J. therm. Biol. Vol. 17, No. 2, pp. 129-133, 1992 0306-4565/92 $5.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1992 Pergamon Press Ltd

THE CIRCADIAN RHYTHM OF BODY TEMPERATURE OF NORMAL A N D TAU-MUTANT GOLDEN HAMSTERS

ROBERTO REFINETTI* and MICHAEL MENAKER

Department of Biology, University of Virginia, Charlottesville, VA 22901, U.S.A.

(Received 27 July 1991; accepted in revised form 12 October 1991)

Abstract--l. Intra-abdominal temperature of 13 normal and 13 tau-mutant golden hamsters maintained in constant light at 22°C was recorded by telemetry. Data for 10 days of continuous recording were analysed for the four parameters of the circadian rhythm of body temperature (CRT): period, amplitude, mean level and shape.

2. The mean period of the CRT was significantly shorter in tau mutant hamsters (20.2 h) as compared to normal hamsters (24.0 h). No significant difference between the two groups was found in the other three parameters.

3. It is concluded that the tau mutation has a specific effect on the period of the CRT without affecting the other parameters of the rhythm.

Key Word Index: Body temperature; circadian rhythm; golden hamster; tau mutation; Mesocricetus auratus

INTRODUCTION

The body temperature of homeothermic animals is regulated at a constant level despite large variations in environmental temperature, but this constant level is subjected to a regular daily oscillation (Cabanac and Simon, 1987). The daily oscillation in body temperature, which is characterized by high tempera- tures during the active phase of the animal and lower temperatures during the inactive phase, is controlled by an endogenous pacemaker in conjunction with environmental factors such as the light-dark cycle (Aschoff, 1970). Under constant environmental con- ditions, the circadian rhythm of body temperature is characterized by four parameters: the period (i.e. the duration of each cycle), the amplitude (i.e. the excur- sion of the variation during each cycle), the mean level (i.e. the average temperature), and the shape (i.e. the wave form of the oscillation) (Refinetti, 1992). The recent discovery of a genetic mutation in the golden hamster, which results in circadian rhythms of activity with considerably shorter periods (Ralph and Menaker, 1988), prompted us to examine the circa- dian rhythm of body temperature of mutant hamsters and compare it with the rhythm of normal (wild-type) hamsters.

As previously described (Ralph and Menaker, 1988), the tau mutation is a single gene autosomai mutation that appeared in a male golden hamster (Mesocricetus auratus, Charles River Laboratories, Wilmington, MA) and has been preserved by selective breeding. As determined by records of running-wheel activity, normal (wild-type) animals maintained in an environment without time cues have a circadian period of approximately 24 h, whereas homozygotic mutants have a period of approximately 20 h. This difference in the period of the circadian rhythm of

*Author for correspondence.

activity prompted us to examine whether an equival- ent difference can be found in the circadian rhythm of body temperature and whether the difference in period is accompanied by differences in the other three parameters of the temperature rhythm. We report here the results of this investigation.

MATERIALS AND METHODS

Thirteen 3-month old normal hamsters (9 females, 4 males) and 13 homozygotic mutants (9 females, 4 males) were used as subjects. Radio transmitters equipped with thermal sensors (Model VM-FH, Mini-Mitter Co., Sunriver, OR) were implanted in- traperitoneally under sodium-pentobarbital anaes- thesia (90mg/kg i.p.) at least 3 days before the beginning of the experiment. The transmitters were calibrated in water baths with an accuracy of 0.2°C before implantation and again at the end of the experiment (after removal from the animal under pentobarbital anaesthesia).

All animals were maintained in individual plastic cages (25 x 46 x 20cm) fitted with 17cm diameter running-wheels and provided with Purina lab chow, water, and wood shavings as bedding. To evaluate the effect of running activity on the rhythm of body temperature, the 4 male mutants and 4 male normals were maintained in cages without running-wheels before being transferred to cages with wheels. In all instances, the cages were placed on top of telemetry receivers (Model RA-1010, Mini-Mitter Co., Sun- river, OR) attached to a computerized data acqui- sition system (Dataquest III, Data Sciences Inc., Roseville, MN). Body temperature values (and wheel revolutions when applicable) were recorded every 6 rain for l0 or more days and saved on disk for later analysis. Constant conditions of ambient temperature (22°C) and illumination (LL, 200 Ix) were maintained throughout the experiment.

129

130 ROBERTO REFINETTI and MICHAEL MENAKER

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D a y s Fig. 1. Body temperature records of a normal (wild-type) female hamster during 14 consecutive days under

constant light. Circadian rhythmicity is clearly seen.

