Environmental Physiology: Proceedings of the 28th International Congress of Physiological Sciences, Budapest, 1980

Physiology"

CIRCADIAN SYSTEM PROPERTIES

Jürgen Aschoff,     Max-Planck-Institut für Verhaltensphysiologie, Andechs, FRG

Publisher Summary

This chapter discusses several basic properties of the circadian system. Circadian rhythms are characterized by their capability to free-run in constant conditions like self-sustaining oscillations and by the way in which they are synchronized—entrained—by periodic factors in the environment, the Zeitgebers. Circadian rhythms are only entrainable by Zeitgebers within a limited range of periods. Within this range of entrainment, the rhythm changes its phase-relationship to the Zeitgeber: the rhythm phase leads a Zeitgeber having a relatively long period, and phase lags a Zeitgeber with a short period. Moreover, when exposed to an appropriate Zeitgeber, for example, a light-dark cycle complemented by regular gong signals, human circadian rhythms can be entrained—within limits—to periods other than 24 h.

Én nem csalódom. Minden szervem ora, mely csillagokhoz igazitva jár.

(I am not misled. All my organs are clocks which run adjusted to the stars.)

Attila József, 1937

When Attila József wrote his poem ‘Majd emlékezni jó lesz’ (It will be good to remember) in which he refers to his inner clocks, the biological basis of such time measuring devices had yet to be discovered. As a true poet, József envisaged what was shown to be true about 25 years later: that there is a biological clock in man which measures time of day, and that it consists of a multiplicity of oscillating units, located in various organs and coacting as a complex entity called ‘the circadian system’. In this lecture, I am attempting to introduce a few basic features of the circadian system to those who have become interested in the subject only recently. To a large extent, the survey is based on data which have been collected by my co-workers E. Gwinner, K. Hoffmann, H. Pohl, U. von Saint Paul, and R. Wever over the last 15 years.

1 FREERUNNING AND ENTRAINED RHYTHMS IN ANIMALS

By evolutionary adaptation to the temporal program of day and night, eucariothic organisms have developed endogenous periodic processes whose natural frequency approximates that of the earth’s rotation and which persist in the absence of any periodic input to the organism. Since the period of the rhythm slightly deviates from 24 h under artificially constant conditions, the prefix circa has been introduced by Halberg (1959). Circadian rhythms, then, are characterized a) by their capability to freerun in constant conditions like self-sustaining oscillations, and b) by the way in which they are synchronized (entrained) by periodic factors in the environment, the Zeitgebers. The two examples provided in Fig. 1 show rhythms in oxygen uptake of two chaffinches, kept initially in light-dark cycles of 12 h light and 12 h darkness (LD 12:12) and thereafter in conditions of constant dim illumination (LL). As dayactive animals, the birds have a high level of oxygen uptake during L, and a low one during D. In LL, the rhythm persists undamped with a period, τ, which is longer than 24 h in the upper record, and shorter than 24 h in the lower record. This difference indicates that there can be a substantial interindividual variability in τ. In addition, τ is known to depend on the physiological state of the organism e.g. with regard to its reproductive functions, as well as on external factors such as intensity of illumination or ambient temperature. The effects of external factors show systematic differences between dayactive and nightactive species. (For a review, cf. Aschoff 1979a).

Fig. 1 Circadian rhythms of oxygen uptake in two chaffinches, Fringilla coelebs, kept first in a light-dark cycle (LD), thereafter in constant dim illumination (LD). τ = Mean circadian period. Shaded area: darkness. (From Pohl, published in Aschoff et al. 1980)

When entrained by a Zeitgeber, the circadian rhythm maintains a distinct phase-relationship with the entraining signals. This phase-angle difference ψ might be measured between an arbitrary phase of the rhythm, e.g. a minimal value, and the time of ‘light-on’ of a LD-cycle. If in case of the records reproduced in Fig. 1 one takes as phase reference the point where oxygen starts to increase from its low D-level, it becomes evident that ψ has a small positive value (i.e. a leading phase) in the upper record, and a large positive value in the lower record. This difference in ψ is correlated with the difference in the τ-values (24.8 h in the upper record, 23.1 h in the lower record) and reflects the general rule that ψ depends on the ratio between the τ of a rhythm (as measured in constant conditions) and the period T of the entraining Zeitgeber. In consequence of this rule, ψ also changes when a rhythm becomes entrained by Zeitgebers with periods other than 24 h (cf. Fig. 3). (For a discussion of these rules, cf. Aschoff 1965a, 1981a).

Fig. 3 Circadian activity rhythms of chaffinches, Fringilla coelebs, kept in light-dark cycles of various period length (T). τ = mean circadian period. Horizontal bars: activity time. Shaded areas: darkness. (Wever, unpubl.)

