Molecular Bases for Circadian Clocks

1. In the first part of the paper, the model of non-parametric entrainment of circadian pacemakers is tested for the case of nocturnal rodents. The model makes use of the available data on freerunning period (τ) in constant darkness and on phase response curves (PRC) for short light pulses. It is tested in experiments using 1 or 2 light pulses per cycle. 2. Mesocricetus auratus and Peromyscus leucopus entrain to Zeitgebers involving 1 pulse (15' or 60') per cycle. The phase angle differences between rhythm and light cycle depends on the periods (τ and T) as predicted by the model. Entrainment of P. leucopus is unstable due to the after effects on τ created by the light pulse. 3. The limiting values of Zeitgeber period to which the animals entrain are much closer to 24 h than in Drosophila pseudoobscura, as the model predicts. However, frequent failures to entrain to T = 23 and T = 25 h are only explained if we take considerable interindividual variation in both τ and PRC into account. 4. With 2 pulses per cycle, the model predicts that entrainment will be more stable when activity is in the longer interval between the pulses than when it is in the shorter interval. This is true in the experimental data, where the phase relationships match predictions for skeleton photoperiods up to ca. 14 h. Increasing asymmetry forces animals into a "phase jump", so that activity shifts from the shorter to the longer interval. These ψ-jumps are accurately predicted in the hamster, but they occur at much longer photoperiods than predicted in P. leucopus. 5. Thus, the unqualified model, using a rigidly fixed species τ and PRC, is surely inadequate to explain entrainment. The extent to which variations in τ and PRC-shape, both "spontaneous" and induced by the entrainment process, can be known or inferred restricts the validity of the predictions. Yet we conclude, from a good deal of agreement between experiment and prediction (i), from the close correspondence between complete and skeleton photoperiods (ii), and on behavioural grounds (iii), that non-parametric entrainment by short light signals has a major share in the entrainment of nocturnal rodent rhythms in nature. 6. With these restrictions in mind, we analyse in the second part of the paper how the empirical regularities concerning τ and PRC contribute to the stabilization of the phase angle difference (ψ) between the pacemaker and the external world. Use is made of computer simulations of artificial pacemakers with variable τ and PRC. 7. ψ is most sensitive to instabilities in τ when ¯τ is close to 24 h. Thus the very circadian nature of these pacemakers helps to conserve ψ. Selection pressure for homeostasis of τ has been large in a species (M. auratus) where ¯τ = 24 h. The effect of ψ-instability is further reduced by entrainment with 2 pulses (dawn and dusk), made possible by the PRC's having both an advance and a delay section. 8. To analyze the contributions to ψ-conservation with seasonally changing photoperiod, we have assumed that it is of functional significance to conserve the phase of activity with respect to dusk (nocturnal animals) or to dawn (diurnal animals). We distinguish three contributions of nocturnal pacemaker behaviour to this type of ψ-conservation: increased amplitude of the PRC (i), asymmetry in the PRC, such that the slope of the delay-part is steeper than the slope of the advance-part (ii), and a short freerunning period in DD (iii). 9. A further contribution must derive from parametric effects of light, which are not traceable by the model, but certainly effective in preventing in complete photoperiods the ψ-jump which is seen in skeleton photoperiods. The existence of parametric effects is further demonstrated by the change of τ with light intensity in LL, described by Aschoffs Rule, which presumably reflects differences in PRC-shape between nocturnal and diurnal animals. 10. The paper concludes with an attempt to distinguish the features of circadian clocks that are analytically necessary for entrainment to occur (i), or have functional meaning, either in the measurement of the lapse of time (ii) or in the identification of local time (iii).

/pdf/a-functional-analysis-of-circadian-pacemakers-in-nocturnal-4uu8zk2j0m.pdf

A Functional Analysis of Circadian Pacemakers in Nocturnal Rodents. I. The Stability and Lability of Spontaneous Frequency

Most physiology and behavior of mammalian organisms follow daily oscillations. These rhythmic processes are governed by environmental cues (e.g., fluctuations in light intensity and temperature), an internal circadian timing system, and the interaction between this timekeeping system and environmental signals. In mammals, the circadian timekeeping system has a complex architecture, composed of a central pacemaker in the brain's suprachiasmatic nuclei (SCN) and subsidiary clocks in nearly every body cell. The central clock is synchronized to geophysical time mainly via photic cues perceived by the retina and transmitted by electrical signals to SCN neurons. In turn, the SCN influences circadian physiology and behavior via neuronal and humoral cues and via the synchronization of local oscillators that are operative in the cells of most organs and tissues. Thus, some of the SCN output pathways serve as input pathways for peripheral tissues. Here we discuss knowledge acquired during the past few years on the complex structure and function of the mammalian circadian timing system.

