59
Journal of Physiology (1991), 433, pp. 59-71 With 6 figures Printed in Great Britain
EFFECT OF HEAT ACCLIMATION ON DIURNAL CHANGES IN BODY TEMPERATURE AND LOCOMOTOR ACTIVITY IN RATS
BY OSAMU SHIDO, SOHTARO SAKURADA AND TETSUO NAGASAKA From the Department of Physiology, School of Medicine, Kanazawa University, Kanazawa 920, Japan
(Received 26 February 1990) SUMMARY
1. The present study was performed to examine the effects of heat exposure for hours at a fixed time once a day on diurnal variations of deep body temperature, heat balance and locomotor activity in rats. 2. The heat-exposed group (HE) was subjected to an ambient temperature of 33-5 °C for about 5 h in the last half of the dark phase daily for at least 10 consecutive days, while the control rats were constantly kept at 24 'C. 3. After the completion of the heat exposure schedule, hypothalamic temperature (Thy), heat loss, heat production and locomotor activity of HE rats significantly decreased for 3-4 h during the period of previous heat exposure time and formed a characteristic trough in the dark phase, which was never observed in the control rats or in the HE rats before the start of the heat exposure schedule. 4. The troughs of Thy, heat loss and heat production in the dark phase were persistent for at least 2 days after the end of the heat exposure schedule. The reductions of locomotor activity in this period were, however, observed only on the first day after stopping the heat exposure schedule. 5. These results suggest that the time memory for heat exposure was formed in heat-acclimated rats and was persistent for at least 2 days after the removal of heat exposure. It is also concluded that the reduction in metabolic heat production contributed to the fall of Thy during the period of heat exposure. INTRODUCTION
Acclimation to intermittent or continuous heat exposure alters the level of daily changes in deep body temperature and threshold temperature for heat loss in human subjects (Fox, Goldsmith, Kidd & Lewis, 1963) and in animals (Shido, Yoneda & Nagasaka, 1989; Shido & Nagasaka, 1990 a, b). In our previous study where rats were subjected to daily heat exposure for several hours at a fixed time of day for 10 consecutive days and then transferred to a constant thermoneutral ambient temperature, deep body temperature appeared to fall during the same hours that the rats had been previously exposed to heat (Shido et al. 1989). The observation, if consistent, suggests that repeated heat exposures at a fixed time of day modify the diurnal cycle of thermoregulatory mechanism. Additionally, it is assumed that heatMS 8305
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OSAMU SHIDO AND OTHERS
acclimated rats memorize the time for temperature stimuli even after the heat acclimation period has ended. However, little is known about this particular issue. A similar phenomenon has been established in a feeding control mechanism in rodents. Schedules of restricted daily meals can substantially modulate the distribution of an animal's behavioural and metabolic activities (Fuller & Diller, 1970; Krieger, 1974; Moberg, Bellinger & Mendel, 1975). When food access is limited to a few hours at a fixed time once a day, for instance, a great increase of locomotion is found during the hours before meal time (Bolles & Stokes, 1965; Boulos, Rosenwasser & Terman, 1980; Nagasaka & Shido, 1989). This anticipatory running activity persists for several days after switching to ad lib. feeding (Mori, Nagai & Nakagawa, 1983). These results suggest the presence of a time memory for meals even under ad lib. feeding following a single meal schedule. It may, therefore, be reasonable to hypothesize that temperature stimuli given daily at a fixed time also modify the patterns of diurnal variation in thermoregulatory mechanisms due to a persistent time memory for heat exposure. The objective of the present study was, thus, to evaluate the hypothesis that repeated heat exposure at a fixed time of day produces a characteristic change in body temperature around the period corresponding to that of the previous exposure and to examine what thermoregulatory mechanisms are involved in the occurrence of body temperature alteration in heat-acclimated rats. METHODS
Animals and heat exposure schedule Male Wistar rats (Std: Wistar/ST) were housed in individual cages and given laboratory rat chow and tap water ad libitum in a 12-12 h light-dark cycle (light on at 15.00 h). They were initially divided into two groups. The control rats (Cn) were maintained at an ambient temperature of 24 +1 °C throughout the experiment. For the heat-exposed group (HE), the ambient temperature was raised to 33 5 + 0 4 °C in 1 h (starting from 09.00 h) and maintained for a subsequent 4 h, and then quickly returned to 24-0 ±0+4 IC. This procedure was repeated for at least 10 consecutive days. During the experiments, cages were cleaned and food and water were supplied once every 2 or 3 days at random during the light phase. After the end of the experiments, the rats were killed by parenteral anaesthetic (pentobarbitone) and, in experiments 1 and 2, a location of the hypothalamic cannula was checked.
Experiment 1 Five Cn and eleven HE rats, initially weighing 280-300 g, were used. A stainless-steel guide cannula was stereotaxically implanted into the median preoptic-anterior hypothalamus under pentobarbitone sodium (50 mg kg-', i.P.) anaesthesia. Six days after the first operation, the rats were lightly anaesthetized with a pentobarbitone sodium (25 mg kg-', i.p.) and a thermocouple covered with polyethylene tube was inserted into the hypothalamus via the guide cannula after passing through a metal spring coil (60 cm long, 5 mm diameter). Then it was fixed to the rat's head with dental cement. The proximal end of the spring coil was held on the ceiling of the cage, which enabled the rats to move freely with the least amount of restraint. After 4 days habituation to the situation, the measurements were started. Hypothalamic temperature (Th,) was recorded every minute through an A/D converter (ADC-12IB, Kanazawa Control Kiki, Kanazawa) connected to a personal computer (PC9801VX, NEC, Tokyo) for 2 days before and after the 10 day heat exposure schedule.
