Circadian And Ultradian Clocks

Little is know of the molecular and cellular mechanisms of event timing. Molecular geneticists have yet to isolate an event timer in animals (see Cevik, this volume). Neuroscientists have the power to isolate function of gross anatomical regions by lesion and transplant, but cell number limits functional understanding of populations of neurons almost completely to neural simulations. Neuroscientists are not readily equipped to investigate genetic and molecular function.

The point of discussing circadian and ultradian clocks is twofold. First, it will allow us to make an important distinction between mechanisms of endogenously controlled time and those of perceived time. Second, it will provide a basic description of a molecular clock as a kind of null model against which we can address future questions.

Animal timing refers to a broad class of behaviors. These include general life history events such as when to stop making sperm and the age of first reproduction. They also include rhythmic behaviors with periods on the order of a year (circan-nual), longer than a day (infradian), a day (circadian), or shorter than a day (ultradian). The suggestion that animals have evolved to organize their lives in time could refer to any of these levels of behavioral timekeeping. Animal event timing, as I use it here, refers to none of these.

The fundamental mechanisms of animal event timing are sensory based. Event timing requires the ability to perceive and remember the duration of an external event. Rhythmic behaviors and the scheduling of large-scale life events are based on endogenously controlled, often genetically predetermined, timetables. Circadian rhythms are 24 h long not because the animal learns a 24-h period in its lifetime, but because it is genetically predisposed to timing an interval approximating the rotation of the earth. On another planet, or in a lab where light-dark (LD) cycles are manipulated, circadian clocks are far from functional in terms of recording the length of the LD cycle. Daily activity cycles appear to fall into the same category. They are driven in well-defined ways by circadian rhythms. It is rare for an animal to learn when to be most active in the LD cycle (but see Section 4.5.4).

This section is also designed to point out that the clock used to measure event duration is not necessarily the same clock that measures time of day. Experiments designed to expose animal event timers can be confounded by this time-of-day clock. For example, intervals of 24 h do not need to be measured, because they can be posted to the circadian phase. I will provide several lines of evidence in this section that the time-of-day clock is inefficient for measuring intervals different from 24 h and show that it is quite distinct from animal event timers.

4.3.1 Circadian Clock Gene mRNA Levels Oscillate with a Circadian Rhythm

Circadian regulation of gene expression is well established in plants (i.e., Arabidopsis — Millar et al., 1995), fungi (i.e., Neurospora — Dunlap, 1996), insects (i.e.,

Drosophila — Iwasaki and Thomas, 1997), unicellular microorganisms (Lloyd, 1998), and vertebrates (Takahashi, 1995). I will describe in some detail only the Neurospora and Drosophila system because they are typical and well-studied examples of circadian dynamics. Our understanding of the molecular details governing the circadian clock is growing rapidly (reviewed in Okamura et al., 2002; Stanewsky, 2002) and is beyond the scope of this review, but I hope to describe the basics of the system in enough detail to provide a foundation for thinking about molecular event timers.

The filamentous bread mold, Neurospora crassa, exhibits a circadian rhythm in its asexual spore formation (conidiation) (Edmunds, 1988). The frq gene encodes a central element in this circadian rhythm. Both the frq gene mRNA and its protein product, FRQ, cycle in amount with a period of approximately 24 h (Dunlap, 1996). In a 24-h LD cycle, frq mRNA and FRQ levels are at their lowest point near the middle of the dark phase. Slowly rising, they peak approximately 10 to 12 h later, with FRQ levels always lagging behind mRNA levels by about 3 h.

The vinegar fruit fly, Drosophila melanogaster, shows oscillations in two of its circadian clock gene transcripts (per and tim) that are exactly out of phase with its own frq (Hardin et al., 1992). Their periods are the same, but when frq mRNA is beginning to rise, per and tim mRNAs are beginning to fall. Like FRQ, PER lags behind its mRNA transcript by several hours (Iwasaki and Thomas, 1997). Interestingly, a recently identified protein in mammals, mPer1, sharing high sequence similarity with the Drosophila PER protein, oscillates in phase with frq (Shigeyoshi et al., 1997), and a mutation in a similar gene in humans is correlated with familial advanced sleep phase syndrome (Toh et al., 2001).