The data were analysed by the methods of periodo- gram and histogram, as described elsewhere (Refinetti, 1992). These methods provide numerical values for the four parameters of the circadian rhythm of body temperature (period, amplitude,

mean level and shape). For each parameter, differ- ences between genotypes (mutant or normal) and genders (male or female) were evaluated by 2 x 2 factorial analyses of variance using the data from all 26 animals tested in cages with running-wheels.

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Fig. 2, Body temperature records of two representative subjects (A) and their analyses by periodograms (B) and histograms [actually, frequency polygons] (C). The dashed line in B indicates a significance level of 0.001 for each value of Qp (approximately 0.02 family-wise). The parameters of the temperature rhythm of the normal and mutant hamsters were, respectively: period =24.1 and 20.2 h, amplitude = 1.5 and

1.2°C, mean level = 37.9 and 37.4°C, and shape == 1.6 and 2.4.

Hamster temperature rhythm 131

Differences between the conditions of wheel and no-wheel were evaluated with t-tests for matched samples.

RESULTS

Body temperature data of a normal (wild-type) female hamster recorded for 14 consecutive days are shown in Fig. 1. Approximately 4% of the measure- ments were spurious (i.e. below 34°C or above 41°C) and were replaced by the measurement immediately preceding them. A robust rhythmicity with a period very close to 24 h can easily be seen. Temperature is approximately 36°C for 2/3 of each cycle and 38°C for 1/3 of the cycle.

Records of body temperature of two other animals (a normal female and a mutant female) are shown in Fig. 2(A). To facilitate visual inspection, only 5 days of raw data are shown for each animal. The periodo- grams and histograms that are also shown in Figs 2(B) and (C) are based on the full 10 days of data (i.e. 2400 temperature measurements). Robust rhythms of body temperature can be observed in the raw data [Fig. 2(A)]. The higher frequency of temperature oscillation in the mutant hamster indicates that its circadian period is shorter than that of the normal animal. Periodogram analysis [Fig. 2(B)] indicates a significant peak at 24 h for the normal hamster and at 20 h for the mutant.

Differences in the amplitude and mean level of the rhythms can also be seen in Fig. 2(A), although these differences reflect inter-subject variability rather than genotypic differences (see below). Numerical values of the amplitude, mean level, and shape of the rhythms can be calculated from the temperature histograms shown in Fig. 2(C) (Refinetti, 1992). The difference between the temperatures at which the two modes of the distribution o c c u r (Thig h -- Tiow) consti- tutes the modal amplitude of the rhythm and is less sensitive to fortuitous variations in temperature than the simple difference between the lowest and the highest measurement in each cycle. For the 26 sub- jects of this experiment (with running-wheel avail- able), the mean modal amplitude (+SE) was 1.4 + 0.1°C, whereas the mean amplitude based on the difference between peaks and troughs was 2.7 + 0.4°C. The weighted mean of the temperatures at which the two modes occur constitutes the modal mean (mean level) of the rhythm, which rarely differs from the arithmetic mean of all measurements by more than 0.1°C. Finally, the ratio of the frequency values of the two modes (f low/fhish) is an index of the

Table 2. Inter-subject variability in the parameters of the body tempera-

ture rhythm

Coefficient N

Period Normals I 13 Mutants 2 13

Amplitude 20 26 Mean level 2 26 Shape 54 26

Values shown are the coefficients of variation (standard deviation divided by the mean, multiplied by 100) derived from the data of animals with access to running wheels.

shape of the rhythm, as it indicates the proportion of low-temperature values in reference to high-tempera- ture values (i.e. it indicates the relative duration of the low-temperature plateau in each cycle).

The mean results for the animals with free access to running-wheels are shown in Table 1. Because analysis of variance revealed no significant effect of gender (P > 0.05) in any of the parameters, the data from males and females were combined. A significant effect of genotype was found for the period (P < 0.001) but not for the other three parameters (P >0.05). Thus, mutant hamsters had shorter periods than normal animals, but did not differ regarding amplitude, mean level, and shape.

Although some degree of inter-subject variability is to be expected in any physiological parameter, some parameters may show considerably more inter- subject variability than others. The data in Table 2 indicate that the standard deviation is only 1 or 2% of the mean for the period and mean level of the temperature rhythm. The amplitude is more variable from subject to subject (20%), and the shape of the rhythm is the least uniform of the four parameters (54%).

Figure 3 shows a section of the body temperature records of an animal before and after introduction of a running wheel. The amplitude of the rhythm was greatly enhanced during the first circadian cycle after the introduction of the wheel and remained so on the following cycles. Smaller alterations in the mean level and shape of the rhythm were also apparent in this animal, but were not consistent across subjects.