Experiment 2 Eight Cn and eight HE rats, initially weighing 280-300 g, were used. A hypothalamic cannula was implanted as in experiment 1. Twelve days after the first operation, the rats were subjected to a 12-14 day heat exposure schedule. They were again anaesthetized with pentobarbitone sodium
MODIFICATION OF TEMPERATURE RHYTHM BY HEAT 61 (25 mg kg-', i.P.) 5 days before the end of heat exposure schedule and a thermocouple protected by the metal spring coil was set and fixed into the hypothalamic guide cannula as in experiment 1. Three days later, they were housed in a wire mesh cage under which an aluminium pan filled with vegetable oil was placed to collect their wastes. Then they were transferred to a gradient type direct calorimeter (sized 35 x 35 x 35 cm) (Nagasaka, Hirata, Sugano & Shibata, 1979; Sugano, 1983) and
the wall temperature was set at 24-0 0C. The rats were reared inside the calorimeter for 2 days under the same light-dark cycle (the light phase was maintained with two tiny electric bulbs producing 2-2 W) in order to get them accustomed to the new environment. For the HE rats, heat exposure was repeated inside the calorimeter during the same period of a day by changing the temperature of jacket water surrounding the calorimeter from 24 to 35 0C. After the last heat exposure was finished, the chamber wall temperature was strictly controlled at 24 'C with an accuracy of +0-02 'C and the photo-cycle was kept in constant darkness. Measurements commenced at 21.00 h the day after the final heat exposure period and were continued for 48 h. Fresh, dry, CO2-free and temperature-controlled air from the outdoors was continuously introduced into the calorimeter chamber at a constant rate of 3 0 1 min-'. Thy and temperatures of the chamber wall and outlet air from the calorimeter were monitored by thermocouples. Non-evaporative heat loss (R + c+K) was measured by a direct calorimeter and evaporative heat loss (E) was calculated from the flow and humidity of the air monitored with a humidity sensor (MHI- 11, Vaisala, Helsinki). A fraction of air withdrawn from the calorimeter chamber was sent into a Zirconia 02 analyser (LC-700E, Toray, Tokyo; 100 ml min-') and an infra-red C02 analyser (LIRA 303NS, Toray, Tokyo; 1000 ml min-') to measure 02 consumption and C02 production, respectively. Metabolic heat production (M) was calculated from 02 consumption and C02 production with the corresponding calorific value at a given respiratory quotient (RQ). The amount of body movement (BM) of rats was measured with piezoelectric ceramics (Z2T4OX90RW C6, Fuji ceramics, Shizuoka) placed underneath the rat's cage. The sensitivity of the piezoelectric ceramics was set so as to detect a very slight locomotion, scratching or grooming but not to count minor movements such as whisker twitching. All parameters were sampled every minute through an A/D converter connected to a personal computer.
Experiment 3 Five Cn and eleven HE rats, initially weighing 230-250 g, were used. They were housed in small cages with a running wheel (30 cm diameter). After 10 days habituation to the running cage, measurements commenced 2 days before the beginning of the 14 day heat exposure period and continued intermittently to the sixth day after the last heat exposure. The numbers of wheel revolutions (WR) were monitored every 5 min by a photoelectric device (LG-916, ONO SOKKI, Tokyo) and saved on digitial cassette tapes through a computer logging system (PS-80, TEAC,
Tokyo).
Data analysis and statistics Total thermal conductance (C), an indicator of activity level in non-evaporative heat loss mechanism (Shido & Nagasaka, 1990a), was calculated as (R+c+K)/(TdY-T ,aii)' where R, c, K and TR,aii are radiation, convection, conduction and the wall temperature of the calorimeter chamber, respectively. The results are presented as means+ or+ S.E.M. and statistical evaluations were assessed by paired and unpaired Student's t tests. The significant level was considered to be P < 0-05. -
RESULTS
Experiment 1: measurements of Thy A clear day-night variation of Thy was observed in all rats tested: lower in the light phase and higher in the dark phase. In the Cn rats kept at 24 'C throughout the experiment, the levels and the pattern of the diurnal fluctuation of Thy were essentially the same before and after the heat exposure schedule, although several significant differences were accidentally observed (Fig. IA). In the HE rats, however, Thy, especially in the light phase, was significantly lowered after acclimation to daily intermittent heat exposure (Fig. 2), which is consistent with our previous
OSAMU SHIDO AND OTHERS
62
observations (Shido et al. 1989; Shido & Nagasaka, 1990 b). In addition, Thy of the HE rats markedly decreased in the last half of the dark phase during which the rats had been previously subjected to heat exposure, forming a characteristic trough of Thy in the dark phase. Such a clear trough of Thy was not observed in the Cn rats (Fig. IA) A 38.5=-38.0 -
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Fig. 1. Mean changes of hypothalamic temperature (Th,; A) and wheel revolutions (WR; B) in the control rats before (left panel) and one and two days after (right panel) heat acclimation period, Values in Thy are means of 5 min in five rats and values in WR are hourly means of five rats. The s.E.M. is not shown in order to simplify the figure. Dots above abscissa, significantly different from corresponding values before heat exposure sohedule. Filled bars on abscissa, dark phases of a day.
or in the HE rats before the heat exposure schedule (Fig, 2). The trough of Thy in the dark phase lasted for 3-4 h and was persistent for at least 2 days after the heat exposure schedule had ended.
Experiment 2: heat balance and body movement The body weights of the Cn and HE rats just after measurements of heat balance with the direct calorimeter were 348 +11 and 349 + 5 g, respectively. Table 1 summarizes the mean values of Thy, total heat loss (H = R + c +K+E), C, M and BM at four periods per day in the Cn and HE groups after the heat exposure schedule had ended. Thy was consistently lower in the HE rats than in the Cn rats in all four periods of the day. A significant reduction of Thy in the HE rats was shown between 09.00 and 15.00 h when the trough of Thy was observed. Particularly on the first day after
MODIFICATION OF TEMPERATURE RHYTHM BY HEAT
63
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Time of day (h) Fig. 2. Mean changes of hypothalamic temperature (Thy) in the heat-exposed rats (HE group) before (left panel) and one and two days after (right panel) heat acclimation. Values are means of 5 min in eleven rats. The S.E.M. is not shown in order to simplify the figure. Dots above abscissa, significantly different from corresponding values before heat acclimation. Filled bars beneath abscissa, dark phases of a day.
TABLE 1. Mean values of thermoregulatory parameters and body movement after heat exposure schedule in control and heat-exposed rats H Time of C M BM thy (W m-2) (W m-2 OC-1) day (W m-2) Group (OC) (count) Cn 37-20+0-09 44-7+1 2 2-958+0i100 43-8+0 8 14912+2273 21.00 to 03.00 HE (1) 37-00+0-07 48.0+0.7* 3.352+0.046* 46-0+0-8 15296+2250 HE (2) 37-08+0-08 45 9+0 9 3-102+0-065 45-7+0 8 16 119+2099 Cn 37-84+0-09 61-1 +03 3-965+0-051 59 0+0 4 41849+4822 03.00 to 09.00 HE (1) 37.58+0.06* 60'9+1.2 4'073+0-079 60-2+1i1 44310+7106 HE (2) 37-72+0-07 61-8+1-6 4-073+0-104 60-9+1-8 44162+7330 Cn 37-91+0-10 61P2+0-5 3-891+0-043 61-3±0-4 43772±4359 09.00 to 15.00 HE (1) 3740+0.07* 56.0+1.5* 3-731+0'098 57.3+1 4* 37753±6061 HE (2) 37.53+0.08* 57-7+1-7 3-798+0'084 57'8± 1-7 41677±7064 Cn 37-08+0-11 41-5±0-7 2-668+0-066 44-4+1'0 15908±2717 15.00 to 21.00 HE (1) 37-05+0-08 46.6±1.2* 3.137+0.093* 48-0+0-8 20605+3735 HE (2) 36-93±+006 44-1 + 1 0 2-962 +0.084* 45-2+0-8 15892+2695 Thy, hypothalamic temperature; H, total heat loss; C, total thermal conductance; M, metabolic heat production; BM, body movement; Cn, values in the control group one day after termination of heat exposure schedule; HE (1), values in the heat-exposed rats (HE) one day after termination of heat exposure; HE (2), values in the HE rats two days after termination of heat exposure. 21.00-03.00 and 15.00-21.00 h, corresponding to the previous light phase; 03.00-09.00 and 09.00-15.00 h, corresponding to the previous dark phase. * Significantly different from values in the Cn.
