period, phase lability and setability, the light and temperature sensitivity rhythms expressed by phase response curves, and the persistence of rhythms in organs isolated from a multicellular animal. Importantly, results arising from studies of this crab–and a few other organisms–resulted in the development of the exogenous timing hypothesis. While philosophically sound, the lack of supporting evidence for this hypothesis has resulted in it being discarded by most chronobiologists; but while still in its prime, it drew great interest, and therefore grant support, to the field in general, stimulated a great deal of research that otherwise might not have been performed, and resulted in the discovery of environmental stimuli previously unsuspected to influence organisms. As could be expected, continuing work with this crab, using modern approaches and statistical techniques, has modified earlier findings and interpretations, has revealed new properties, and has resulted in the creation of new hypotheses. The review and update is a synthesis of 45 years of this work.     

Sleep and circadian rhythms: key components in the regulation of energy metabolism

In this review, we present evidence from human and animal studies to evaluate the hypothesis that sleep and circadian rhythms have direct impacts on energy metabolism, and represent important mechanisms underlying the major health epidemics of obesity and diabetes. The first part of this review will focus on studies that support the idea that sleep loss and obesity are "interacting epidemics." The second part will discuss recent evidence that the circadian clock system plays a fundamental role in energy metabolism at both the behavioral and molecular levels. These lines of research must be seen as in their infancy, but nevertheless, have provided a conceptual and experimental framework that potentially has great importance for understanding metabolic health and disease.     

Circadian regulation of cardiovascular function: a role for vasoactive intestinal peptide

The circadian system, driven by the suprachiasmatic nucleus (SCN), regulates properties of cardiovascular function. The dysfunction of this timing system can result in cardiac pathology. The neuropeptide vasoactive intestinal peptide (VIP) is crucial for circadian rhythms in a number of biological processes including SCN electrical activity and wheel running behavior. Anatomic evidence indicates that SCN neurons expressing VIP are well positioned to drive circadian regulation of cardiac function through interactions with the autonomic centers. In this study, we tested the hypothesis that loss of VIP would result in circadian deficits in heart rate (HR) and clock gene expression in cardiac tissue. We implanted radiotelemetry devices into VIP-deficient mice and wild-type (WT) controls and continuously recorded HR, body temperature, and cage activity in freely moving mice. Under light-dark conditions, VIP-deficient mice displayed weak rhythms in HR, body temperature, and cage activity, with onsets that were advanced in phase compared with WT mice. Similarly, clock gene expression in cardiac tissue was rhythmic but phase advanced in mutant mice. In constant darkness, the normal circadian rhythms in HR were lost in VIP-deficient mice; however, most mutant mice continued to exhibit circadian rhythms of body temperature with shortened free-running period. The loss of VIP altered, but did not abolish, autonomic regulation of HR. Analysis of the echocardiograms did not find any evidence for a loss of cardiac function in VIP-deficient mice, and the size of the hearts did not differ between genotypes. These results demonstrate that VIP is an important regulator of physiological circadian rhythmicity in the heart.     