In Drosophila, the PER and FRQ proteins are localized to the cytoplasm. In short order, FRQ enters into a binding complex with (at least) itself and PER enters into a 1:1 heterodimeric complex with TIM, as suggested by studies in the yeast two-hybrid system (Dunlap, 1996). These relationships appear to stabilize FRQ and PER in the cytosol. They also appear to be necessary for translocation to the nucleus, where they suppress their own expression (Aronson et al., 1994; Dunlap, 1996). This is the negative feedback mechanism that generates the cyclical rise and fall of protein products, allowing the animal to keep track of time.

In the absence of light (dark-dark (DD)), all of these gene products show free-running periods of about 24 h. The phase of gene expression can also be directly entrained by light, but this response is limited to certain intervals of the cycle. For example, in Neurospora, when FRQ levels are low, a pulse of light leads to rapid transcription of the frq mRNA, moving the phase of oscillation forward in time (Crosthwaite et al., 1995). Given the swiftness of the response, it is believed that light acts directly on the frq promoter (Dunlap, 1996). mPer1 shows a similar response to light (Shigeyoshi et al., 1997). In Drosophila, light has little effect on per mRNA but reduces levels of TIM. Constant light breaks down the entire rhythm (Power et al., 1995). Given this evidence, it is not surprising that manipulations by light are unable to change the period of the circadian rhythm, but are able to adjust its phase.

Light is not the only source of entrainment for the circadian clock. Temperature is also a cue (Iwasaki and Thomas, 1997). There is recent evidence that the circadian clock can also be affected by conditioned stimuli (Amir and Stewart, 1996). Some disagreement exists about the extent of this phenomenon. There is evidence that social contacts in humans can synchronize circadian pacemakers (Hastings, 1997), but recent research on blind subjects and manipulated light schedules in sighted subjects supports the necessity of light as an entrainment cue (Czeisler, 1995). Pioneering work by Winfree (1980) led him to predict strong resetting of the circadian clock by light across a wide array of species, and this appears to hold true.

4.3.2 Circadian Clocks Are Temperature Compensated

Although there are physiological temperature limits for the maintenance of circadian rhythms, there is no effect of stable temperatures within these limits (Iwasaki and Thomas, 1997). However, temperature changes can elicit rises or falls in gene expression (Edery et al., 1994; Rensing et al., 1995). The mechanism by which this temperature compensation operates has been worked out in detail for Neurospora.

Within the first 100 codons of the frq gene there are three methionine codons (AUG) at codons 1, 11, and 100 (Liu et al., 1997). Codon 11 is not used to initiate the sequence under normal conditions, but codons 1 and 100 are. Thus, in wild-type Neurospora there are two FRQ proteins, FRQ100-989 and FRQ1-989, and either of them alone is sufficient for circadian rhythms. At low temperatures, FRQ100-989 is preferentially transcribed. At high temperatures, FRQ1-989 is preferentially transcribed. The ratio between the absolute levels of the two gene products is thus controlled by temperature. Removal of either form disturbs the ability of the clock to compensate for physiological temperature extremes (Liu et al., 1997).

It is possible that other mechanisms operate to compensate for temperature. Both per and frq encode an internally repetitive array of Thr-Gly codons. In D. melano-gaster this region is polymorphic in length (Kyriacou et al., 1992). Work done by Rosato et al. (1996) shows a significant latitudinal cline in this repeat sequence ranging from Europe to North Africa. These sequences are directly related with the ability of the flies to maintain a compensated circadian rhythm at different temperatures (Sawyer et al., 1997). Furthermore, deleting the Thr-Gly region produces flies that have temperature-sensitive circadian periods (Kyriacou et al., 1992).