the heat exposure schedule, H, C and M of HE rats were higher than those of the Cn rats between 15.00 and 21.00 h and 21.00 and 03.00 h, corresponding to the previous light phase of a day. From 09.00 to 15.00 h, on the contrary, H and M were lower in the HE rats than in the Cn rats. The HE rats appeared to be less active than the Cn rats between 09.00 and 15.00 h, but there were no significant differences in the counts of BM between the Cn and HE groups throughout the day.
OSAMU SHIDO AND OTHERS
64
In order to compare patterns of diurnal variation of thermoregulatory parameters in constant darkness, relative changes from minimal values in each variable were computed as the percentage of the maximum amplitude in a day. Figure 3 shows the percentage changes calculated hourly in Thy, H, M and BM in the Cn and HE rats 80 1
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after the heat exposure schedule had ended. In the Cn rats, all these variables showed clear and similar diurnal variations. High levels of Thy, M, H and BM were maintained from 03.00 to 15.00 h corresponding to the previous dark phase of a day. In the HE rats, however, the levels of Thy from 09.00 to 13.00 h when the rats had been exposed to heat were significantly lower than those of the Cn rats. Thy, then, showed a distinct trough in the last half of the previous dark phase as observed in experiment 1 under the 12-12 h light-dark cycle. A similar pattern was shown in the variations of H and M. The diurnal changes in BM were rather varied, but the trough of BM in the HE rats was consistently seen. This characteristic pattern of diurnal thermoregulatory parameters in the HE rats persisted for at least 2 days after acclimation to the heat exposure schedule. The diurnal changes of these thermoregulatory parameters in absolute values
65 MODIFICATION OF TEMPERATURE RHYTHM BY HEAT calculated hourly were almost the same as the relative changes shown in Fig. 3. Significant differences in Thy, (R + c +K), H, C and M between the Cn and HE groups were obtained between 09.00 and 15.00 h on the first and second day after the heat exposure schedule. However, the absolute value of BM in the HE rats did not differ 4.5
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Fig. 4. Mean total thermal conductance (C) as a function of body movement between 03.00 and 09.00,09.00 and 15.00,21.00 and 03.00, and 15.00 and 21.00 h after termination of the heat exposure schedule. The former two periods correspond to the previous dark phase and the latter two correspond to the previous light phase. Body movements were divided into six parts at a step of 200 counts. Open bars, values in the control rats (Cn); hatched bars, values in the heat-exposed rats (HE) on the first day after heat exposure schedule; vertical bars, S.E.M. Numbers above each bar, number in group. * Significantly different from corresponding values in the Cn rats.
from that of the Cn rats during any period on the second day after the heat exposure schedule. Figure 4 shows the mean values of C in the four periods per day as a function of BM (divided into six ranges at a step of 200 counts) in the Cn and HE rats after the end of the heat exposure schedule. C in the HE rats was higher than that of the Cn group 3
PHY 433
OSAMU SHIDO AND OTHERS
66
at any level of BM between 03.00 and 09.00 h, the early half of the previous dark phase, and significantly during 21.00-03.00 h and 15.00-21.00 h, corresponding to the previous light phase of a day. From 09.00 to 15.00 h, however, C in the HE rats became smaller than that of the Cn rats regardless of the BM magnitude. Similar 70
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Body movement (15.00-21.00 h) Body movement (21.00-03.00 h) Fig. 5. Mean metabolic heat production (M) as a function of body movement between 21.00 and 03.00, 03.00 and 09.00, 09.00 and 15.00, and 15.00 and 21.00 h after the heat exposure period had ended. Other explanations are the same as in Fig. 4.
results were obtained in the relationship between M and BM (Fig. 5). From 09.00 to 15.00 h, M was smaller in the HE rats than in the Cn rats at any BM level, although the HE rats showed greater M in the other periods of the day.
Experiment 3: mecaurements of locomotor activity As is well known, rats turn the running wheel frequently in the dark phase and rarely in the light phase. In the Cn rats, the hourly numbers of WR were not significantly different during the experimental days in any period per day, although there was a great intra- and interindividual variety of the WR activity distributions in a day (Fig. 1B). Figure 6 shows the mean numbers of WR before, during and after
MODIFICATION OF TEMPERATURE RHYTHM BY HEAT 67 acclimation to daily heat exposure in the HE rats. On the first day of heat exposure, WR increased during the heat exposure time (09.00-15.00 h). It gradually decreased and the reduction of WR became significant from the sixth day after the start of the heat exposure schedule. On the ninth day of heat exposure, WR both before and after 250250 -200X 150-o 10050-
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Time of day (h) Fig. 6. Wheel revolution (WR) changes in the heat-exposed rats (HE) before, during and after the heat exposure schedule. Values are hourly means of eleven rats. The S.E.M. is not shown in order to simplify the figure. Filled bars beneath abscissa, dark phases of a day; open bars above abscissa, heat exposure time. Pre, before heat exposure; numbers, days after the onset of heat exposure; numbers with Po, days after the end of heat exposure. * Significantly different from corresponding values before the start of the heat exposure. The number of rats was six from 3 days after the termination of heat exposure (on days of Po3, Po4 and Po5).
