Chronobiology E. ROBERT BUP~NS*

Department of Anatomy, University of Arkansas Medical Center, Little Rock, Arkansas

Abstract-Several fundamental principles of chronobiology are discussed. The importance of biological rhythmicity in (1) the every day interpretation of data, both descriptive and experimental, and (2) the design of scientifically accurate experiments, is the main theme of this presentation. Examples of circadian rhythms in a variety of physiological, psychological, and biochemical variables of the rodent and man are given.

IT IS AN HONORfor me to address the members of the Pavlovian Society on the occasion of its 15th Annual Meeting. My purpose is to tell you something about Chronobiology in general and to explain several fundamental concepts. I sincerely hope to convince you of the importance of Chronobiology and the important role it plays in almost every type of research endeavor. Time governs our lives. We have rhythmic behavior in our periods of wakefulness and sleep. The seasons change. Women of reproductive age have a monthly menstrual cycle. Indeed, "oscillation is an innate, endogenous, fundamental characteristic of all living systems" (Scheving and Pauly, 1974). Oscillation exists "at all levels of organization within an organism," and in a wide range of frequencies. The rutting of deer is an example of circannual rhythm or one with a frequency of 1 cycle/year 4-- 2 months. The monthly menstrual rhythm of the human female is an example of a circatrigintan rhythm or one with a frequency of 1 cycle/30 --- 5 days. The heart rate, respiratory and pulse rhythms are examples * Recipient of a Research Career Development Award (1KO4CA70594) from the National Cancer Institute of the National Institutes of Health. Associate Professor of Anatomy, Department of Anatomy, University of Arkansas Medical Center, Little Rock, Arkansas 72201. 161

Pay. J. Biol. Sci. July-Sept. 1975

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of ultradian rhythms, ones with frequencies which are higher than circadian. A circadian variation is a frequency of 1 cycle/24 -+-- 4 hours. (See Halberg and Katinas, 1973 for a complete glossary of chronobiological terms. ) Circadian rhythms in many physiological variables are not always as obvious as the respiratory, menslrual, and sleep-wake cycles. In fact, to look for circadian fluctuation in any particular variable requires that it be examined at frequent intervals, such as every 3 hours, over one or more 24-hour spans. It is the additional amount of work and c~st required to document circadian fluctuation (perhaps 8 times that of doing the experiment only once during a 24-hour span) that discourages some investigators; consequently, the acceptance of its importance in experimental biology and medicine is hampered. Although I will deal primarily with circadian rhythms, the reader should be aware of the fact that a great deal of research currently is being conducted on ultradian, circannual and other frequencies. The first concept I would like to discuss is the '~aours of changing resistance and susceptibility" (see reviews by Haus, 1964; Reinberg, 1967; Reinberg and Halberg, 1971; Scheving, yon Mayersbach and Pauly, 1974). Almost everyone in biology has experienced the frustration of trying to anesthetize a rat or a mouse correctly for

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the purpose of conducting an experiment. A standard dose (per gram body weight) of the anesthetic is injected into a mouse or rat, but the animal does not become anesthetized to the desired degree. The anesthesia is either too light or too heavy. One explanation for this phenomenon is that there is a circadian rhythm in an animal's resistance and susceptibility to the anesthetic agent (Fig. 1). In other words, a standard dose of a drug like pentobarbitol will cause a deeper and longer anesthesia when given during the phase o~ the circadian rhythm in which the animal is most susceptible to this particular drug. Likewise a mild, short lasting anesthesia results when the same dose of the same drug is given during the phase of the circadian rhythm in which the animal is most resistant. A long list of drugs, toxins, etc. (E. coli endetoxin, Ouabain, noise, librium, ethanol, methepyrapone, dimethylbenzanthracene, to mention a few) has accumulated for which rhythms in susceptibility and resistance have been documented (see reviews by Haus, 1964; Reinberg, 1967; Reinberg and Halberg, 1971; Scheving, yon Mayersbach and Pauly, 1974). The LDs0 of any particular drug is, therefore, a LDs0 only at one or two points in the animal's circadian rhythm in resistance and susceptibility to that particular drug.

