Environmental synchronizers of squirrel monkey circadian rhythms.

Environmental synchronizers monkey circadian rhythms FRANK M. SULZMAN, Department of Physiology,

CHARLES Harvard

A. FULLER, AND MARTIN C. MOORE-EDE Medical School, Boston, Massachusetts 02115

SULZMAN, FRANK M., CHARLES A. FULLER, AND MARTIN C. MOORE-EDE. EnvironmentaL synchronizers of squirreZ monkey circadian rhythms. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 43(5): 795800, 1977. - Various temporal signals in the environment were tested to determine if they could synchronize the circadian timing system of the squirrel monkey (Saimiri sciureus). The influence of cycles of light and dark, eating and fasting, water availability and deprivation, warm and cool temperature, sound and quiet, and social interaction and isolation was examined on the drinking and activity rhythms of unrestrained monkeys, In the absence of other time cues, 24-h cycles of each of these potential synchronizers were applied for up to 3 wk, and the periods of the monkey’s circadian rhythms were examined. Only light-dark cycles and cycles of food availability were shown to be entraining agents, since they were effective in determining the period and phase of the rhythmic variables. In the presence of each of the other environmental cycles, the monkey’s circadian rhythms exhibited free-running periods which were significantly different from 24 h with all possible phase relationships between the rhythms and the environmental cycles being examined. zeitgebers; primates; biological dark cycles; feeding schedules

rhythms;

entrainment;

light-

IN NATURAL ENVIRONMENTS are synchronized with solar time so that specific activities are confined to characteristic phases of the day and thus show 24.0-h periodicities. In the absence of temporal information from the environment, free-running patterns are observed with continuously varying phase angles with respect to solar time, and with periods which are approximately, but not exactly 24.0 h in length. The environmental signals that have been reported to entrain circadian rhythms are light and dark (4), temperature (12, 14, 21, 23, 26), social interaction (1, 10, 15, ZO), sound (2, 7, 9, 15, 16), electrostatic fields (6), and barometric pressure (11). Because of the paucity of information on zeitgebers in nonhuman primates we have screened and compared a wide range of environmental signals which might act as synchronizers of squirrel monkey circadian rhythms. We tested cycles of light and dark (LD), eating and fasting (EF), water availability and deprivation (thirst) (WT), hot and cold temperature (HC), noise and quiet (NQ), and social interaction and isolation (SI). Three criteria were used to evaluate the ability of the various environmental cycles to entrain free-running circadian ANIMALS

of squirrel

rhythms. First, the period of the animal’s rhythm must be equal to the period of the environmental cycles. Second, a stable and reproducible phase relationship between the rhythm and the environmental cycle must be evident, with the rhythm’s phase being independent of solar time and dependent on the time of the environmental cycle. Third, upon cessation of the zeitgeber cycle the initial phase of the ensuing free-running rhythm must be determined by the phase of the environmental cycle. By these criteria, only light-dark cycles and cycles of food availability were shown to be effective entraining agents. METHODS

The animals used in this study were adult squirrel monkeys (Saimiri sciureus). These small (600-1200 g) daytime active South American primates were individually housed in cages (35 x 35 x 50 cm). Unless otherwise noted these experiments were conducted in temperature-controlled isolation chambers. The circadian rhythms of drinking and activity were followed in this work. The drinking pattern of each animal was monitored by means of an electrical circuit which provided a switch closure every time the monkey touched the spout of the water bottle. Gross motor activity was recorded by an ultrasound motion detector (Alton Electronics Co.) or by placing the animal in a tilt cage. In the latter situation, a bar was placed under the center of the cage and positioned so that the animal’s movement from one side of the cage to the other caused the cage to tilt and thereby activate a microswitch. Activity and drinking were recorded on cumulative recorders (Gerbrands model C-3) or event recorders (Gerbrands model PZ-C). The data from the event recorders were digitalized by estimating the number of events which occurred during each hour of the experiment. The data from these experiments were plotted by a Complot plotter. The plotting program represents the level of activity or drinking at each hour by a series of vertical pen strokes, with the number of strokes per hour being proportional to the amount of activity or drinking. Missing data are represented by a dashed horizontal line. To determine a phase reference point for each cycle, a PDP-12 computer was used to fit a sine wave to 24-h segments of data and the phase of the maximum of the 795