4.3.3 Circadian and Ultradian Rhythms Are Connected, But Not with Event Timing

Ultradian behaviors appear to oscillate in circadian time. They consist of such behaviors as the defecation cycle in Caenorhabditis elegans (Iwasaki et al., 1995) and wheel-running behavior in mice (Antoch et al., 1997). The relationship between circadian and ultradian behaviors is best understood in D. melanogaster. In a screen looking for mutants of the pupal-adult eclosion phenotype, Konopka and Benzer (1971) identified three mutants of the per gene: pers showed a 19-h cycle, perL1 showed a 29-h cycle, and per01 appeared to be arrhythmic. pers and perL1 mutations are due to single amino acid substitutions. per01 encodes a stop codon at the 460th residue (Yu et al., 1987). The amazing thing about these mutants is that their ultradian behaviors, like activity and courtship song cycles, are proportional to their circadian behaviors (Kyriacou et al., 1992). Where wild-type male flies have 60-sec courtship song cycles, per flies have 40-sec cycles, perL1 flies have 80-sec cycles, and per01 flies show little evidence of cycling at all.

D. melanogaster females show a preference for 55-sec songs, and D. simulans females prefer 35-sec songs. It is a reasonable assumption then that the various per mutations would show changes in female preference for male song duration. In fact, just the opposite is true (Kyriacou et al., 1992). D. melanogaster females, regardless of their per genotype, prefer 55-sec songs. To the extent that flies are not able to use other cues, this provides a fundamental difference between ultradian transmitter and receiver mechanisms. It also suggests that the event timer in Drosophila is not driven by an underlying circadian oscillation. This may be one of the more telling observations about circadian and event timing, explaining the peculiar inability of bees to discriminate short intervals despite their mastery of circadian time.

The Zeitgedachtnis (time sense) of honeybees is widely reported (Saunders, 1971; Seeley, 1995; Wilson, 1971). Bees can relocate almost anything provided it is presented at 24-h intervals (Saunders, 1971; Moore et al., 1989). As well, "marathon" dancers who return to the hive dance floor to dance for hours following a fruitful foraging trip compensate for the sun's motion as the day passes (Wilson, 1971). However, in FI trials, honeybees show no evidence that they can learn anything about a 2-min interval (Richelle and Lejeune, 1980).

Despite this evidence for a distinction between perceived time and circadian time, the relationship between ultradian and circadian behaviors is a profound one. The Syrian hamster tau mutant has a circadian rhythm that is shortened by 4 h from the wild type. Wild-type females have approximately 30-min periods of cortisol and luteinizing hormone fluctuations that are slightly shortened in the tau mutant (Loudon et al., 1994). The recently cloned circadian Clock gene in the mouse was isolated in a massive screen for mutants that exhibited altered wheel-running activity rhythms in constant darkness (King et al., 1997a, 1997b). Close inspection of King et al.'s (1997a) wheel-running data shows that the wheel-running activity cycle is very cyclical in its ultradian oscillations.

The pineal gland's circadian rhythm and direct control of numerous hormonally controlled behaviors is further evidence for this relationship between circadian and ultradian time. At the receiving end of the suprachiasmatic nucleus (see Section 4.3.4), the pineal gland is located at the posterior dorsal aspect of the diencephalon. The rhythmic release of melatonin is its most obvious circadian feature, as melatonin is directly responsible for transmitting the circadian LD signal to the rest of the organism (Menaker, 1997). Among other things, melatonin is known to affect human thermoregulation. Given the definitive relationship between body temperature and sleep (Wever, 1992), the pineal gland has a clear role in the sleep-wake cycle. In an experiment reported by Lavie (1992), eight young male adults experienced 20-min "days" for 48 h in a sleep-wake cycle of 7 and 13 min. In this experiment subjects were instructed to attempt to fall asleep and to resist sleep for 7 and 13 min, respectively, every 20 min. The results show a well-defined sleep-wake cycle at 24 h, despite the best efforts of the subjects to overcome this interval.