/pdf/the-mammalian-circadian-timing-system-organization-and-54vfplckht.pdf

The mammalian circadian timing system: organization and coordination of central and peripheral clocks.

1

Phase response curves for 15′ bright light pulses of four species of nocturnal rodents are described. All show delay phase shifts early in the subjective night, advance shifts in the late subjective night, and relative insensitivity during the subjective day.




2

The broad scatter in measured phase-shifts is largely due to error of measurement: the response of the pacemakers to light stimuli is more accurate than we observe.




3.

Indications are found that the response to a resetting stimulus at a given phase of the rhythm is correlated with the individual\(\bar \tau \) (freerunning period). Fast pacemakers (short\(\bar \tau \)) tend to be more delayed or less advanced by the light than slow pacemakers (long\(\bar \tau \)).




4.

Within individual mice (Mus musculus) the circadian pacemaker adjusts its resetting response to variations in its frequency: when τ is long (induced as after-effect of prior light treatment) light pulses at a defined phase of the oscillation (ct 15) produce smaller delay phase shifts than when τ is short.




5.

Among species there are conspicuous differences in the shape of the phase response curve: where\(\bar \tau \) is long, advance phase shifts are large and delay phase shifts small (Mesocricetus auratus); where\(\bar \tau \) is short, advance shifts are small, and delay shifts are large (Mus musculus;Peromyscus maniculatus).




6.

The functional meaning of the interrelationships of τ and PRC is briefly discussed.

A functional analysis of circadian pacemakers in nocturnal rodents

In a search for genes that regulate circadian rhythms in mammals, the progeny of mice treated with N-ethyl-N-nitrosourea (ENU) were screened for circadian clock mutations. A semidominant mutation, Clock, that lengthens circadian period and abolishes persistence of rhythmicity was identified. Clock segregated as a single gene that mapped to the midportion of mouse chromosome 5, a region syntenic to human chromosome 4. The power of ENU mutagenesis combined with the ability to clone murine genes by map position provides a generally applicable approach to study complex behavior in mammals.

/pdf/mutagenesis-and-mapping-of-a-mouse-gene-clock-essential-for-3635gtsmds.pdf

Mutagenesis and Mapping of a Mouse Gene, Clock, Essential for Circadian Behavior

Three mutants have been isolated in which the normal 24-hour rhythm is drastically changed. One mutant is arrhythmic; another has a period of 19 hr; a third has a period of 28 hr. Both the eclosion rhythm of a population and the locomotor activity of individual flies are affected. All these mutations appear to involve the same functional gene on the X chromosome.

https://www.pnas.org/content/pnas/68/9/2112.full.pdf

Clock Mutants of Drosophila melanogaster

The circadian oscillators of genetically short-period and long-period Drosophila exhibit reciprocal behaviour in four distinct ways: (1) with respect to the dependence of period on temperature, (2) in the change of period during constant darkness after ten days of constant light, (3) in the change of period during the second ten days of darkness as compared with the period during the first ten days, and (4) in the period change resulting from exposure to low-intensity constant light. The homeostatic control of the dependence of period length on temperature is impaired in the mutants as compared with wild-type flies. The normal Drosophila pacemaker may comprise two mutually coupled oscillators, whereas the mutants may represent a reduction in activity of one or the other constituent oscillator.