Next to a LD-cycle which is the prime Zeitgeber for most organisms, a cycle of low and high temperature can entrain circadian rhythms at least in poikilothermic animals. In lizards, a cycle with a range of only 0.9°C suffices to entrain the activity rhythm of 1/3 of the animals tested (Hoffmann 1969). Homeiothermic animals are less easily entrained by temperature cycles. In squirrel monkeys, a range of 17°C has been found to be effective in about 50% of the animals tested (Fig. 2, left diagram). If entrainment is not achieved, the rhythm continues to freerun with a mean period which is continuously modulated by the signals from the Zeitgeber. This relative coordination (Enright 1965) between the rhythm and a Zeitgeber of insufficient strength is illustrated in the right diagram of Fig. 2. The period of the non-entrained activity rhythm is close to that of the Zeitgeber when onset of activity coincides with the warm half of the temperature cycle, and it is lengthened when the onset of activity falls in the cold half of the cycle. Relative coordination also plays its role when various components of a freerunning circadian system loose their mutual coupling as in the case of internal desynchronization (cf. section 4, Fig. 9 and 10).

Fig. 2 Circadian activity rhythms in two squirrel monkeys, Saimiri sciureus, kept alternatively in constant temperature and in a temperature cycle. Original record duplicated along the abscissa. Within rectangles: time of higher temperature. (Tokura and Aschoff, unpubl.)

Fig. 9 Freerunning circadian rhythms of a singly isolated human subject, showing spontaneous internal desynchronization on day 14 of the experiment. White bars: sleep time. Triangles: minima of rectal temperature. Data plotted twice along the abscissa. (Data from Wever 1979)

Fig. 10 Interaction between the rhythm of rectal temperature and the sleep-wake cycle during internal desynchronization. Range of temperature (upper ordinate) and duration of sleep (lower ordinate) drawn as a function of the time at which sleep onset occurs in the temperature cycle. The abscissa represents a full circadian cycle of rectal temperature with a mean period of 25 h. (Data from Zulley 1980 and Zulley et al. 1981)

Circadian rhythms are only entrainable by Zeitgebers within a limited range of periods. Within this range of entrainment the rhythm changes its phase-relationship to the Zeitgeber according to the rule mentioned above: the rhythm phase leads a Zeitgeber which has a relatively long period, and phase lags a Zeitgeber with a short period. This is illustrated in Fig. 3 by the activity records from chaffinches. When kept in a LD-cycle with a period T = 24 h, the bird is dayactive but a late riser (onset of activity about 2 h after light-on). When T is changed to 25 h, the bird becomes an early riser, while in T = 23 h the normally dayactive bird becomes mainly nightactive. When T is lengthened to 26 h, or shortened to 22 h, entrainment is lost and the freerunning rhythm only shows relative coordination. It should be noted that a LD-cycle with 5 lux in L and 1 lux in D has been used in these experiments. Such a small range in intensity of illumination provides only a weak Zeitgeber; hence the range of entrainment is very small, and the changes in ψ with changing T are large (cf. Aschoff and Pohl 1978).

2 A SYSTEM OF COUPLED OSCILLATORS

So far, circadian rhythms have been treated as being controlled by one basic oscillator, a single circadian clock. A growing body of data indicates that such a one-oscillator model does not account for all the facts. Some evidence for a multi-oscillator system comes from the observation that free-running rhythms of locomotor activity can be dissociated into two components which, for some time, run with different frequencies until they have reached a phase difference of 180°. Such splitting often occurs after a change in the intensity of illumination, as illustrated in Fig. 4. A for the tree shrew (Hoffmann 1971). Splitting was first described by Pittendrigh (1960) for the arctic ground squirrel Spermophilus undulatus, and for the golden hamster Mesocricetus auratus (Pittendrigh 1967). Together with the fact that many activity records show two major peaks at the beginning and the end of each activity time (Aschoff 1957, 1966), the phenomenon of splitting prompted Pittendrigh (1974) to suggest that the circadian pacemaker is comprised of two separable oscillators which mutually entrain each other and whose period differentially depends on light intensity. An expansion of this idea and its implications can be found in Pittendrigh and Daan (1976).

Fig. 4 Splitting of circadian activity rhythms in constant light; records duplicated along the abscissa. A) Tree shrew Tupaia belangeri (from Hoffmann 1971). B) European starling Sturnus vulgaris; arrows: injection of testosterone (from Gwinner 1974).