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sine determined. The period of the rhythm was then determined by linear regression using the phase points calculated for each day, so that the slope of the line is equal to the change of phase per day. This value was added to 24.0 h to give the period in hours.

agents were examined for their ability to entrain the drinking rhythm. Figure 1 shows the result of one such experiment. To facilitate visual examinatioii, the data in this figure (and subsequent figures) are double-plotted so that the first line shows days 1 and 2, the second line days 2 and 3, etc. From days 1 to 20, the animal was in constant light, with all other environmental RESULTS factors held at constant levels, and a free-running environmental1 1 factors1 could pro- 1 Dattern was evident with a period of 24.9 t 0.2 h (mean . To 1 determinea- what A vlde temporal information to squirrel monkeys, several Ak SD).

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FIG. 1. Drinking rhythm of a monkey plotted as a function of time of day. To facilitate visual inspection, data are double-plotted as described in text. The following environmental cycles were examined with all other environmental factors held constant with LL at 600 lx: days 21-32, cycles of 12 h of noise (0800-2000 h) and 12 h of quiet (2000-0800 h) (NQ 12:12); days 40-51, cycles of 8 h of social interaction (09001700 h) and 16 h of isolation (1700-0900 h) (SI 8:16); days 71-84, hot and cold temperature cycles with 28 * 1°C from 0300 to 1500 h, and 20 + 1°C from 1500 to 0300 h (IX 12:12); days 97-134, eating and fasting cycles in which the animal could eat for 3 h (0900-1200 h) and was deprived of food for 21 h (12000900 h) (EF 3:21); and days 153-X0, light-dark cycles with light from 0800 to 2000 h and dark from 2000 to 0800 h (LD 12:12). Missing data are represented as dashed horizontal Zincs.

EF

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ENVIRONMENTAL

SYNCHRONIZERS

OF

MONKEY

CIRCADIAN

797

RHYTHMS

NQ

Noise and quiet. The ability of sound cycles to entrain was tested by alternating periods of 12 h of noise with 12 h of quiet (NQ 12:12) for days 21-32. The 12-h noise consisted of 15-min cycles composed of 2 min of 100 dB of white noise followed by 13 min of ambient noise (-70 dB). During the quiet portion of the day, the 100-dB white noise was discontinued and only the ambient noise was provided. Although entrainment did not occur during the NQ portion of this experiment, there was an apparent shortening of the free-running period (7) to 24.2 t 0.1 h. However, the other two times this NQ cycle was imposed on animals no effect on 7 was elicited. The results of one of these experiments is given in Fig. 2 where the amount of activity is plotted as a function of time of day. Light (600 lx) and noise (-70 dB) were maintained at constant levels from days I to 22 and the free-running period was 25.7 t 0.3 h. Beginning on day 23 the animal was exposed to NQ 12:12, and this continued until day 46. Although all phase relationships between the rhythm and the NQ cycle were examined no entrainment was evident and the period was 25.7 t 0.3 h. The drinking rhythm for this animal showed the same results. Social interaction and isolation. The role that social factors play in synchronizing squirrel monkey circadian rhythms was next examined. For 8 h each day from days 40 to 51 (Fig. 1) the free-running monkey was allowed social interaction with another monkey which was entrained to an LD 12:12 cycle. This social interaction-isolation cycle (SI 8:16) was accomplished by opening the isolation chamber at 0900 h each day and placing a cage containing an LD 12:12 synchronized female squirrel monkey in sight, smell, and hearing of the monkey in the isolation chamber. Eight hours later the female was returned to the colony room (on an LD

FIG. 2. Activity rhythm of a monkey in constant light (600 lx> plotted as a function of time of day. From days 23 to 46 the animal was in a NQ 1212 cycle similar to that used in Fig. 1, and from days 50 to 66 a WT 3121 cycle was applied in which the monkey had water available for 3 h (08004100 h) and was deprived of water for 21 h (1100-0800 h).