TIDAL RHYTHMS: THE CLOCK CONTROL OF THE RHYTHMIC PHYSIOLOGY OF MARINE ORGANISMS

1. A great number of vital processes are rhythmic and the rhythms quite often persist in constant conditions. The best-known rhythms are circadian; much less is known about circalunadian rhythms, and this review was prepared in an attempt to rectify this deficiency. All through the article comparisons are drawn between circalunadian and circacian rhythms. 2. Activity rhythms. (a) The activity patterns of 28 intertidal animals are discussed. All describe a periodicity with a basic component of 24.8 hours, and this approximate period persists in the laboratory in constant light and temperature and in the absence of the tides. The duration of persistence ranges from a few cycles to months, and is a function of the species studied, the conditions imposed, and individual tenacity. (b) In those few cases where relatively long-term observations have been made, there is a trend for the period of the rhythm to become circatidal, or better, circalunadian. (c) The ‘desired’ phase relationship between rhythm and tidal cycle is species-specific. Geographical translocation experiments have shown that the phase is set by the local tides. (d) In some cases the amplitude of the persistent rhythm mimics the semidiurnal inequality of the tides. (e) In about a third of the species discussed, a circadian component has been found combined with the tidal component. Many of the other studies were of such short duration that a low-amplitude circadian component would have gone unnoticed. (f) The tidal rhythm is innate. However, the rhythm is (i) sometimes lacking in organisms living in non-tidal habitats, or (ii) fades after a spell of incarceration in constant conditions. Various treatments — some aperiodic — can induce the expression of the missing tidal rhythm. (g) In the green crab, removal of the eyestalks destroys the activity rhythm. 3. Vertical migration rhythms. (a) A rather surprisingly large number of intertidal animals have been found to undergo migration rhythms between the upper layers of the substratum and its surface. The movements are synchronized with the tides in nature, but most species have either been shown to be diurnal in constant conditions, or in cases where adequate testing has not been done, suspected of being so. (b) In only one species has confirming work shown that the fundamental frequency is truly tidal. This finding is especially important as it shows that tidal rhythms need only the single-cell level of organization for expression. Even at this level there appears to be a dictatorial override by a circadian clock. 4. Colour change. Low-amplitude tidal rhythms in colour change — superimposed on a more dominant circadian change — have been reported to be intrinsic in four species and inducible in a fifth. 5. Oxygen consumption. Tidal rhythms in oxygen consumption have been described for seven invertebrates and one alga; six of the species have superimposed solar-day rhythmic components also. 6. Translocation. A total of five geographical translocation experiments, in which the organisms were maintained in constant conditions throughout, have been tried. Unequivocally in one case, and possibly in a second, the test organisms rephased spontaneously to the times commensurate with local tidal conditions. In two other cases, the pretranslocation phase was retained. The fifth experiment has not been reproducible. 7. Determination of phase. (a) The tidal cycle on the home shoreline sets the phase of the inhabitant's rhythms. Even the location of a crab's burrow on the beach incline can play a determining role. (b) Paradoxically, the periodic wetting by inundation is not an important entraining factor for most intertidal organisms. Instead, the effective portions of the tidal cycle include one or more of the following. (i) Mechanical agitation, especially for animals living in an uprush zone where they are periodically subjected to the pounding surf, (ii) Temperature cycles, though they have not yet been systematically investigated, have very pronounced entraining roles in crabs. (iii) Pressure is probably not a generally important entraining agent for most intertidal organisms, but it is so for the green crab. (c) Light-dark cycles in general, whether daily or tidal in length, have no effect on the entrainment or phase setting of many tidal rhythms. There are two exceptions: (i) a 24-hour light-dark cycle is known to keep a tidal locomotor rhythm (one that becomes circalunadian in constant conditions) at a strict tidal frequency. (ii) In rhythms with both daily and tidal components, when the former is shifted by light stimuli, the latter is affected in a nearly identical manner. 8. Temperature. (a) The role of temperature on tidal rhythms is compared with its role on circadian rhythms. (b) The effects of different constant temperatures have so far been studied on only four tidal rhythms. All studies indicate a lack of any permanent change in period, which is not so with most circadian rhythms; the latter having temperature coefficients around 1.1. In two of the studies the rhythms under test temperatures were followed for less than a day, and a third study cannot be repeated. (c) Short exposure to very cold temperature pulses produced a response that may be interpreted as a temporary stoppage of the clock. Exposure to relatively less-cold pulses appear simply to reset the hands of the clock. The same responses have been demonstrated with circadian rhythms. (d) In the case of green crabs, which had become arrhythmic during prolongued captivity in the laboratory, a tidal rhythm could be reinitiated by a single short cold treatment. The cold pulse also set the phase of the rhythm. (e) A few superficial studies employing temperature steps or pulses have produced results which suggest that a phase-change sensitivity rhythm — just like that found associated with circadian rhythms — may underlie tidal rhythms. Certainly a determined search for this rhythm should be made in the near future. 9. Clock control of rhythms. (a) An argument is constructed claiming that tidal rhythms have a basic period of about 24–8 hours rather than the more expected tidal interval of 12.4 hours. In constant conditions, a circalunadian period is usually displayed. (b) After speculating that a frequency-transforming coupler may function between the clock and the overt rhythm, reasons are given that lead to the further speculation that both circadian and circalunadian rhythms could be generated by a single clock, via specific coupling mechanisms. (c) Two current hypotheses concerning the nature of the clockworks are reviewed and discussed. (d) Suggestions are made for future investigations.     