Reciprocal Behaviour Associated with Altered Homeostasis and Photosensitivity of Drosophila Clock Mutants

P-element-mediated transformations involving DNA fragments from the period (per) clock gene of Drosophila melanogaster have shown that several subsegments of the locus restore rhythmicity to per0 or per- mutants. Such fragments overlap in a genomic region complementary to one transcript, a 4.5-kb RNA which is probably the per message, in that it is necessary and (in terms of expression from this X-chromosomal locus) sufficient for the fly's circadian rhythms. It is also at least necessary for the high-frequency oscillations normally produced by courting males as they vibrate their wings. The entirety of the 4.5-kb transcript is not necessary for rather strong rhythmicity; nor does it seem to be sufficient, in transformants, for wild-type behavioral phenotypes. A 0.9-kb RNA, homologous to genomic region immediately adjacent to the source of the 4.5-kb species, oscillates in its abundance over the course of a day; but coverage of this transcript source in several transformants carrying a per0 mutation--which eliminates the 0.9-kb RNA's oscillation--does not restore rhythmicity. All of the independently isolated arrhythmic mutations tested were covered by the same array of overlapping per+-derived DNA fragments, implying that the only portion of the locus which has mutated to arrhythmicity is complementary to the 4.5-kb transcript.

Germ-line transformation involving DNA from the period locus in Drosophila melanogaster: overlapping genomic fragments that restore circadian and ultradian rhythmicity to per0 and per- mutants.

A MAJOR question in the physiology of activity rhythms is the nature of the coupling between the pacemaker and the motor system. There is good evidence for humoral coupling in several species. In the sparrow, pineal organs from donor birds maintained in a light–dark cycle different from that of the host were transplanted into the anterior chamber of the eye of recipient birds made arrhythmic by pinealectomy. In this case the activity rhythm of the host was restored and its phase was determined by a diffusible humoral factor from the implanted pineal1. In the mollusc Aplysia humoral coupling between the circadian pacemaker and the output cells is thought to control locomotor activity, although electrical coupling has not been ruled out2. In insects, however, experiments involving cockroaches and silkmoths indicate that an intact electrical connection between the brain and thoracic ganglia is required for rhythmic activity. The pacemaker controlling the locomotor activity rhythm in the cockroach appears to reside in the optic lobes, and disruption of the electrical pathway between the optic lobes and thoracic ganglia results in arrhythmicity3–6. Initial transplant experiments with this insect indicated that activity could be hormonally controlled by the sub-oesophageal ganglion7. While these results have not been repeatable by other laboratories8–10, evidence from parabiosis experiments does suggest some humoral influence on the activity rhythm11. Surgical experiments using giant silkmoths have also demonstrated the need for an intact neural connection between brain and thorax for expression of the flight activity rhythm12. Thus, until now, there has been no unambiguous evidence for humoral control of activity rhythms in insects. Here we show that a short-period mutant brain can produce a short-period activity rhythm when implanted into the abdomen of a genetically arrhythmic host. In this instance, there were no functional neural connections between the implanted brain and the locomotor system of the recipient, so the action of the brain must be mediated by humoral influences.

Transplantation of a circadian pacemaker in Drosophila

The normal 24-h period of the circadian rhythms of locomotor activity and eclosion of Drosophila melanogaster is altered by changes in per gene dosage. Females with only one dose of per+ or pers (the 19-h short-period mutant allele) or per1 (the 29-h long-period mutant allele) have periods which are about 1–2 h longer than the corresponding females with 2 doses. Females with 3 doses of per+ and males with 2 doses of per+ or pers have periods which are 1/2 to 1 h shorter than the corresponding individuals without the extra dose. Males with three per+ doses have periods which are about 1.5 h shorter than wild-type males; additional per+ doses do not shorten period further. The observation that decreased per dosage lengthens period while increased dosage shortens period suggests that the long- and short-period mutations alter period by respectively decreasing and increasing per gene or gene product activity. The per+ dosage results and the complementation behavior of pers indicate that the hypermorphic phenotype of pers results from increased activity of the pers gene product rather than an overproduction of per+ product. This is the first report of such a mutant action in Drosophila.

Ronald J. Konopka

Papers

Clock Mutants of Drosophila melanogaster

Reciprocal Behaviour Associated with Altered Homeostasis and Photosensitivity of Drosophila Clock Mutants

Germ-line transformation involving DNA from the period locus in Drosophila melanogaster: overlapping genomic fragments that restore circadian and ultradian rhythmicity to per0 and per- mutants.

Transplantation of a circadian pacemaker in Drosophila

Effects of dosage alterations at the per locus on the period of the circadian clock of Drosophila