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A dose considered to be a LDs0 might well be a LDr5 at the phase of most susceptibility to the drug and a LDI~ at the phase of most resistance. Another example of the hours of changing resistance is seen in Figure 2. In two separate studies done exactly 1 month apart (January and February, 1971) identical doses of arabinosyl cytosine (ara-C, a cancer chemotherapeutic agent) were iniected at the same point in the mouse circadian system for 5 consecutive days. One group of 15 mice received ara-C only at 0800 for 5 days; a different group of 15 mice received the same dose of the drug only at 1400, etc. The resulting mortality from such a schedule demonstrates a circadian rhythm in susceptibility (mortality) to ara-C (Seheving, et al. 1974). The same dose of ara-C kills 75g of the animals treated at midnight and only 15g of the animals treated at noon! (If you were a mouse and were to be treated once/day with this dose of ara-C, when would you want your injection, at 1200 or 24007) The practical advantages of an understanding of rhythms in susceptibility or resistance can be demonstrated by examining the use of ara-C as the therapeutic agent in the treatment of the lethal, transplantable mouse neoplasm, leukemia L1210. Without knowledge or use of the circadian rhythm in susceptibility to ara-C, a dosage schedule of 30 mg/kg body weight every 3 hours during one 24-hour span (total of 240 mg/kg) was devised as the best therapeutic schedule for the treatment of this leukemia (Skipper, Sehabel and Wilcox, 1967). Employing the knowledge of the circadian rhythm in resistance and susceptibility to ara-C, as measured by mortalities, a sinusoidal schedule was devised which gave the same total dosage (240 mg/kg) over the 24-hour span (Haus, eta/. 1972). The highest doses (67.5 mg/kg) were given near 1200 (the most resistant time) and the lowest doses (7.5 mg/kg) near 2400 (the most susceptible time), with doses of 15 and 30 mg/kg bridging the gap between the low and the high doses on both the ascend~,g and descending limbs of the known rhythm. Thus, by taking advantage of the host's rhythm in resistance and susceptibility to ara-C (use a sinusoidal schedule instead of an unchanging schedule), the mean survival time of the leukemia bearing mice was doubled (Hans, et al. 1972; this concept has been extensively reviewed by Halberg, et d. 1973). Changes in the wave form of a rhythm may determine the kind of experimental results one obtains. More importantly, the interpretation of the data may be erroneous ff the investigator uses single time point sampling and is unaware of rhythmicity in the variable being investigated. The example of a change in the wave

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Number 3

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FIG. 3. Circadian pattern in the uptake of ~H-TdR into the mouse kidney (solid line) and the effect of a single injection of isoproterenol (dashed line) when administered 28 hours previously. The time of sacrifice is plotted against the uptake of 3H-TdR. All animals received 3H-TdR 30 minutes before sacrifice. Each point is the mean of 6 mice • the standard error of the mean. (From Burns, Scheving and Tsai, 1972, courtesy of Science.)

form of a rhythm I will use is the effect that isoproterenol has on DNA synthesis and mitosis in the kidney and corneal epithelium of the mouse. Isoproterenol (IPR) was injected into different groups of mice every 4 hours in one 24-hour span and the mice were sacrificed exactly 28 hours later (one group received IPR at 0900 on day one and was sacrificed at 1300 on day two; a second group received IPR at 1300 on day one and was sacrificed at 1700 on day two, etc.). Saline injected mice served as the controls. The variable studied was the uptake of tritiated thymidine (aH-TdR) (Burns, Scheving and Tsai, 1972). The data appear in Figure 3. If this particular experiment had been done with smnpling at only a single time point (0900), the conclusion might have been that IPR significantly stimulated the uptake of ~H-TdR. In the groups sacrificed at 1300, 1700 and 2100, there was no statistical difference between the IPR and saline treated mice. In these instances the conclusion would have been that IPR had no effect on DNA synthesis. Surprisingly, in the groups sacrificed at 0100 and 0500, the conclusion might be that IPR significantly inhibits DNA synthesis. Three