Acute exposure to 2G phase shifts the rat circadian timing system

The circadian timing system (CTS) provides internal and external temporal coordination of an animal's physiology and behavior. In mammals, the generation and coordination of these circadian rhythms is controlled by a neural pacemaker, the suprachiasmatic nucleus (SCN), located within the hypothalamus. The pacemaker is synchronized to the 24 hour day by time cues (zeitgebers) such as the light/dark cycle. When an animal is exposed to an environment without time cues, the circadian rhythms maintain internal temporal coordination but exhibit a "free-running" condition in which the period length is determined by the internal pacemaker. Maintenance of internal and external temporal coordination are critical for normal physiological and psychological function in human and non-human primates. Exposure to altered gravitational environments has been shown to affect the amplitude, mean, and timing of circadian rhythms in species ranging from unicellular organisms to man. However, it has not been determined whether altered gravitational fields have a direct effect on the neural pacemaker, or affect peripheral physiological systems that express these circadian parameters. In previous studies, the ability of a stimulus to phase shift circadian rhythms was used to determine whether a stimulus has a direct effect on the neural pacemaker. The present experiment was performed in order to determine whether acute exposure to a hyperdynamic field could phase shift circadian rhythms.     

Adverse effects of chronic circadian desynchronization in animals in a "challenging" environment

Continuous disruption of circadian rhythms, as seen in human shift workers, has been associated with the development of a number of adverse mental and physiological conditions. However, scientific evidence linking circadian disruption to overall health, particularly in animal models, is not well documented. In this study, we have demonstrated that exposing C57BL/6J mice to 12-h phase shifts every 5 days for 3 mo had no effect on body weight or intestinal physiology. However, when animals were further challenged with dextran sodium sulfate to induce colitis, chronic shifting of the light-dark cycle led to a dramatic increase in the progression of the colitis as indicated by reduced body weight, abnormal intestinal histopathology, and an exacerbated inflammatory response. These data indicate that circadian disruption is an important predisposing factor that may provoke the onset or worsening of various disease states such as inflammatory disorders. This study provides further evidence for continued investigations using animal models of circadian disruption to examine the consequences of circadian disruption on health when organisms are faced with a "challenging" environment.     

Review of the Dual-Clock Control of Tidal Rhythms and the Hypothesis that the Same Clock Governs Both Circatidal and Circadian Rhythms

Discoveries first published in 1986 did not fit the de rigueur working hypothesis that the clocks governing tide-associated rhythms had a fundamental period of 12.4 h, a value equal to the average interval between successive tides on most coastlines of the world. To explain the results a dual-clock schema was fashioned that envisioned two clocks, strongly coupled together 180° antiphase, each running at a basic rate of 24.8 h (the interval of a lunar day), as the driving agents of tide-associated rhythms (details are given in the text). This elaboration has been named the circalunidian-clock hypothesis, a hypocorism used in some armchair ruminations back in 1973. In the decade since 1986, a goodly amount of evidence has been garnered that is consistent with this hypothesis—suggesting that first-call divination appears to have been visionary. Acceptance of this hypothesis leads to further cerebration that a 24.8-h clock, its circa periods in constant conditions, and other properties—which fully overlap with our perception of the circadian clock that drives daily rhythms—may indicate that circadian and circalunidan timepieces are not different entities. The known properties of both daily and lunar clock-types are compared and contrasted, and, with the exception of one feature (for which there is at least a philosophical explanation), it is concluded that the same clock that drives tidal rhythms could also motor daily rhythms, i.e., there may be no such thing as a 12.4-h horologue.     