In discussing his findings with tree shrews, Hoffmann (1971) suggested that the endocrine system might be involved in the processes which result in splitting. This hypothesis is supported by Gwinner’s (1975) observation that, in the European starling, the period of the freerunning rhythm and the activity time are subject to seasonal changes in correlation with the growth and regression of the testes, and that activity time can be lengthened by the administration of testosterone. At the time of maximal testes growth, Gwinner (1974) observed a splitting of the starling’s activity rhythm, and was able to induce splitting in castrated birds by repeated injections of testosterone (Fig. 4 B). These results suggest that testosterone affects the mutual coupling of circadian oscillators.

Other data which, more indirectly, support the multi-oscillatory concept refer to the temporal interrelationship between various rhythmic functions. If an organism is fully entrained by a Zeitgeber, all rhythms usually maintain a distinct phase-relationship with each other. As has been shown by Pohl (1971) for three species of fringillid birds, these relationships can be altered when the birds are placed in constant conditions, and they are subject to changes when the period of the freerunning rhythm is altered due to a change in light intensity. This dependence of internal phase-relationships on experimental conditions will be discussed in more detail below (cf. section 3, Fig. 5 and 6). Drastic changes in the temporal patterns of circadian systems also occur during entrainment after a sudden shift of the Zeitgeber. It usually takes for the system several days to regain its normal phase relationship to the shifted Zeitgeber. Since different rhythmic functions are re-entrained at a different rate (cf. Aschoff et al. 1975), the system is temporarily out of order, a state which has been called transitory dissociation (Aschoff 1965b; transient internal desynchronization, Moore-Ede et al. 1977). This kind of internal dissociation is to be distinguished from true internal desynchronization, a state in which different rhythmic functions continue to run with different frequencies, and hence continuously change their mutual phase-relationship (cf. section 4, Fig. 8 to 10).

Fig. 5 Circadian rhythms of a subject exposed to the natural Zeitgebers for the first and last 7 days, and isolated from day 8 to 24. Black and white bars represent wakefulness and sleep, triangles the maxima of oral temperature; τ = mean circadian period (± standard deviation). Right two diagrams: patterns of rhythms, averaged over 14 days under entrainment and 12 days during isolation. Shaded area: sleep. The abscissa represents 24 h in the upper diagram, and 26.1 h in the lower diagram. (Data from Kriebel 1974).

Fig. 6 Circadian rhythms of a subject exposed to an artificial Zeitgeber, with reading lamp available. Left: Black and white bars represent wakefulness and sleep, triangles maxima (above bars) and minima (below bars) of rectal temperature; shaded area = darkness; τ = mean circadian period; T = Zeitgeber period. Right two diagrams: patterns of rhythms, averaged over several periods under T = 24 and T = 26.67 h; shaded area = sleep (From Aschoff et al. 1969).

Fig. 8 Circadian rhythms of two singly isolated subjects under the influence of a strong Zeitgeber (i.e. a light-dark cycle without reading lamp) whose period T is steadily shortened (above) or lengthened (below). Black and white bars represent wakefulness and sleep, triangles the maxima and minima of rectal temperature. τ= mean circadian period of the temperature rhythm. Shaded area: darkness. (From Wever 1980).

It finally should be mentioned that, in confirmation of József’s antizipation, circadian rhythms have been documented in organ cultures in vitro, e.g. for the adrenal gland of hamsters (Andrews 1968; Shiotsuka et al. 1974) and for the chicken pineal gland (Binkley et al. 1978; Deguchi 1979)

3 FREERUNNING AND ENTRAINED RHYTHMS IN MAN

When enclosed in an isolation unit without time cues, human subjects usually show freerunning rhythms with periods somewhat longer than 24 h. In studies with 137 singly isolated subjects and 12 groups of two subjects each, a mean period (± standard deviation) of 25.0±0.50 h has been found (Wever 1979). The transition from the entrained to the free-running state is accompanied by changes in the internal phase relationships. The left diagram of Fig. 5 exemplifies this for the rhythm of wakefulness and sleep and of oral temperature in a subject who was exposed to the natural Zeitgebers for the first and last 7 days of the experiment, and was isolated from day 8 to 24. The rhythm of oral temperature remained close to 24 h during the first few days of isolation even though the activity rhythm was shortened for two cycles and then assumed a mean period of 26.1 h. As a consequence of this transitory internal dissociation (Aschoff and Wever 1976) the maxima of oral temperature were advanced relative to the sleep-wake cycle until, in the steady state of the freerunning rhythm, they occurred at the beginning instead of at the end of wakefulness. Similar changes in internal phase-relation ships were observed in other autonomic functions, as shown in the right half of Fig. 5. In addition to the well expressed shifts in maxima and minima of the rhythms, the two diagrams indicate that the wave form was changed: the curves are skewed to the right in the entrained rhythm, and skewed to the left in the freerunning rhythm.