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FIc. 4. Mean mitotic index and -+- the standard error of the mean in the corneal epithelium of saline (solid line) or IPR (dashed line) injected mice. The points plotted are sacrifice times, not injection times. The mice were injected at 0500, 1300, 1700, 2100, 0100 and 0500. The mice were sacrificed exactly 28 hours after injection (e.g., mice injected at 1300 were sacrificed at 1700 on the next day). (From Burns and Scheving, 1973, courtesy of The ]ournal of Cell Biology.) different results and conclusions might be obtained from the same experimental design depending on the point in the circadian system of the mouse that the experiment was initiated! Imagine the confusion that exists in the literature, simply because investigators have worked (1) in ignorance of biological rhythms and (2) with only single time point sampling. Figure 4 shows the mitotic index data obtained from the corneas of the same IPR or saline treated mice. The solid line represents the circadian rhythm in the mitotic index of the corneal epithelium of the saline treated mice. The peak occurred at 0900 and the trough at 2100 (approximately a 10-12 fo!d difference in mean values). The dashed line is the mitotic index data obtained from the groups of mice injected with IPR 28 hours previously. The tempting conclusion is that IPR abolishes the prominent circadian rhythm in mitotic index. Remember, however, that this is still a form of single time point sampling, i.e., each point represents the data obtained exactly and only 28 hours after treatment with IPR. There is, therefore, another time dimension in this experimental situation which must be investigated.

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FIc. 5. Mean mitotic index -4- the standard error of the mean in the corneal epitheliumof mice injected with saline (solid line) or IPR (dashed line) at 0900 and sacrificed in subgroups of 5 mice each every 4 hours for 3 days beginning 12 hours (2100) after injection of saline or IPR. Light from 0600-1800. (From Bums and Scheving, 1973, courtesy of The Journal of Cell Biology.) What is happening to the rhythm in the mitotic index in the time span after IPR treatment, i.e., not only at 28 hours, but at frequent intervals after IPR treatment? To investigate this problem, 100 mice were injected with IPR at 0900 and sampled at 4 hour intervals for 3 days (dashed line) beginning 12 hours after injection. A different 100 mice were injected with saline (solid line) at 0900 and sampled at the same times. Subgroups of mice were sacrificed every 4 hours for 3 consecutive days and the mitotic indices o{ the corneal epithelium were plotted (Fig. 5). The solid line (controls) again demonstrates the circadian rhythm in mitotic index with a peak on each day at 0900 and a trough at 2100. These data also demonstrate the remarkable reproducibility of this rhythm. A study of the dashed line (IPR treated mice) reveals that the circadian rhythm was not abolished by IPR; such might have been the conclusion if this phase of the experiment had not been completed. The rhythm was present, but the wave form has been altered or shifted. Note that the peak in the mitotic index occurred 8 hours ahead of schedule on day 1, and 4 hours ahead of schedule on days 2 and 3. The

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troughs in this rhythm were broadened, but occurred when one would expect them in the normal animal. A scientist working with the rhythm in the mitotic index of the cornea and the change in wave form induced in the rhythm by IPR would lead to erroneous conclusions ff single time point sampling was used. For example, let us suppose that the animals treated with IPR or saline at 0900 were sacrificed at only 0900 during the 3 days after the treatment. An examination of Figure 5 indicates that the conclusion, in this ease, would have been that IPR significantly inhibited mitosis on days i and 2 with a return to normal on the 3rd day. If 2100 was selected as the single t~ne point of sampling, the conclusion would be that IPR had no effect on corneal mitotic index for 4 consecutive clays (2100 sampling occurs 4 times on this graph). The same conclusion would be reached if 1300 was the single time point of sampling. If 0500 was used as the single time point of sampling, the conclusion would be that there was no effect on day i and a stimulation of mitosis on days two and three. It is obvious that single time point sampling is exceedingly dangerous, especially when one is totally unaware of circadian rhythmicity. Pitfalls also await the investigator who has mapped the rhythmicity of the variable being studied, but does not understand that changes in the wave form of the rhythm may occur upon treatment with a wide variety of compounds or situations. Although there is agreement among most chronobiologists that the social routine of man is his primary synchronizer, biological rhythms in rodents can be synchronized to a specific environmental stimulus, such as an artificial light-dark cycle. This is external synchronization (see Halberg and Katinas, 1973), and it serves to lessen the inherent variation in any particular variable in a group or colony of experimental animals. It does this by synchronizing individuals to the environmental stimulus and, therefore, to each other. In the absence of external synchronization biological rhythms may free-run. Free-nmning is a desynchronization "in the sense of exhibiting a continually and systematically changing phase relation to the schedule of a known synchronizer following the removal of the synchronizer" (see Ha!berg and Katinas, 1974). The artificial light-dark (LD) cycle is the most widely employed synchronizer. In rodents kept under an artificial light-dark cycle (light from 0600-1800) for approximately 7 days the cornea mitotic index rhythm consistently peaks around 0900 and reaches a trough at 2100. Figure 6 shows eight different 24 hour rhythms in the mitotic index of the rat cornea obtained over a span of years from 1965 to 1969 (Seheving and Pauly, 1974) in rats standardized to