The mean amplitude, mean level and shape of the rhythm of the 4 mutants and 4 normal hamsters during 10 days with access to running wheels and 10

Table 1. Parameters of the circadian rhythm of body tem- perature of hamsters with free access to running-wheels

Normal Mutant

Mean SE Mean SE Period (h) 24.04 0.04 20.15 0.12" Amplitude (°C) 1.39 0.10 1.49 0.06 Mean level (°C) 37.13 0.16 37.08 0.16 Shape 2.65 0.21 3.19 0.59 Each value is the mean (and SE) of 13 subjects (males and

females combined). Ten days of data (2400 measure- ments) were used for each subject.

*Significant (P < 0.01) effect of genotype (normal vs mu- tant), as determined by analysis of variance.

Table 3. Parameters of the circadian rhythm of body temperature of hamsters with and without access to running-

wheels With wheels Without wheels

Mean SE Mean SE

Amplitude (°C) 1.64 0.09 0.g4 0.03* Mean level (°C) 36.85 0.25 36.63 0.16 Shape 3.19 0.36 3.46 0.82 Each value is the mean (and SE) of 8 subjects (mutants and

normals combined). Ten days of data (2400 measure- ments) were used for each subject.

*Significant (P < 0.01) difference between With wheel and Without wheel, as determined by t-tests for matched samples.

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ROBERTO REFINETTI and MICHAEL MENAKER

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Days Fig. 3. Body temperature records of a representative wild-type hamster before and after the introduction of a running-wheel, as indicated by the vertical dashed line. An enhancement of the amplitude of the

rhythm after the introduction of the wheel is especially clear.

days without it are shown in Table 3. Significant differences were observed in the amplitude (P <0.001) but not in the other two parameters (P >0.05). Animals with access to wheels had rhythms with larger amplitude than animals without access to wheels, but the mean level and the duration of the high-temperature plateau were not affected. No effect of the availability of running wheels on the period of the temperature rhythm was observed, but the animals were studied for only 10 days in each situation. A shortening or elongation of the circadian

period might be observed after many weeks with access to running-wheels.

Running activity amounted to the equivalent of a linear displacement of 3 kin/day. Mean number of revolutions per circadian cycle was 5953 for normals (~24-h cycle) and 4824 for mutants (~ 20-h cycle). Number of revolutions per hour was almost identical in both groups (normais: 248, mutants: 240). Examples of simultaneous recordings of body tem- perature and running-wheel activity of a normal and a mutant hamster are shown in Fig. 4.

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Fig. 4. Body temperature and running-wheel activity records of a normal (wild-type) and a tau-mutant hamster during 5 consecutive days. In both animals the two rhythms free-ran in synchrony.

Hamster temperature rhythm 133

DISCUSSION

Although Moiler and Bojsen (1974) failed to detect a daily rhythm of body temperature in single-housed golden hamsters, such rhythms have been observed in other laboratories (Chaudhry et al., 1958; Connet al., 1990). We have shown recently that the body tem- perature rhythm of the hamster free runs in the absence of external time cues and, therefore, is a true circadian rhythm (Refinetti and Menaker, 1992). The results presented here indicate that the rhythm is enhanced when the hamsters have free access to running wheels, which is consistent with previous observations (Conn et al., 1990) and may partially explain Moiler and Bojsen's (1974) failure to detect it, as their animals did not have access to running- wheels.

The comparison between normal hamsters and tau-mutant hamsters revealed differences only in the period of the rhythm. The difference in the periods of the body temperature rhythms is consistent with the difference in periods of the activity rhythms pre- viously reported (Ralph and Menaker, 1988). Nor- mals and mutants did not differ regarding the amplitude, mean level and shape of the rhythm. Irrespective of genotype, the mean amplitude in animals without access to running-wheels was 0.8°C (modal amplitude) or 2.2°C (peak minus trough). The mean level of body temperature was 36.6°C, which is very close to values obtained in temperature regu- lation studies (Gordon et al., 1986; Jones et al., 1976) but over I°C lower than the values obtained in a previous telemetry study conducted at the same en- vironmental temperature (Conn et al., 1990). The reasons for this discrepancy are not clear. The mean value of the shape of the rhythm was approximately 3, which indicates that the low-temperature plateau is about 3 times as long as the high-temperature plateau. This is consistent with observations that hamsters are active only during a small fraction of the circadian cycle (Refinetti et al., 1991). In humans, who are active for 2/3 of the cycle, the high-tempera- ture plateau is longer than the low-temperature plateau (Refinetti, 1992).

The fact that the temperature rhythm of tau- mutant hamsters differs from that of normal hamsters only in respect to the period indicates that the tau

mutation is specific to the determination of period- icity. Clearly, alterations in one of the parameters of the rhythm did not result in alterations in the other parameters. This suggests that the different par- ameters of the circadian rhythm of body tempera- tures may be controlled by separate mechanisms.

Acknowledgements--This work was supported by the NSF Science and Technology Center for Biological Timing, by NIH NRSA Award MH-10146 to R. Refinetti, and by NIH grant HD-13162 to M. Menaker.

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