heat exposure time significantly increased. The subsequent rise of WR to heat exposure time quickly stopped but the high activity before daily heat exposure appeared to be maintained through the end of the heat acclimation schedule. After the heat exposure schedule had ended, the significant reduction of WR in the last half of the dark phase lasted for only one day, although the numbers of WR in the period appeared to decrease during the subsequent 5 days. DISCUSSION
The present results clearly confirm that the diurnal variations in deep body temperature and locomotor activity are modified by acclimation to heat exposure for about 5 h daily at a fixed time in the dark phase for at least 10 consecutive days. The Thy of heat-acclimated rats definitely decreased during the period corresponding to the previous heat exposure time and the characteristic trough of Thy was formed in 3-2
68
OSAMU SHIDO AND OTHERS
the dark phase. This trough of Thy was consistent under both the 12-12 h light-dark cycle and the constant darkness. During heat exposure, heat loss mechanisms are activated and metabolic heat production is suppressed to prevent heat accumulation in the body. These physiological responses to heat stress occurred daily during the same period per day in rats on a heat exposure schedule. This fall in Thy of the HE rats during the period corresponding to the previous heat exposure time, therefore, may be attributed to the persistence of the thermoregulatory responses to heat even without actual heat exposure. Similar to the observation of the time memory for meals in rats subjected to a restricted feeding schedule (Boulos et al. 1980; Mori et al. 1983), the present results show that a time memory for heat exposure could be formed in the present rats acclimated to heat exposure and that the memory persists for at least 2 days after the heat exposure schedule ended. In the HE rats, (R+c+K), E, H and C in both absolute and relative values significantly decreased either before or during the period corresponding to the time of the previous heat exposure schedule even after the heat exposure schedule had ended. These results provide no evidence that the activation of heat loss mechanisms contribute to the formation of the trough of Thy in the dark phase. In freely moving rats, however, body movement strongly affects heat transfer from body to environment through altering heat convection and changing effective body surface area for dissipating heat. Under uncontrolled body movement conditions, the activity level of the autonomic heat loss mechanism cannot be evaluated. To eliminate such influences, C was assessed as a function of body movement (Fig. 3). C from 21.00 to 03.00 h, 15.00 to 21.00 h and 03.00 to 09.00 h was consistently greater in the HE rats than in the Cn rats regardless of the intensity of body movement. The results may agree with the report that non-evaporative heat loss capacity is improved by acclimation to intermittent heat exposure (Shido & Nagasaka, 1990b). Between 09.00 and 15.00 h corresponding to the previous heat exposure period, however, C of the HE rats never exceeded C of the Cn rats at any intensity of body movement. The decrease of C may not indicate the facilitation of heat loss responses but the occurrence of activation in the heat conservation mechanism. It is, therefore, unlikely that heat dissipation mechanisms contributed to the fall in body temperature during the period of previous heat exposure after the heat exposure schedule had ended in heat-acclimated rats. Reduction of M is another potent cause of the decrease in body temperature. After the heat exposure schedule in the HE rats, M significantly decreased between 09.00 and 15.00 h, which implies the predominant contribution of metabolic heat production mechanism to the formation of the Thy trough in the dark phase. The change of M obtained in the present system involves changes of muscular work intensity associated with behaviour and of resting metabolic rate. The acclimation to an intermittent heat exposure schedule obviously modulated the distributions of body movement determined by piezoelectric ceramics and of WR in a day. The HE rats showed less body movement during the previous heat exposure time on the first day after heat exposure schedule termination. However, the reduction of activity became unclear on the second day after heat exposure termination where the fall in Thy and the definite reduction of M were constantly observed. Similarly, WR decreased significantly during the last half of the dark phase, but the low locomotor
MODIFICATION OF TEMPERATURE RHYTHM BY HEAT
69
activity lasted only for one day after the heat exposure schedule had ended. Thus the suppression of M in the HE rats cannot be explained by only the redistribution of locomotor activity. When assessed as a function of body movement (Fig. 4), M from 03.00 to 09.00 h, 15.00 to 21.00 h and 21.00 to 03.00 h was consistently greater in the HE rats than in the Cn rats at all intensities of body movement except between 15.00 and 21.00 h at the range of body movement from 401 to 800 counts. From 09.00 to 15.00 h corresponding to the previous heat exposure period, however, M of the HE rats became smaller than that of the Cn rats regardless of body movement magnitude. Furthermore, mean M during this period of the HE rats was significantly lower than that of the Cn rats, although the amount of body movement was not different between the two groups. These results may suggest that resting metabolic rate, heat production without muscular movement, was suppressed during a particular period of a day in the HE rats. The characteristic trough of Thy formed during the previous heat exposure period after termination of the heat exposure schedule was, therefore, attributed to both the reduction of heat production accompanied by behavioural activity and to the suppression of resting metabolic rate. The detailed mechanism of the suppression of resting heat production during the previous heat exposure period is not yet clear. At the same level of locomotor activity, M before meals was markedly lower than that after feeding in rats on restricted feeding regimes (Boyle, Storlien, Harper & Keesey, 1981; Nagasaka & Shido, 1989). The reduction of postprandial thermogenesis was possibly involved in the low M of the previous heat exposure period in the HE rats. In this case, the distributions of feeding activity in a day are expected to be modified by daily heat exposure at a fixed time as observed in the alterations in diurnal variation of locomotor activity. Acclimation to chronic heat exposure brings about several adjustments of thermogenic pathway (Cassuto, 1968) and of endocrine functions (Collins & Weiner, 1968; Cassuto, Chayoth & Rabi, 1970; Arieli & Chinet, 1986). Some reports give evidence of modulations in circadian rhythm of endocrine systems by chronic heat exposure (Cure, 1989; Brandenberger, Follenius, Di Nisi, Libert & Simon, 1989). It is, therefore, possible that the pattern of diurnal cycle of hormones related to metabolism or the activity of thermogenic organs was influenced by the heat exposure schedule adopted in this study and the persistence of re-established rhythm contributed to the reduction of M during the period of previous heat exposure.