Phase resetting of the mammalian circadian clock by DNA damage

To anticipate the momentum of the day, most organisms have developed an internal clock that drives circadian rhythms in metabolism, physiology, and behavior [1]. Recent studies indicate that cell-cycle progression and DNA-damage-response pathways are under circadian control [2-4]. Because circadian output processes can feed back into the clock, we investigated whether DNA damage affects the mammalian circadian clock. By using Rat-1 fibroblasts expressing an mPer2 promoter-driven luciferase reporter, we show that ionizing radiation exclusively phase advances circadian rhythms in a dose- and time-dependent manner. Notably, this in vitro finding translates to the living animal, because ionizing radiation also phase advanced behavioral rhythms in mice. The underlying mechanism involves ATM-mediated damage signaling as radiation-induced phase shifting was suppressed in fibroblasts from cancer-predisposed ataxia telangiectasia and Nijmegen breakage syndrome patients. Ionizing radiation-induced phase shifting depends on neither upregulation or downregulation of clock gene expression nor on de novo protein synthesis and, thus, differs mechanistically from dexamethasone- and forskolin-provoked clock resetting [5]. Interestingly, ultraviolet light and tert-butyl hydroperoxide also elicited a phase-advancing effect. Taken together, our data provide evidence that the mammalian circadian clock, like that of the lower eukaryote Neurospora[6], responds to DNA damage and suggest that clock resetting is a universal property of DNA damage.     

Evidence for the adaptive significance of circadian rhythms

Circadian (∼24 h) clock regulated biological rhythms have been identified in a wide range of organisms from prokaryotic unicellular cyanobacteria to higher mammals. These rhythms regulate an enormous variety of processes including gene expression, metabolic processes, activity and reproduction. Given the widespread occurrence of circadian systems it is not surprising that extensive efforts have been directed at understanding the adaptive significance of circadian rhythms. In this review we discuss the approaches and findings that have resulted. In studies on organisms in their natural environments, some species show adaptations in their circadian systems that correlate with living at different latitudes, such as clines in circadian clock properties. Additionally, some species show plasticity in their circadian systems suggested to match the demands of their physical and social environment. A number of experiments, both in the field and in the laboratory, have examined the effects of having a circadian system that does not resonate with the organism's environment. We conclude that the results of these studies suggest that having a circadian system that matches the oscillating environment is adaptive.     

Aging human circadian rhythms: conventional wisdom may not always be right

This review discusses the ways in which the circadian rhythms of older people are different from those of younger adults. After a brief discussion of clinical issues, the review describes the conventional wisdom regarding age-related changes in circadian rhythms. These can be summarized as four assertions regarding what happens to people as they get older: 1) the amplitude of their circadian rhythms reduces, 2) the phase of their circadian rhythms becomes earlier, 3) their natural free-running period (tau) shortens, and 4) their ability to tolerate abrupt phase shifts (e.g., from jet travel or night work) worsens. The review then discusses the empirical evidence for and against these assertions and discusses some alternative explanations. The conclusions are that although older people undoubtedly have earlier circadian phases than younger adults, and have more trouble coping with shift work and jet lag, evidence for the assertions about rhythm amplitude and tau are, at best, mixed.     

The Ultradian Clocks of Eukaryotic Microbes: Timekeeping Devices Displaying a Homeostasis of the Period