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and Chronobiologia.)

the same artificial light-dark cycle (light from 0600-1800). Barring the possible influence of a circannual frequency superimposed on the circadian frequency; one can see that anyone, anywhere in the world should be able to obtain this rhythmic pattern in cornea mitotic index simply by synchronizing the research animals to the same light-dark cycle. The same should be true for human circadian

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rhythms. The mitotic index rhythm for adult human epidermis and its reproducibility is shown in Figure 7. Note that this human rhythm is not exactly the reverse of the rodent rhythm. What do you think would happen to the mitotic index rhythm in corneal epithelium of rodents ff the light-dark cycle was reversed, i.e., light from 1800-0600 and darkness from 0600-1800 (DL instead of LD)? If enough time was allowed for the animals to synchronize to the new environmental stimulus (a DL cycle), the peak in the cornea mitotic rhythm will occur shortly after "lights-on" and the trough will occur shortly after "lights-out" (Fig. 8). Thus, the animals will have completely resynchronized to the new external synchronizer. It takes approximately 7 days to reverse the rhythm in corneal epithelium, but a longer time is required for some other rhythms, for example those in enzymatic activity. In the work involving the corneal mitotic index the rhythm which was synchronized and then resynchronized by reversing the light-dark cycle from LD to DL was actually a colony rhythm derived from synchronized individual rhythms (Scheving, Pauly, yon Mayersbach and Dunn, 1974). Since the animals are sacrificed to acquire data on this variable, only one time point per animal can be obtained.

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Variables which do not require sacrificing the animals, such as rectal temperature, individual motor activity, etc., permit an analysis of colony data as well as individual data. If rodents are kept in continuous illumination (LL) instead of on a LD cycle, the animals 'have nothing to which they may synchronize, and each individual will begin to free-run at its own innate, endogenous frequency. To demonstrate this directly, one needs to study a variable which can be serially sampled in the living animal. Such serial sampling was done using the response of the same guinea pig to identical doses of histamine along a 24 hour time scale (Scheving, et al. 1973). The data appear in Figure 9. The panel on the left demonstrates the circadian colony rhythm in resistance (small erythema) and susceptibility (large erythema) to a standard dose of histamine under LD conditions. The middle panel demonstrates what happens to this colony rhythm ff the animals are kept in continuous illumniation (LL). Note the apparent loss of the rhythmicity. There is (1) a flattening of the rhythm to the point of its statistical abolishment or (2) the phasing of the

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rhythm of the colony changes from one study to the next. One might be tempted to hastily conclude that aU research animals should be kept on continuous illumination (LL), because this abolishes circadian rhythmicity. The important point, however, is that LL does not abolish individual rhythmicity, only the colony rhythm. The panel on the right of Figure 9 demonstrates the data from each individual guinea pig under the LL condition. Note that each animal does have a rhythm in its response to histamine, but there was a definite desy~chronization between some, but in this case, not all of the animals. Thus, five animals under LL demonstrated a peak response at 1600; whereas, under LD conditions, the trough occurs at 1600. One animal reached its peak response at 2200, and two animals demonstrated a peak at 0400. Taking the means from such partially desynchronized, free-running animals at the selected time intervals results in a plot of the data which does not show any evidence of rhythmicity. A second example of the effect of LL is seen in Figure 10. Here a LD synchronized rhythm in the mitotic index of corneal epithelium is plotted with the data obtained from two colonies of animals