Repeated temperature stimuli have been well known to shift the thermoneutral zone and set-point for temperature regulation in mammals (Fox et al. 1963; Shido et al. 1989; Shido & Nagasaka, 1990 a, b). Tokura & Aschoff (1983) have demonstrated that a free-running period of circadian activity rhythm was elongated by a rise in
ambient temperature from 17 to 33 °C in pig-tailed macaques. A similar observation in the effect of ambient temperature on a free-running period of spontaneous activity was made in house finch (Enright, 1966). The results clearly suggest that the internal time-keeping system can also be modified by external temperature stimuli. On the other hand, circadian rhythms of heat production, heat loss, body temperature and activity are considered to be driven by potentially independent oscillators (Aschoff & Pohl, 1970; Aschoff, Biebach, Heise & Schmidt, 1974; Wever, 1975) and can
70
OSAMU SHIDO AND OTHERS
desynchronize either spontaneously (Aschoff, Gerecke & Wever, 1967; Sulzman, Fuller & Moore-Ede, 1977) or when forced by manipulations of zeitgeber period (Wever, 1975). To date, there was no available information with regard to the effect of repeated heat exposure on oscillation of heat balance, body temperature or spontaneous activity. However, if heat stress could cause the internal desynchronization between heat gain and loss, the pattern of diurnal changes in body temperature should be altered, which might result in the formation of the characteristic trough in the dark phase in the present rats acclimated to repeated daily heat exposure. In summary, heat exposure for about 5 h at a fixed time once a day modified diurnal changes of body temperature after the heat exposure schedule termination, which had been characterized by a fall in body temperature during the period of previous heat exposure. Since this distinct trough of body temperature persisted for at least 2 days after heat exposure termination, it is suggested that a time memory for heat exposure or for thermoregulation was formed in rats through repeated daily heat exposure at a fixed time. The predominant contributor to the fall in body temperature was the reduction of metabolic heat production associated with and without locomotor activity. The authors gratefully acknowledge the contribution made by Naotoshi Sugimoto in performing the experiments. The authors are also grateful to Akio Yachi and Shigeyuki Tabata for their technical assistance in building the calorimeter system. REFERENCES
ARIELI, A. & CHINET, A. (1986). Thyroid status and noradrenaline-induced regulatory thermogenesis in heat acclimated rats. Hormonal Metabolic Research 18, 103-106. ASCHOFF, J., BIEBACH, H., HEISE, A. & SCHMIDT, T. (1974). Day-night variation in heat balance. In Heat Loss from Animals and Man, ed. MONTEITH, J. C. & MOUNT, L. E., pp. 147-172. Butterworths, London. ASCHOFF, J., GERECKE, V. & WEVER, R. (1967). Desynchronization of human circadian rhythms. Japanese Journal of Physiology 17, 450-457. ASCHOFF, J. & POHL, H. (1970). Rhythmic variations in energy metabolism. Federation Proceedings 29, 1541-1552. BOLLES, R. C. & STOKES, L. W. (1965). Rat's anticipation of diurnal and a-diurnal feeding. Journal of Comparative and Physiological Psychology 60, 290-294. BoULOS, Z., ROSENWASSER, A. M. & TERMAN, M. (1980). Feeding schedules and the circadian organization of behavior in the rat. Behavioral Brain Research 1, 39-65. BOYLE, P. C., STORLIEN, L. H., HARPER, A. E. & KEESEY, R. E. (1981). Oxygen consumption and locomotor activity during restricted feeding and realimentation. American Journal of Physiology 241, R392-397. BRANDENBERGER, G., FOLLENIUS, M., Di Nisi, J., LIBERT, J. P. & SIMON, C. (1989). Amplification of nocturnal oscillation in PRA and aldosterone during continuous heat exposure. Journal of Applied Physiology 66, 1280-1286. CASSUTO, Y. (1968). Metabolic adaptations to chronic heat exposure in the golden hamster. American Journal of Physiology 214, 1147-1151. CASSUTO, Y., CHAYOTH, R. & RABI, T. (1970). Thyroid hormone in heat-acclimated hamsters. American Journal of Physiology 218, 1287-1290. COLLINS, K. J. & WEINER, J. S. (1968). Endocrinological aspects of exposure to high environmental temperatures. Physiologial Reviews 48, 785-839. CURE, M. (1989). Plasma corticosterone response in continuous versus discontinuous chronic heat exposure in rat. Physiology and Behavior 45, 1117-1122.
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ENRIGHT, J. T. (1966). Temperature and free-running circadian rhythm of the house finch. Comparative Biochemistry and Physiology 18, 463-475. Fox, R. H., GOLDSMITH, R., KIDD, D. J. & LEWIS, H. E. (1963). Blood flow and other thermoregulatory changes with acclimatization to heat. Journal of Physiology 166, 548-562. FULLER, R. W. & DILLER, E. R. (1970). Diurnal variations of liver glycogen and plasma free fatty acids in rats fed ad libitum or single daily meal. Metabolism 19, 226-229. KRIEGER, D. T. (1974). Food and water restriction shifts corticosterone, temperature, activity and brain amine periodicity. Endocrinology 95, 1195-1201. MOBERG, G. P., BELLINGER, L. L. & MENDEL, V. E. (1975). Effect of meal feeding on daily rhythm of plasma corticosterone and growth hormone in the rat. Neuroendocrinology 19, 160-169. MORI, T., NAGAI, K. & NAKAGAWA, H. (1983). Dependence of memory of meal time upon circadian biological clock in rats. Physiology and Behavior 30, 259-265. NAGASAKA, T., HIRATA, K., SUGANO, Y. & SHIBATA, H. (1979). Heat balance during physical restraint in rats. Japanese Journal of Physiology 29, 383-392. NAGASAKA, T. & SHIDO, 0. (1989). Locomotor activity and heat production of rats on restricted two-hour feeding regimes. Japanese Journal of Biometeorology 26, 85-90. SHIDO, 0. & NAGASAKA, T. (1990a). Thermoregulatory responses to acute body heating in rats acclimated to continuous heat exposure. Journal of Applied Physiology 68, 59-65. SHIDO, 0. & NAGASAKA, T. (1990b). Heat loss responses in rats acclimated to heat loaded intermittently. Journal of Applied Physiology 68, 66-70. SHIDO, O., YONEDA, Y. & NAGASAKA, T. (1989). Changes in body temperature of rats acclimated to heat with different acclimation schedule. Journal of Applied Physiology 67, 2154-2157. SUGANO, Y. (1983). Heat balance of rats acclimated to diurnal 2-hour feeding. Physiology and Behavior 30, 289-293. SULZMAN, F. M., FULLER, C. A. & MOORE-EDE, M. C. (1977). Spontaneous internal desynchronization of circadian rhythms in the squirrel monkey. Comparative Biochemistry and Physiology 58A, 6367. TOKURA, H. & ASCHOFF, J. (1983). Effects of temperature on the circadian rhythm of pig-tailed macaques Macaca nemestrina. American Journal of Physiology 245, R800-808. WEVER, R. (1975). The circadian multi-oscillator system of man. International Journal of Chronobiology 3, 19-55.