12:lZ cycle) and the door to the isolation chamber was closed. As can be seen in Fig. 1, the free-running drinking rhythm continued, with 7 = 25.3 2 0.4 h. Nonvisual aspects of social interaction were also tested as synchronizers of squirrel monkey circadian rhythms. Animals were maintained in the colony room but within a light-tight compartment so that they were visually screened from the other animals in the room. The experimental animals were kept in constant light (LL), while the rest of the colony room was in LD 12:X All of the animals were free to communicate with each other by sound and smell. The seven experimental animals we tested in this protocol all showed freerunning rhythms. Hot and cold temperature. The synchronizing ability of ambient temperature cycles was assayed by holding the environmental temperature at 28°C for 12 h, and then switching the temperature to 20°C for 12 h (HC 12:12). From days 71 to 84 of the record in Fig. 1, the HC 12:12 cycle was applied but no entrainment was evident (T = 25.0 t 0.3 h). Alternating cycles of 28 and 20°C were tested on two other animals, neither of which showed entrainment. The temperature of 28’C was chosen because it is a typical daytime temperature in the monkeys’ natural habitat (27), and the low temperature of 20°C was picked because it is below the thermal neutral zone for these animals and heat conservation and production mechanisms must be invoked for temperature regulation. In one additional animal, a HC 12:12 cycle with H = 32°C and C = 17OCwas tested for 4 wk, and this animal also showed no entrainment. Eating and fasting. The ability of food to act as a zeitgeber was examined in the following way. Every day at a fixed time the chamber was entered and food was placed in the food cup. Three hours later the


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chamber was reentered and any remaining food was removed. Thus, the monkey could eat for 3 h, and then fasted for 21 h each day (EF 3:21). As can be seen in Fig. 1, from days 97 to 134, this EF 3:21 cycle was applied from 0900 to 1200 h and the animal’s drinking rhythm was entrained (7 = 24.1 t 0.1 h). However, it can be seen that for several days after the EF cycle was initiated the drinking rhythm slowly moved into its new phase relationship with the EF cycle. These transients further support the conclusion that food is actively entraining the drinking rhythm and not merely eliciting a passive response. The activity rhythm of this animal was also entrained by the EF 3:21 cycle as can be seen in Fig. 3 (7 = 24.1 t 0.1 h). Here, the activity of the animal used in Fig, 2 is shown from days 90 to 160 of the experiment. Further evidence that the EF 3:21 cycle entrains these circadian rhythms was that when the EF cycle was discontinued and food was continuously available, both these rhythms freeran from a phase determined by the EF cycle. Six other animals that we have examined in EF 3:21 cycles similarly showed entrainment. However, in a seventh animal, besides the dominant 24.0-h periodicity in the data, visual inspection of the record indicated that there was a splitting of the drinking pattern with the major component entrained to the EF cycle and a minor component free-running. Water availability and thirst. We have also examined whether cycles of water availability and deprivation (thirst) can entrain squirrel monkeys. This was accomplished by entering the chamber at a fixed time each day and providing the monkey with a water bottle. Three hours later the chamber was reentered and the bottle was removed. Thus the animals had water available for 3 h, and for the remaining 21 h of the day water was removed (WT 3:21). The data from one of RCTIVITY

TIME

OF DAY

FULLER,

AND

MOORE-EDE

these experiments are shown in Fig. 2. The WT 3:21 cycle was tested from days 50 to 66, but the animal’s activity rhythm free-ran (7 = 25.7 t 0.4 h) through the WT cycles demonstrating that the WT cycles did not entrain. WT cycles were tested on three other animals, two of which showed no entrainment to the WT cycle, while the third showed partial entrainment with a splitting of the drinking pattern into an entrained component and a free-running component. Light and dark. Light-dark cycles entrain circadian rhythms of squirrel monkeys. The activity rhythm and the drinking rhythm of one animal maintained in isolation is shown in Fig. 4, top and bottom, respectively. During days 1 to 10, the animal was exposed to alternating periods of 12 h of light (600 lx) and 12 h of dark (~1 lx) (LD 12:12). It is apparent that the vast majority of drinking and activity are confined to the light portion of the LD cycle. The period of the drinking rhythm in the ED 12:12 cycle was 24.0 t 0,l h and the period of the activity rhythm was 24.0 t 0.1 h. From days 11 to 24 the monkey was placed in constant light (600 lx) (LL). Free-running patterns developed for both rhythms, with periods of 25.4 t 0.4 h for drinking and 25.5 t 0.4 h for activity. These results are typical of the 50 squirrel monkeys that we have examined in similar conditions. DISCUSSION