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maintained on LL. Again we see the flattening or disappearance of the colony rhythm. Remember, however, that this observation does not mean that each animal has lost its biological rhythmicity. In fact, the individual animals are still oscillating, but with a freerunning pattern. It has been demonstrated that the light-dark cycle is a good synchronizer. Is food a good synchronizer? If so, will it dominate the LD cycle? To test this possibility, mice were permitted to eat only during a 4 hour span each day; and these feeding periods (dark vertical bars in Figures 11 and 12) occurred at different clock hours for 4 different groups of mice (Scheving, et al. 1974). A LD, ad libitum group was included as the control group. After 5 weeks of this standardization to restricted food in the presence o~ a lightdark cycle (LD or DL), a variety of variables were studied. Figure 11 demonstrates the mitotic index in the corneas from all 5 groups. The group fed ad libitum is represented by the dashed line in all panels; it is the standard circadian rhythm in the mitotic index of corneal epithelium with ~t peak soon after "lights-on" and a trough soon after "lights-out." In all four experimental groups, the rhythm in the mitotic index of corneal epithelium occurred with exactly the

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Fro. 13. Twenty - four hour periodicity in non-stress (dashed line) and ether stress (solid line) levels of plasma corticosterone in female rats. The stippled area indicates the standard error of the mean. Vertical lines with numbers indicate differences between non-stress and stress values. The horizontal black bar indicates the period of darkness. Non-stress animals were sacrificed in less than 20 seconds after cage opening. Ether stressed animals were exposed to ether vapor for 3 minutes, placed in a holding cage for 12 additional minutes and then sacrificed. (From Dunn, Schevhag and Millet, 1972, courtesy of the authors and the

American Journal of Physiology. )

same timing and phasing as the ad libitum rhythm. T h e conclusion reached was that this particular r h y t h m was not changed; i.e., cannot be synchronized to a restricted food schedule. The lightdark cycle is the obvious predominating synchronizer for this r h y t h m (Seheving, et al. 1974). Figure 12 shows the data on the n u m b e r of eosinophils found in the peripheral blood of the same mice used for the afore-

Fro. 14. Twenty - four hour period/cityin non-stress and ether stress levels of plasma eorticosterone in male rats (see legend to Figure 13).

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Fxc. 15. Twenty - four hour periodicity in nons t r e s s and novelty stress levels of plasma corticosterone in male rats (see legend to Figure 13).

mentioned restricted-feeding experiment. The circadian rhythm in the ad Iibitum group is the dashed line on each of the panels. Unlike the rhythms in the mitotic index of the cornea which did not demonstrate any change in phasing in response to restricted feeding, the rhythm in the number of eosinophils in peripheral blood did demonstrate a change in phasing which can be correlated with the feeding period. The trough in the eosinophil rhythm always occurred during the feeding period no matter when, during the 24 hour span, the four hour feeding period occurred. Apparently food, rather than the light-dark cycle, is the primary synchronizer for the rhythm in eosinophiles and possibly other rhythms (Pauly, et al. 1975). Other rhythms, however, such as the one in the mitotic index of the corneal epithelium, may be primarily synchronized to the lightdark cycle and not to food intake. In addition, other rhythms may be partially controlled by both food and light and, therefore, will not synchronize to either of these environmental conditions, they may instead demonstrate a synchrony that is the result of a complex interaction between the light-dark cycle and food intake. Another important area of chronobiology which has a significant impact on certain .types of biological research is the effect that stress may have on circadian rhythms. The example I will discuss is the circadian variation in stress-evoked increases in plasma corticosterone levels in rats (Dunn, Scheving and Millet, 1972). Figure 13 shows both the circadian rhythm in plasma corticosterone (dashed line) from non-stressed female rats (sacrificed immediately) and that from animals stressed by exposure to ether vapor. The increases evoked by ether stress in the level of plasma corticosterone differed in magnitude depending on the phase when the stress was applied.

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corticosterone levels occurred when the non-stress rhythm was near trough levels (0800). The smallest increases were obtained when the non-stress rhythm was near peak levels (1700). Female rats had peak plasma corticosterone levels and a mean 24-hour concentral:ion of plasma corticosterone which were markedly higher than those in males. Ether stress evoked a similar type of response in males (Fig. 14), but the quantity of the increase in the plasma corticosterone was always m u c h less than that found in females (compare Figs. 13 and 14). Novelty-stress (animal removed from its cage and placed in a different cage 15 minutes prior to sacrifice) evoked a response in female rats w h i c h was very similar to the one

following ether-stress. However, in males, novelty-stress only increased the plasma corticosterone levels when the non-stress rhythm

4, FIc. 16. Circadian variation in man showing rhythmic variables in vital signs. Meal times: 0830, 1430 and 1630. Rest or sleep time: 2245 to 0700. (From Scheving and Pauly, 1974, courtesy of the authors and Chronobiologia).