Temperature compensation of their period is one of the canonical characteristics of circadian rhythms, yet it is not restricted to circadian rhythms. This short review summarizes the evidence for ultradian rhythms, with periods from 1 minute to several hours, that likewise display a strict temperature compensation. They have been observed mostly in unicellular organisms in which their constancy of period at different temperatures, as well as under different growth conditions (e.g., medium type, carbon source), indicates a general homeostasis of the period. Up to eight different parameters, including cell division, cell motility, and energy metabolism, were observed to oscillate with the same periodicity and therefore appear to be under the control of the same central pacemaker. This suggests that these ultradian clocks should be considered as cellular timekeeping devices that in fast-growing cells take over temporal control of cellular functions controlled by the circadian clock in slow-growing or nongrowing cells. Being potential relatives of circadian clocks, these ultradian rhythms may serve as model systems in chronobiolog-ical research. Indeed, mutations have been found that affect both circadian and ultradian periods, indicating that the respective oscillators share some mechanistic features. In the haploid yeast Schizosaccharomyces pombe, a number of genes have been identified where mutation, deletion, or overex-pression affect the ultradian clock. Since most of these genes play roles in cellular metabolism and signaling, and mutations have pleiotropic effects, it has to be assumed that the clock is deeply embedded in cellular physiology. It is therefore suggested that mechanisms ensuring temperature compensation and general homeostasis of period are to be sought in a wider context. (Chronobiology International, 14(5), 469–479, 1997)     

The mammalian circadian system: models and physiology

Mammalian circadian rhythms have been studied in great detail using primarily two different methods. One method is usually referred to as the formal analysis of rhythms. Its goal is to describe the properties of both rhythms and their underlying mechanisms, and it aims at the development of adequate mathematical models of the circadian system. The other method is the physiological analysis of the mechanisms that generate and entrain rhythms. Its goal is the identification of the anatomical components of the circadian system and the elucidation at a cellular and molecular level of how these components work. This paper reviews how the formal analysis of circadian systems, primarily in rodents, set the agenda for physiological studies, and the degree to which this agenda has been fulfilled. It then discusses how physiological analyses of the system have helped to redefine issues such as the nature and identity of the pacemaker, the nature of the entrainment process, the roles of photic and nonphotic cues, and the role of feedback in the circadian system. The continued commerce between these two approaches has led to a sophisticated appreciation of the complexities and subtleties of circadian organization in mammals. The further integration of formal and physiological analyses remains a challenging goal for the future.     

Alteration of period and amplitude of circadian rhythms in shift workers. With special reference to temperature, right and left hand grip strength

48 male shift workers in various industries volunteered to document circadian rhythms in sleeping and working, oral temperature, grip strength of both hands, peak expiratory flow and heart rate. All physiological variables were self-measured 4 to 5 times a day for 2 to 4 weeks. Individual time series were analyzed according to several statistical methods (power spectrum, cosinor, chi squares, ANOVA, correlation, etc.) in order to estimate rhythm parameters such as circadian period (tau) and amplitude (A), and to evaluate subgroup differences with regard to tolerance to shift work, age, duration of shift work, speed of rotation and type of industry. The present study confirms for oral temperature and extends to other variables (grip strength of both hands, heart rate) that intolerance to shift work is frequently associated with both internal desynchronization and small circadian amplitude. The internal desynchronization among several circadian rhythms supports the hypothesis that these latter are driven by several oscillators. Many differences were observed between circadian rhythms in right and left hand grip strength: circadian tau in oral temperature was correlated with that in the grip strength of the dominant hand but not with that of the other hand; changes in tau s of the non-dominant hand were age-related but did not correlate with temperature tau; only the circadian A of the non-dominant hand was associated with a desynchronization. Thus, circadian rhythms in oral temperature and dominant hand grip strength may be driven by the same oscillator while that of the non-dominant hand may be governed by a different one. Internal desynchronization between both hand grip rhythms as well as desynchronization of performance rhythms reported by others provide indirect evidence that circadian oscillator(s) may be located in the human cerebral cortex.     