Journal of Physiology (1991), 433, pp. 59-71 With 6 figures Printed in Great Britain
EFFECT OF HEAT ACCLIMATION ON DIURNAL CHANGES IN BODY TEMPERATURE AND LOCOMOTOR ACTIVITY IN RATS
BY OSAMU SHIDO, SOHTARO SAKURADA AND TETSUO NAGASAKA From the Department of Physiology, School of Medicine, Kanazawa University, Kanazawa 920, Japan
(Received 26 February 1990) SUMMARY
1. The present study was performed to examine the effects of heat exposure for hours at a fixed time once a day on diurnal variations of deep body temperature, heat balance and locomotor activity in rats. 2. The heat-exposed group (HE) was subjected to an ambient temperature of 33-5 °C for about 5 h in the last half of the dark phase daily for at least 10 consecutive days, while the control rats were constantly kept at 24 'C. 3. After the completion of the heat exposure schedule, hypothalamic temperature (Thy), heat loss, heat production and locomotor activity of HE rats significantly decreased for 3-4 h during the period of previous heat exposure time and formed a characteristic trough in the dark phase, which was never observed in the control rats or in the HE rats before the start of the heat exposure schedule. 4. The troughs of Thy, heat loss and heat production in the dark phase were persistent for at least 2 days after the end of the heat exposure schedule. The reductions of locomotor activity in this period were, however, observed only on the first day after stopping the heat exposure schedule. 5. These results suggest that the time memory for heat exposure was formed in heat-acclimated rats and was persistent for at least 2 days after the removal of heat exposure. It is also concluded that the reduction in metabolic heat production contributed to the fall of Thy during the period of heat exposure. INTRODUCTION
Acclimation to intermittent or continuous heat exposure alters the level of daily changes in deep body temperature and threshold temperature for heat loss in human subjects (Fox, Goldsmith, Kidd & Lewis, 1963) and in animals (Shido, Yoneda & Nagasaka, 1989; Shido & Nagasaka, 1990 a, b). In our previous study where rats were subjected to daily heat exposure for several hours at a fixed time of day for 10 consecutive days and then transferred to a constant thermoneutral ambient temperature, deep body temperature appeared to fall during the same hours that the rats had been previously exposed to heat (Shido et al. 1989). The observation, if consistent, suggests that repeated heat exposures at a fixed time of day modify the diurnal cycle of thermoregulatory mechanism. Additionally, it is assumed that heatMS 8305
60
OSAMU SHIDO AND OTHERS
acclimated rats memorize the time for temperature stimuli even after the heat acclimation period has ended. However, little is known about this particular issue. A similar phenomenon has been established in a feeding control mechanism in rodents. Schedules of restricted daily meals can substantially modulate the distribution of an animal's behavioural and metabolic activities (Fuller & Diller, 1970; Krieger, 1974; Moberg, Bellinger & Mendel, 1975). When food access is limited to a few hours at a fixed time once a day, for instance, a great increase of locomotion is found during the hours before meal time (Bolles & Stokes, 1965; Boulos, Rosenwasser & Terman, 1980; Nagasaka & Shido, 1989). This anticipatory running activity persists for several days after switching to ad lib. feeding (Mori, Nagai & Nakagawa, 1983). These results suggest the presence of a time memory for meals even under ad lib. feeding following a single meal schedule. It may, therefore, be reasonable to hypothesize that temperature stimuli given daily at a fixed time also modify the patterns of diurnal variation in thermoregulatory mechanisms due to a persistent time memory for heat exposure. The objective of the present study was, thus, to evaluate the hypothesis that repeated heat exposure at a fixed time of day produces a characteristic change in body temperature around the period corresponding to that of the previous exposure and to examine what thermoregulatory mechanisms are involved in the occurrence of body temperature alteration in heat-acclimated rats. METHODS
Animals and heat exposure schedule Male Wistar rats (Std: Wistar/ST) were housed in individual cages and given laboratory rat chow and tap water ad libitum in a 12-12 h light-dark cycle (light on at 15.00 h). They were initially divided into two groups. The control rats (Cn) were maintained at an ambient temperature of 24 +1 °C throughout the experiment. For the heat-exposed group (HE), the ambient temperature was raised to 33 5 + 0 4 °C in 1 h (starting from 09.00 h) and maintained for a subsequent 4 h, and then quickly returned to 24-0 ±0+4 IC. This procedure was repeated for at least 10 consecutive days. During the experiments, cages were cleaned and food and water were supplied once every 2 or 3 days at random during the light phase. After the end of the experiments, the rats were killed by parenteral anaesthetic (pentobarbitone) and, in experiments 1 and 2, a location of the hypothalamic cannula was checked.
Experiment 1 Five Cn and eleven HE rats, initially weighing 280-300 g, were used. A stainless-steel guide cannula was stereotaxically implanted into the median preoptic-anterior hypothalamus under pentobarbitone sodium (50 mg kg-', i.P.) anaesthesia. Six days after the first operation, the rats were lightly anaesthetized with a pentobarbitone sodium (25 mg kg-', i.p.) and a thermocouple covered with polyethylene tube was inserted into the hypothalamus via the guide cannula after passing through a metal spring coil (60 cm long, 5 mm diameter). Then it was fixed to the rat's head with dental cement. The proximal end of the spring coil was held on the ceiling of the cage, which enabled the rats to move freely with the least amount of restraint. After 4 days habituation to the situation, the measurements were started. Hypothalamic temperature (Th,) was recorded every minute through an A/D converter (ADC-12IB, Kanazawa Control Kiki, Kanazawa) connected to a personal computer (PC9801VX, NEC, Tokyo) for 2 days before and after the 10 day heat exposure schedule.
Experiment 2 Eight Cn and eight HE rats, initially weighing 280-300 g, were used. A hypothalamic cannula was implanted as in experiment 1. Twelve days after the first operation, the rats were subjected to a 12-14 day heat exposure schedule. They were again anaesthetized with pentobarbitone sodium
MODIFICATION OF TEMPERATURE RHYTHM BY HEAT 61 (25 mg kg-', i.P.) 5 days before the end of heat exposure schedule and a thermocouple protected by the metal spring coil was set and fixed into the hypothalamic guide cannula as in experiment 1. Three days later, they were housed in a wire mesh cage under which an aluminium pan filled with vegetable oil was placed to collect their wastes. Then they were transferred to a gradient type direct calorimeter (sized 35 x 35 x 35 cm) (Nagasaka, Hirata, Sugano & Shibata, 1979; Sugano, 1983) and
the wall temperature was set at 24-0 0C. The rats were reared inside the calorimeter for 2 days under the same light-dark cycle (the light phase was maintained with two tiny electric bulbs producing 2-2 W) in order to get them accustomed to the new environment. For the HE rats, heat exposure was repeated inside the calorimeter during the same period of a day by changing the temperature of jacket water surrounding the calorimeter from 24 to 35 0C. After the last heat exposure was finished, the chamber wall temperature was strictly controlled at 24 'C with an accuracy of +0-02 'C and the photo-cycle was kept in constant darkness. Measurements commenced at 21.00 h the day after the final heat exposure period and were continued for 48 h. Fresh, dry, CO2-free and temperature-controlled air from the outdoors was continuously introduced into the calorimeter chamber at a constant rate of 3 0 1 min-'. Thy and temperatures of the chamber wall and outlet air from the calorimeter were monitored by thermocouples. Non-evaporative heat loss (R + c+K) was measured by a direct calorimeter and evaporative heat loss (E) was calculated from the flow and humidity of the air monitored with a humidity sensor (MHI- 11, Vaisala, Helsinki). A fraction of air withdrawn from the calorimeter chamber was sent into a Zirconia 02 analyser (LC-700E, Toray, Tokyo; 100 ml min-') and an infra-red C02 analyser (LIRA 303NS, Toray, Tokyo; 1000 ml min-') to measure 02 consumption and C02 production, respectively. Metabolic heat production (M) was calculated from 02 consumption and C02 production with the corresponding calorific value at a given respiratory quotient (RQ). The amount of body movement (BM) of rats was measured with piezoelectric ceramics (Z2T4OX90RW C6, Fuji ceramics, Shizuoka) placed underneath the rat's cage. The sensitivity of the piezoelectric ceramics was set so as to detect a very slight locomotion, scratching or grooming but not to count minor movements such as whisker twitching. All parameters were sampled every minute through an A/D converter connected to a personal computer.