When exposed to an appropriate Zeitgeber, e.g. a light-dark cycle complemented by regular gong signals (Wever 1970), human circadian rhythms can be entrained, within limits, to periods other than 24 h. In Fig. 6 the results are reproduced of an experiment in which the subject was able to make use of an additional reading lamp at will; hence, he was not forced to adher to the cycle of the main lights controlled from outside the chamber. Nevertheless, he remained entrained when the artificial 24-h day was lengthened to 26.67 h (left diagram of Fig. 6). At the same time he changed from a late riser to an early riser, a change in the external phase relationship that was to be expected in view of the rule mentioned above (cf. Fig. 3). When the Zeitgeber period was shortened to 22.67 h, after an interlude of some 24-h days, entrainment was lost, and the subject’s rhythms started to freerun with a period of 25:2 h.

The lengthening of the Zeitgeber period from 24 to 26.67 h not only forced the subject to become an early riser, but was also accompanied by changes in the internal phase-relationships. This is illustrated in the right two diagrams of Fig. 6 by the patterns of 3 rhythmic variables, individually averaged over several periods under the respective condition. Compared with the normal day (above), in the long day (below) all phases were advanced relative to the sleep-wake cycle.

A summary of data from 6 subjects who were entrained to various Zeitgeber periods is given in Fig. 7. The horizontal dashed line representing the middle of light time of the Zeitgeber is used as a reference phase. Within the range of periods from about 23 to 27 h, all rhythms change their phase angle difference from large negative values (lagging phases) to less negative or even positive values. The slope of the curve is steeper for the autonomic functions than for the sleep wake cycle. This indicates that a smaller range of entrainment is to be expected for the autonomic functions than for the activity rhythm (Aschoff and Pohl 1978; Wever 1979). The results obtained from 12 subjects in these and other experiments support this view: when exposed to a 26.67-h day or a 22.67-h day, some of the subjects remained entrained only with their sleep-wake cycle while the rhythms of body tempereture and urinary excretion started to freerun (Aschoff et al. 1969)

Fig. 7 Phase-angle differences between circadian rhythms and Zeitgeber, derived from 6 singly isolated subjects who were entrained to various Zeitgeber periods. Regression lines drawn through acrophases (computed maxima) as a function of Zeitgeber period (From Wever 1972).

4 INTERNAL DESYNCHRONIZATION

That the entrainment of autonomic rhythmic functions is limited to a narrow range of periods can be easily demonstrated in subjects who are exposed to light-dark cycles but do not have access to reading lamps. Under those conditions, the subject can hardly do anything as long as the room is dark, and, hence, is more or less forced to adjust his activity-rest cycle to the cycle of light and dark. Consequently, it is possible to entrain the activity rhythm beyond the limits found with weaker Zeitgebers as described above (cf. Fig. 6). The two examples provided in Fig. 8 show entrainment to light-dark cycles whose period is either steadily shortened (above) or lengthened (below) in small daily increments. In the upper record, the rhythm of rectal temperature (indicated by the triangles) becomes desynchronized from the activity rhythm when the Zeitgeber has reached a period of about 22 h; in the lower record the same happens when the Zeitgeber period approaches 27 h. Using this technique of forced internal desynchronization, Wever (1980) has been able to demonstrate that different overt rhythms split away from the activity cycle at different periods, indicating differences in the range of entrainment between various functions. One further comment has to be made regarding the two records reproduced in Fig. 8. As indicated in the diagrams, the τ-values of the freerunning temperature rhythms differ by 0.5 h. This should not be interpreted as indicating after-effects of the shortened or lengthened Zeitgeber period on τ; at least, the data collected so far do not produce evidence for systematic effects of that kind (Wever 1980).

Internal desynchronization can always be enforced by applying a strong Zeitgeber, but it also occurs spontaneously in freerunning rhythms. As shown in Fig. 9, a sleep-wake cycle (indicated by the sleep times only) may first freerun with a period of approximately 25.7 h and then suddenly lengthen to a mean period of 33.4 h. When this happens, the rhythm of rectal temperature desynchronizes from the activity rhythm and continues to freerun with a period of about 25.1 h. Several points should be noted: a) When coupled to each other (internal synchronization, day 1 to 13), the two rhythms maintain a distinct phase-relationship, the minima of temperature occurring at the beginning of each sleep time (cf. Fig. 5); b) During internal desynchronization, the minima of rectal temperature continuously change their phase-relationship to the sleep wake cycle; c) The duration of sleep continuously changes as the rhythm of rectal temperature crosses through the rhythm of sleep and wakefulness. The last observation points to the fact that, although synchronization between the two rhythms is lost there still is an interaction due to the coupling forces which are no longer strong enough to achieve entrainment. This persisting interaction between the uncoupled oscillators is indicated by the continuous modulation of the mean period, i.e. by relative coordination (Wever 1968; Aschoff