We have shown that 24-h light-dark or eating-fasting cycles are capable of entraining free-running rhythms of both drinking and activity in squirrel monkeys. Daily cycles of water availability and deprivation, hot and cold temperature, sound and quiet, and social interaction and isolation were also examined, but these were unable to synchronize squirrel monkey rhythms.

CI-RSI

FIG. 3. Simultaneously recorded activity rhythm of the monkey shown in Fig. 1 during days 90-160 of experiment.


ENVIRONMENTAL

SYNCHRONIZERS

DRINKING OS'

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FIG. 4. Comparison of the entrained and free-running rhythms of activity (top) and drinking (bottom) of a monkey maintained in isolation. Animal was in an LD 12:12 cycle from days 1 to 10 and in LL (600 lux) from days 11

CI-RSI 111

08

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Each potential environmental synchronizer was tested several times in different animals for up to 3 wk, and all possible phase relationships between the animals’ rhythms and the environmental cycles were examined. While our results on these ineffective entraining agents do not necessarily mean that they provide no temporal information to animals in nature, we can say that under the conditions tested, they are ineffective as zeitgebers. Twenty-four-hour illumination cycles or feeding cycles however, are capable of capturing freerunning rhythms and holding the period constant at 24 h so that there is a fixed phase relationship between the zeitgeber and the measured overt rhythm. It has long been known that light and dark is the major entraining agent for most animals (4), and the ability of LD cycles to entrain circadian rhythms of squirrel monkeys has been known for some time (17, 22). In other nonhuman species maintained in the absence of changes in illumination, several environmental factors have been shown to be capable of entraining activity patterns. Social interactions can be an effective zeitgeber. Blind mice housed in the same room with sighted mice may be synchronized to LD cycles (after several months), presumably through social cues (10). Additionally, Menaker and Eskin (15) have shown that two sparrows housed in constant conditions in one cage and separated by a screen can entrain one another so that both free-run with the same period. Cycles of conspecific sound have been shown to entrain activity patterns of birds (9, 15) and hamsters (16). Even a 6-h intermittent buzz 25 dB above ambient noise levels can entrain birds (7). Temperature cycles are known to be effective zeitgebers for poikilotherms (12, 21, 23, 26), but with one exception (14) they are ineffective in rodents (4, 5, 24). Wheel-running activity of mice can be entrained by cycles of electrostatic field strength (6). Another geophysical variable shown to be a zeitgeber is atmospheric pressure. Cycles of pressure changes (12 h

20-

1 1 , , , ,

to24. 38

of 1.00 atm alternating with 12 h of 1.09 atm) can entrain the rhythm of body temperature in mice (11). However, cycles of electrostatic field strength and barometric pressure are only effective at levels much larger than those seen in normal environments. Periodic availability of food (3, 8, 13, 18, 19) and water (13) has been shown to modulate the phases of expressed rhythms in animals concurrently exposed to LD cycles. Apart from a preliminary communication from this laboratory (25), no rigorous experiments have been published which have tested whether or not periodic availability of food is capable of entraining circadian rhythms in the absence of any other temporal information. Although both food and water were provided in the same manner in the current studies, the periodic availability of food entrained the circadian rhythms while the periodic availability of water did not. This suggests that there is some active agent in the food which functions as a zeitgeber. Man, the only other primate in which a variety of entraining agents has been examined, shows quite different results from those we report here for the squirrel monkey. It has been reported that light is not a strong zeitgeber in man since human rhythms may free-run in 24 h lighting cycles which are capable of entraining the rhythms of other species. Social stimuli have been suggested to play a more important role in synchronizing human circadian rhythms (1, 20). For squirrel monkeys, light is a strong zeitgeber, while social factors are not. In humans, concurrent LD and sound cycles can synchronize circadian rhythms (Z), whereas NQ cycles do not synchronize monkey rhythms. While EF cycles can entrain monkey circadian rhythms, they have not been tested in man. Definitive comparisons of different zeitgebers for human and nonhuman primates are tenuous, however, because of different experiment protocols. For example, monkeys have had no control over the light-dark cycles