Experiment 3 Five Cn and eleven HE rats, initially weighing 230-250 g, were used. They were housed in small cages with a running wheel (30 cm diameter). After 10 days habituation to the running cage, measurements commenced 2 days before the beginning of the 14 day heat exposure period and continued intermittently to the sixth day after the last heat exposure. The numbers of wheel revolutions (WR) were monitored every 5 min by a photoelectric device (LG-916, ONO SOKKI, Tokyo) and saved on digitial cassette tapes through a computer logging system (PS-80, TEAC,
Tokyo).
Data analysis and statistics Total thermal conductance (C), an indicator of activity level in non-evaporative heat loss mechanism (Shido & Nagasaka, 1990a), was calculated as (R+c+K)/(TdY-T ,aii)' where R, c, K and TR,aii are radiation, convection, conduction and the wall temperature of the calorimeter chamber, respectively. The results are presented as means+ or+ S.E.M. and statistical evaluations were assessed by paired and unpaired Student's t tests. The significant level was considered to be P < 0-05. -
RESULTS
Experiment 1: measurements of Thy A clear day-night variation of Thy was observed in all rats tested: lower in the light phase and higher in the dark phase. In the Cn rats kept at 24 'C throughout the experiment, the levels and the pattern of the diurnal fluctuation of Thy were essentially the same before and after the heat exposure schedule, although several significant differences were accidentally observed (Fig. IA). In the HE rats, however, Thy, especially in the light phase, was significantly lowered after acclimation to daily intermittent heat exposure (Fig. 2), which is consistent with our previous
OSAMU SHIDO AND OTHERS
62
observations (Shido et al. 1989; Shido & Nagasaka, 1990 b). In addition, Thy of the HE rats markedly decreased in the last half of the dark phase during which the rats had been previously subjected to heat exposure, forming a characteristic trough of Thy in the dark phase. Such a clear trough of Thy was not observed in the Cn rats (Fig. IA) A 38.5=-38.0 -
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Fig. 1. Mean changes of hypothalamic temperature (Th,; A) and wheel revolutions (WR; B) in the control rats before (left panel) and one and two days after (right panel) heat acclimation period, Values in Thy are means of 5 min in five rats and values in WR are hourly means of five rats. The s.E.M. is not shown in order to simplify the figure. Dots above abscissa, significantly different from corresponding values before heat exposure sohedule. Filled bars on abscissa, dark phases of a day.
or in the HE rats before the heat exposure schedule (Fig, 2). The trough of Thy in the dark phase lasted for 3-4 h and was persistent for at least 2 days after the heat exposure schedule had ended.
Experiment 2: heat balance and body movement The body weights of the Cn and HE rats just after measurements of heat balance with the direct calorimeter were 348 +11 and 349 + 5 g, respectively. Table 1 summarizes the mean values of Thy, total heat loss (H = R + c +K+E), C, M and BM at four periods per day in the Cn and HE groups after the heat exposure schedule had ended. Thy was consistently lower in the HE rats than in the Cn rats in all four periods of the day. A significant reduction of Thy in the HE rats was shown between 09.00 and 15.00 h when the trough of Thy was observed. Particularly on the first day after
MODIFICATION OF TEMPERATURE RHYTHM BY HEAT
63
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TABLE 1. Mean values of thermoregulatory parameters and body movement after heat exposure schedule in control and heat-exposed rats H Time of C M BM thy (W m-2) (W m-2 OC-1) day (W m-2) Group (OC) (count) Cn 37-20+0-09 44-7+1 2 2-958+0i100 43-8+0 8 14912+2273 21.00 to 03.00 HE (1) 37-00+0-07 48.0+0.7* 3.352+0.046* 46-0+0-8 15296+2250 HE (2) 37-08+0-08 45 9+0 9 3-102+0-065 45-7+0 8 16 119+2099 Cn 37-84+0-09 61-1 +03 3-965+0-051 59 0+0 4 41849+4822 03.00 to 09.00 HE (1) 37.58+0.06* 60'9+1.2 4'073+0-079 60-2+1i1 44310+7106 HE (2) 37-72+0-07 61-8+1-6 4-073+0-104 60-9+1-8 44162+7330 Cn 37-91+0-10 61P2+0-5 3-891+0-043 61-3±0-4 43772±4359 09.00 to 15.00 HE (1) 3740+0.07* 56.0+1.5* 3-731+0'098 57.3+1 4* 37753±6061 HE (2) 37.53+0.08* 57-7+1-7 3-798+0'084 57'8± 1-7 41677±7064 Cn 37-08+0-11 41-5±0-7 2-668+0-066 44-4+1'0 15908±2717 15.00 to 21.00 HE (1) 37-05+0-08 46.6±1.2* 3.137+0.093* 48-0+0-8 20605+3735 HE (2) 36-93±+006 44-1 + 1 0 2-962 +0.084* 45-2+0-8 15892+2695 Thy, hypothalamic temperature; H, total heat loss; C, total thermal conductance; M, metabolic heat production; BM, body movement; Cn, values in the control group one day after termination of heat exposure schedule; HE (1), values in the heat-exposed rats (HE) one day after termination of heat exposure; HE (2), values in the HE rats two days after termination of heat exposure. 21.00-03.00 and 15.00-21.00 h, corresponding to the previous light phase; 03.00-09.00 and 09.00-15.00 h, corresponding to the previous dark phase. * Significantly different from values in the Cn.
the heat exposure schedule, H, C and M of HE rats were higher than those of the Cn rats between 15.00 and 21.00 h and 21.00 and 03.00 h, corresponding to the previous light phase of a day. From 09.00 to 15.00 h, on the contrary, H and M were lower in the HE rats than in the Cn rats. The HE rats appeared to be less active than the Cn rats between 09.00 and 15.00 h, but there were no significant differences in the counts of BM between the Cn and HE groups throughout the day.