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in plasma corticosterone was not near or at its peak (Fig. 15). If single point sampling was being used with this system, two different conclusions could be reached: (1) novelty stress does increase plasma corticosterone levels in males (result when sampling was done at non-peak times in the non-stress rhythm) or (2) novelty stress has no effect on plasma corticosterone levels (result when sampling was done at the time of the peak in the non-stress rhythm).

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CI-IRONOBIOLOGY GROUP AND INDIVIDUAL - -

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I have been discussing circadian rhythms and their role in experimental design and the interpretation of data in research animals. Now, I would like to demonstrate a few of the many variables in humans which have been shown to be characterized by circadian rhythmicity (Kanabrochi, et aI. 1973; Scheving and Pauly, 1974). Keep in mind, however, that similar fundamental concepts are applicable in the interpretation of human data and similar pitfalls exist as they did for experimental animals. Figure 16 is a chronogram of the circadian variation in oral temperature, systolic blood pressure and minute ventilation. Although oral or rectal temperature may vary only two degrees (F ~) over a

182

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24 hour span, the pronounced effect that such a temperature change has on metabolic activity is well known. Figure 17 is a chronogram of the circadian variation in the concentrations of epinephrine, 17-hydroxycorticoids and cyclic AMP in urine. Figure 18 is a chronogram of the circadian variation in the ability to perform the addition Of a sequence of random numbers, eye-hand coordination tasks, and personal ratings of mood and vigor. Figure 19 is a ehronogram of the circadian variation in serum triglycerides. It demonstrates the group rhythm and two cases of individual variation. It is clear that some individuals have a very high range of change, others have relatively minor fluctuations, and the majority have oscillations occurring between these two extremes. Similar extreme variations have been documented for blood and intraocular pressure. From the practical viewpoint, it is possible to obtain an early diagnosis of hypertension or glaucoma, if the measurement is made at the time of the peak of the rhythm in an individaul with

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that acrophase was determined as a % of amplitude

FIG. 2"1. An acrophase map of 13 young, healthy soldiers. The acrophase, represented by a dot, approximates the crest of the circadian cycle and is shown in reference to the activity-rest schedule. The horizontal bar extending from the acrophase represents the 95% confidence interval. The acrophases which did not show a significant fit to a 24-hour cosine curve do not have confidence limits. The extent of total change along the 24-hour scale is shown in column 2. (From Scheving and Pauly, 1974, courtesy of the authors and Chronobiologia.)

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a natural wide range of change in his or her rhythmicity. Figure 9.0 is a chronogram of rat plasma eorticosterone compared to human serum cortisol. Because man is a dinmally active animal, and the rat is a noeturually active animal, it is tempting to simply reverse peak and trough times when predicting human data profiles from established rat and mouse data. Such a procedure will work for some rhythmic variables; however, many of those in man are not the exact reverse of the same rhyflm!ic variables in the rodent, and one must not generalize when data are availabh from only one type of animal and not the other. Chronobiology uses computerized statistical methods for the analysis of time series data. Among many methods of analyzing time series data is one developed by Halberg, et al. (1967). It is an inferential statistical method which involves the fitting of 24 hour and other cosine curves to the data by the method of least squares. This analysis provides a variety of information from the data such as (1) the mesor, M, the rhythm-adiusted, computer-determined, overall 24-hour location index which is equal to the arithmetic mean only if the data are equidistant and cover an integral number of periods; (2) amplitude, A, representing the distance from the mesor to the peak of the cosine curve; (3) acrophase, the timing of the peak of the cosine curve which best approximates the data (see also Halberg and Katinas, 1973). Some chronobiologieal data are displayed in the form of acrophase maps instead of in the form of a chronogram. An aerophase map of 40 different variables studied in healthy young men, appears in Figure 21. Condusion

Oscillation or rhythmicity is a fundamental property of life. It is a scientist's responsibility not to ignore biological rhythmicity, but to understand it and work with it. This paper is by no means a review article. It has only dealt with a few of the concepts and applications of chronobiology. It should serve as an introduction to the field and its importance in modem science. For further details the reader is referred to the following journals: Chronobiologia, Internat'.wnal lournal of Chronobiology and lournal of Interdisciplinary Cycle Research, and to the book Chronobiology, edited by L. E. Seheving, F. Halberg and J. E. Pauly, Igaku Shoin Ltd., Tokyo, 1974. Acknowledgments I would like to express my appreciation and thanks to Drs. Roscoe A. Dykman and William G. Beese for inviting me to speak before the