800

SULZMAN,

in which they have been studied, while humans have had access to other source of illumination (e.g., reading and bathroom lights) during the dark period of the light-dark cycle. The effect of a true light-dark cycle has never been reported for man when no other time cues were present. Therefore it now seems appropriate to reexamine the efficacy of LD cycles in humans, and to examine the role that meal timing plays in human circadian organization, We gratefully

acknowledge

the contributions

made

by Dr.

Linda

FULLER,

AND

MOORE-EDE

Hiles and Dr. Janet Zimmerman in discussing these results; MS. Meredith McLaughlin, Mr. Michael DeLeo, Mr. John Jacobs, and Mr. Alfred Wallace in conducting these experiments; Mr. Billy Mock, Mr. Mark Anthony, and Mr. Louis DeToma in processing these data; and Ms. Margaret Harrigan in preparing this manuscript. This work was supported by National Aeronautics and Space Administration Grant NAS-9-14249, National Science Foundation Grant PCM-6-19943 and National Institutes of Health Grant GN22085. Received

for publication

4 March

1977.

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by sound in Passer domesticus. Science 154: 1579-1581, 1966. von Schall auf die tagesperiodische Aktivitst 16. MEYER, A. Einfluss des Goldhamsters. Naturwissenshaften 55: 234-235, 1968. 17. MOORE-EDE, M. C., D. A. KASS, AND J. A. HERD. Transient internal desynchronization after light-dark phase shift in monkeys. Am. J. Physiol. 232: R31-R37, 1977. 18. MOURET, J. R., AND P. BOBILLIER. Diurnal rhythms of sleep in the rat: augmentation of paradoxical sleep following alterations of the feeding schedule. Intern. J. Neurosci. 2: 265-270, 1971. 19. NELSON, W., L. SCHEVING, AND F. HALBERG. Circadian rhythms in mice fed a single daily meal at different stages of lighting regimen. J. Nutr. 105: 171-184, 1975. 20. POPPEL, E. Desynchronisationen circadianer Rhythmen innerhalb einer isolierten Gruppe. Pfluegers Arch. 299: 364-370, 1968. 21. RENCE, B., AND W. LOHER. Arrhythmically singing crickets: thermoperiodic reentrainment after bilobectomy. Science 190: 385-387, 1975. 22. RICHTER, C. P. Inherent twenty-four hour and lunar clocks of a primate -the squirrel monkey. Corn mun. Behavioral BioZ. 1: 305-332, 1968. 23 ROBERTS, S. K. Circadian activity rhythm in cockroaches: entrainment and phase-shifting. J. Cellular Comp. Physiol. 59: 175-186, 1962. 24 STEWART, M. C., AND W. C. REEDER. Temperature and light synchronization experiments with circadian activity rhythms in two color fdrms of the rock pocket mouse. Physiol. Zool. 41: 149156, 1968. 25. SULZMAN, F. M., C. A. FULLER, AND M, C. MOORE-EDE. Feeding time synchronizes primate circadian rhythms. Physiol. Behau. 18: 775-779, 1977. 26. SWEENEY, B. M., AND J. W. HASTINGS. Effect of temperature upon diurnal rhythms. Cold Spring Harbor Symp. Quant. Biol. 25: 87-104, 1960. 27. THORINGTON, R. W. Observations of squirrel monkeys in a columbian forest. In: The SquirreZ Monkey, edited by L. A. Rosenblum and R. W. Cooper New York: Academic, 1969, p. 87145. 28. ZIMMERMAN, W. F., C. S. Pittendrigh, AND T. PAVLJDIS. Temperature compensation of the circadian oscillation in Drosophila pseudoobscura and its entrainment by temperature cycles. J. Insect Physiol. 14: 669-684, 1968.


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