Studies of interval timing also reveal a circadian influence on attention and memory. Meck (1991) tested rats for their ability to discriminate 2- and 8-sec durations (temporal bisection procedure) over the course of the day. There was no effect of circadian phase on the clock rate — the PSE did not change. However, the overall sensitivity to time (measured by the variability of the response) was highest during the dark phase and lowest during the light phase. This relationship has also been demonstrated for honeybee arrival times, with more accuracy in the morning than later in the day (Moore et al., 1989). If there is a clear relationship between circadian clocks and event timing, this is likely to be it: circadian phase influences the accuracy of event timers. As an example, experiments on human isolation in Antarctica, submarines, and underground are typically difficult to interpret because subjects often lose their abilities to concentrate; it is as if the circadian mechanisms controlling attention are somehow lost (Harrison et al., 1989).

Unfortunately, the majority of psychophysical literature fails to mention the time of day at which experiments were performed or any salient features of the LD cycle. Ecological studies on animal behavior are seldom better, the unspoken assumption being that the behavior, once initiated, is stereotypical. Variance is regarded as "noise" in the form of genetic variance or sensitivity of the animal to subtle environmental factors. However, given Meck's (1991) results, it may be predictable circadian oscillations in the animal's sensitivity or general attention that is responsible for the behavioral variance. If multiple animals are observed over a given day, then time-of-day effect must be considered in the analysis.

A second issue with respect to the importance of recording LD cycles is that animals that are not reared in 24-h LD oscillations may not exhibit circadian rhythms (Richelle and Lejeune, 1980). For example, if the LD cycle at a particular geographical location or in a particular lab is highly unpredictable, then the activity rhythms observed in animals reared there may be quite arrhythmic or may be based on other cues, such as temperature or resource availability. These may lead to uncontrolled or unpredictable behavioral results that are not replicable under other LD cycles.

Finally, animals that exhibit circadian rhythms may be biased toward remembering 24-h event times. Honeybees are exceptionally good at following the daily p eaks in pollen and nectar production by visiting only certain species of flowers at certain times of day (Saunders, 1971; Seeley, 1995). However, bees are unable to learn to return at non-24-h periods. Kestrels (Falco tinnunculus) and starlings (Stur-nus vulgaris) have also been observed to follow food abundance in time, and both can be trained to return to a feeder at specific times of theday(Bell, 1991). Food-anticipatory activity rhythms have also been observed for rats fed at 24-h intervals ( Rosenwasser, 1984). Rats show a clear inclination for remembering feeding times set at 24-h intervals over much shorter (3 to 14 h) or longer (34 h) times, as shown by Crystal (2001). Given the nature of temporal memory, which I discuss further in "How Event Timers Might Work" (Section 4.4), it is probably also the case that there is an annual memory bias, as animals may be able to remember more clearly their seasonal context simultaneously with important events.

More work needs to be done to verify the distinction between circadian rhythms and event timers (see Crystal, this volume). Molecular examinations of mechanisms i nvolved in event timing require a phenotype for the isolation and cloning of relevant genes. A designer behavior would be useful in this respect. Tim Tully (DeZazzo and Tully, 1995) has made superb use of electrical stimulus to dissect memory formation in Drosophila. To determine if flies are capable of learning temporal durations, experimenters could train flies in a periodic shocking regime. For example, every 60 sec the experimenter runs a current through the cage. If flies have event timers, they may learn to associate flight at specific temporal intervals with the absence of shock. Reward paradigms might also be useful, in which flies are rewarded with food at consistent intervals. Once the phenotype is established, various per mutants could be assayed for defects in event timing (see Cevik, this volume). These mutants could then be assayed for more ecologically relevant behaviors associated with fitness. C. elegans might also be a useful subject for molecular study of event timers, as their nervous system and genome are amenable to neural and genetic investigation.

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