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Pavlovian Society. A special note of appreciation goes to Dr. W. Horsley Gantt for his interest in and support of this undertaking. I would also like to acknowledge the advice, help, criticism, support and intellectual motivation supplied by Drs. Lawrence E. Scheving and John E. Pauly during the preparation of this manuscript.

References Bums, E. R., and Scheving, L. E.: Isoproterenol-induced phase shifts in circadian rhythm of mitosis in murine corneal epithelium. 1. Cell Biol., 56:605-608, 1973. Burns, E. R., Scheving, L. E., and Tsai, T. H.: Circadian rhythm in uptake of tritiated thymidine by kidney, parotid, and duodenum of isoproterenoltreated mice. Science, 175:71-73, 1972. Dunn, J., Scheving, L. E., and Millet, P.: Circadian variation in stress-evoked increases in plasma corticosterone. Am. 1. Physiol., 223:402-406, 1972. Halberg, F., and Katinas, G. S.: Chronobiologic glossary. Intern. 1. Chronobiol., 1:31-63, 1973. Halberg, F., Tong, Y. L., and Johnson, E. A.: Circadian system phase-An aspect of temporal morphology; Procedures and illustrative examples. In H. yon Mayersbach (Ed.): The Celhdar Aspects of Biorhythms. Berlin, Germany, Springer-Verlag, 1974; pp. 20-48. Haus, E., Halbert, F., Scheving, L. E., Pauly, J. E., Cardosa, S., Kuhl, J. F. W., Sothern, R. B., Shiotsuka, R. N., and Hwang, D. S.: Increased tolerance of leukemic mice to arabinosyl cytosine with schedule adjusted to circadian system. Science, 177:80-82, 1972. Kanabrocki, E. L., Scheving, L. E., Halberg, F., Brewer, R. L. and Bird, T. J.: Circadian variations in presumably healthy men under conditions of peace-time army reserve unit training. Space Life Sci., 4:258-270, 1973. Reinberg, A.: The hours of changing responsiveness or susceptibility. Persp. Biol. Med., 11:111-128, 1967. Reinberg, A., and Halberg, F.: Circadian chronopharmacology. Ann. Rev. Pharmacol., 11:455-492, 1971. Scheving, L. E., Cardosa, S. S., Pauly, J. E., Halberg, F., and Hans, E.: Variation in susceptibility of mice to the carcinostatic agent arabinosyl cytosine. In L. E. Scheving, F. Halberg, and J. E. Pauly (Eds.): Chronobiology, Tokyo, Japan, Igaku Shoin Ltd., 1974. Scheving, L. E., and Pauly, J. E.: Circadian rhythms: Some examples and comments on clinical application. Chronobiologia, 1:3-21, 1974. Scheving, L. E., v. Mayersbach, H., and Pauly, J. E.: An overview of chronopharmacology. Europ. 1. Toxicol., 7:203-227, 1974. Scheving, L. E., Pauly, J. E., v. Mayersbach H., and Dunn, J. E.: The effect of continuous light or darkness on the rhythm of the mitotic index in the corneal epithelium of the rat. Acta Anat., 88:411-423, 1974. Scheving, L. E., Sohal, G. S., Enna, C. D., and Pauly, J. E.: The persistence of a circadian rhythm in histamine response in guinea pigs maintained under continuous illumination. Anat. Rec., 175:1-6, 1973. Scheving, L. E., Vedral, D. F., and Pauly, J. E.: A circadian susceptibility rhythm in rats to pentobarbitol sodium. Anat. Rec., 160:741-750, 1968. Scheving, L. E.: Mitotic activity in the human epidermis. Anat. Rec., 135: 7-20, 1959. Skipper, H. E., Schabel, F. M., and Wilcox, W. S.: Experimental evaluation of potential anticancer agents. XXI. Scheduling of arabinosyl cytosine to take advantage of its S-phase specificity against leukemia cells. Cancer Chemo. Rep., 51:125-165, 1967.