Circadian Locomotor Activity Under Artificial Light in the Freshwater Crab Pseudothelphusa americana

Long-term recordings of locomotor activity were obtained from intact freshwater crabs, Pseudothelphusa americana in constant darkness (DD), constant light (LL) and different light-dark (LD) protocols. Bimodal rhythms were typically observed in this crab when subjected to DD or LD, with bouts of activity anticipating lights-on and lights-off, respectively. Freerunning circadian rhythms were expressed in both DD and LL for longer than 30 days. In DD, we observed that some animals presented different period lengths for each activity component. During LL, activity was primarily unimodal, however spontaneous splitting of the rhythms were observed in some animals. When activity was recorded under artificial long days, the morning bouts maintained their phase relationship but the evening bouts changed their phase relationship with the Zeitgeber. Our results indicate that, bimodal locomotor activity rhythm in the crab Pseudothelphusa americana is variable among organisms. The characteristics of phase relationship with LD and responses to LL for morning and evening bouts, suggest that, locomotor activity could be driven by multiple oscillators, and that coupling between these oscillators may be regulated by light.     

Circadian Locomotor Activity Under Artificial Light in the Freshwater Crab Pseudothelphusa americana

Long-term recordings of locomotor activity were obtained from intact freshwater crabs, Pseudothelphusa americana in constant darkness (DD), constant light (LL) and different light-dark (LD) protocols. Bimodal rhythms were typically observed in this crab when subjected to DD or LD, with bouts of activity anticipating lights-on and lights-off, respectively. Freerunning circadian rhythms were expressed in both DD and LL for longer than 30 days. In DD, we observed that some animals presented different period lengths for each activity component. During LL, activity was primarily unimodal, however spontaneous splitting of the rhythms were observed in some animals. When activity was recorded under artificial long days, the morning bouts maintained their phase relationship but the evening bouts changed their phase relationship with the Zeitgeber. Our results indicate that, bimodal locomotor activity rhythm in the crab Pseudothelphusa americana is variable among organisms. The characteristics of phase relationship with LD and responses to LL for morning and evening bouts, suggest that, locomotor activity could be driven by multiple oscillators, and that coupling between these oscillators may be regulated by light.     

Reply To-Healy D. and Waterhouse J.M.: the Circadian System and Affective Disorders: Clocks or Rhythms? Chronobiologic Disorders, Social Cues and the Light-Dark Cycle

In their monograph (1), Healy and Waterhouse quite thoughtfully distinguish between the altered shapes of circadian rhythms and their entrainment (synchronization). Although there is a great deal of evidence that various influences can alter the shape of circadian rhythms (“masking”), the literature on multiple time cues (zeitgebers) entraining different pacemakers is less convincing. In humans, evidence for nonphotic (social and activity-rest cycle) zeitgebers is restricted to human studies of anchor sleep (2), to data from Wever (3) and Czeisler (4) and to animal studies by Mrosovksy (5) and Turek (6). Until proven otherwise, it seems most likely that social cues primarily affect the sleep-wake cycle (activity-rest cycle), which- being loosely coupled to the endogenous circadian pacemaker-can be dissociated from it and the overt rhythms that are driven by it.     

Review of the Dual-Clock Control of Tidal Rhythms and the Hypothesis that the Same Clock Governs Both Circatidal and Circadian Rhythms

Discoveries first published in 1986 did not fit the de rigueur working hypothesis that the clocks governing tide-associated rhythms had a fundamental period of 12.4 h, a value equal to the average interval between successive tides on most coastlines of the world. To explain the results a dual-clock schema was fashioned that envisioned two clocks, strongly coupled together 180° antiphase, each running at a basic rate of 24.8 h (the interval of a lunar day), as the driving agents of tide-associated rhythms (details are given in the text). This elaboration has been named the circalunidian-clock hypothesis, a hypocorism used in some armchair ruminations back in 1973. In the decade since 1986, a goodly amount of evidence has been garnered that is consistent with this hypothesis—suggesting that first-call divination appears to have been visionary. Acceptance of this hypothesis leads to further cerebration that a 24.8-h clock, its circa periods in constant conditions, and other properties—which fully overlap with our perception of the circadian clock that drives daily rhythms—may indicate that circadian and circalunidan timepieces are not different entities. The known properties of both daily and lunar clock-types are compared and contrasted, and, with the exception of one feature (for which there is at least a philosophical explanation), it is concluded that the same clock that drives tidal rhythms could also motor daily rhythms, i.e., there may be no such thing as a 12.4-h horologue.