OSAMU SHIDO AND OTHERS
64
In order to compare patterns of diurnal variation of thermoregulatory parameters in constant darkness, relative changes from minimal values in each variable were computed as the percentage of the maximum amplitude in a day. Figure 3 shows the percentage changes calculated hourly in Thy, H, M and BM in the Cn and HE rats 80 1
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after the heat exposure schedule had ended. In the Cn rats, all these variables showed clear and similar diurnal variations. High levels of Thy, M, H and BM were maintained from 03.00 to 15.00 h corresponding to the previous dark phase of a day. In the HE rats, however, the levels of Thy from 09.00 to 13.00 h when the rats had been exposed to heat were significantly lower than those of the Cn rats. Thy, then, showed a distinct trough in the last half of the previous dark phase as observed in experiment 1 under the 12-12 h light-dark cycle. A similar pattern was shown in the variations of H and M. The diurnal changes in BM were rather varied, but the trough of BM in the HE rats was consistently seen. This characteristic pattern of diurnal thermoregulatory parameters in the HE rats persisted for at least 2 days after acclimation to the heat exposure schedule. The diurnal changes of these thermoregulatory parameters in absolute values
65 MODIFICATION OF TEMPERATURE RHYTHM BY HEAT calculated hourly were almost the same as the relative changes shown in Fig. 3. Significant differences in Thy, (R + c +K), H, C and M between the Cn and HE groups were obtained between 09.00 and 15.00 h on the first and second day after the heat exposure schedule. However, the absolute value of BM in the HE rats did not differ 4.5
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from that of the Cn rats during any period on the second day after the heat exposure schedule. Figure 4 shows the mean values of C in the four periods per day as a function of BM (divided into six ranges at a step of 200 counts) in the Cn and HE rats after the end of the heat exposure schedule. C in the HE rats was higher than that of the Cn group 3
PHY 433
OSAMU SHIDO AND OTHERS
66
at any level of BM between 03.00 and 09.00 h, the early half of the previous dark phase, and significantly during 21.00-03.00 h and 15.00-21.00 h, corresponding to the previous light phase of a day. From 09.00 to 15.00 h, however, C in the HE rats became smaller than that of the Cn rats regardless of the BM magnitude. Similar 70
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0 0
(CD
00
0
0 (0
q
o 0
C
Body movement (15.00-21.00 h) Body movement (21.00-03.00 h) Fig. 5. Mean metabolic heat production (M) as a function of body movement between 21.00 and 03.00, 03.00 and 09.00, 09.00 and 15.00, and 15.00 and 21.00 h after the heat exposure period had ended. Other explanations are the same as in Fig. 4.
results were obtained in the relationship between M and BM (Fig. 5). From 09.00 to 15.00 h, M was smaller in the HE rats than in the Cn rats at any BM level, although the HE rats showed greater M in the other periods of the day.
Experiment 3: mecaurements of locomotor activity As is well known, rats turn the running wheel frequently in the dark phase and rarely in the light phase. In the Cn rats, the hourly numbers of WR were not significantly different during the experimental days in any period per day, although there was a great intra- and interindividual variety of the WR activity distributions in a day (Fig. 1B). Figure 6 shows the mean numbers of WR before, during and after
MODIFICATION OF TEMPERATURE RHYTHM BY HEAT 67 acclimation to daily heat exposure in the HE rats. On the first day of heat exposure, WR increased during the heat exposure time (09.00-15.00 h). It gradually decreased and the reduction of WR became significant from the sixth day after the start of the heat exposure schedule. On the ninth day of heat exposure, WR both before and after 250250 -200X 150-o 10050-
~Pre
1
_
2
*
6
9
8
7
*~~~~~~~~~~~r
* __ I
0 0
N
o 250--
0 o
200 150 0 -~100 5000
N
0
~13
N
0
0
0
N
0
C14
0
N
0
(N
~0
0
0iV--io;C4
0
1o
0
N
(N
0
Po4
Po3
00
0
0
o c 0
Po2
Pol
14
0
0
N
Po5
D
0
0
0
0
0
0
N
0
N
0
N
Time of day (h) Fig. 6. Wheel revolution (WR) changes in the heat-exposed rats (HE) before, during and after the heat exposure schedule. Values are hourly means of eleven rats. The S.E.M. is not shown in order to simplify the figure. Filled bars beneath abscissa, dark phases of a day; open bars above abscissa, heat exposure time. Pre, before heat exposure; numbers, days after the onset of heat exposure; numbers with Po, days after the end of heat exposure. * Significantly different from corresponding values before the start of the heat exposure. The number of rats was six from 3 days after the termination of heat exposure (on days of Po3, Po4 and Po5).
heat exposure time significantly increased. The subsequent rise of WR to heat exposure time quickly stopped but the high activity before daily heat exposure appeared to be maintained through the end of the heat acclimation schedule. After the heat exposure schedule had ended, the significant reduction of WR in the last half of the dark phase lasted for only one day, although the numbers of WR in the period appeared to decrease during the subsequent 5 days. DISCUSSION
The present results clearly confirm that the diurnal variations in deep body temperature and locomotor activity are modified by acclimation to heat exposure for about 5 h daily at a fixed time in the dark phase for at least 10 consecutive days. The Thy of heat-acclimated rats definitely decreased during the period corresponding to the previous heat exposure time and the characteristic trough of Thy was formed in 3-2
68
OSAMU SHIDO AND OTHERS
the dark phase. This trough of Thy was consistent under both the 12-12 h light-dark cycle and the constant darkness. During heat exposure, heat loss mechanisms are activated and metabolic heat production is suppressed to prevent heat accumulation in the body. These physiological responses to heat stress occurred daily during the same period per day in rats on a heat exposure schedule. This fall in Thy of the HE rats during the period corresponding to the previous heat exposure time, therefore, may be attributed to the persistence of the thermoregulatory responses to heat even without actual heat exposure. Similar to the observation of the time memory for meals in rats subjected to a restricted feeding schedule (Boulos et al. 1980; Mori et al. 1983), the present results show that a time memory for heat exposure could be formed in the present rats acclimated to heat exposure and that the memory persists for at least 2 days after the heat exposure schedule ended. In the HE rats, (R+c+K), E, H and C in both absolute and relative values significantly decreased either before or during the period corresponding to the time of the previous heat exposure schedule even after the heat exposure schedule had ended. These results provide no evidence that the activation of heat loss mechanisms contribute to the formation of the trough of Thy in the dark phase. In freely moving rats, however, body movement strongly affects heat transfer from body to environment through altering heat convection and changing effective body surface area for dissipating heat. Under uncontrolled body movement conditions, the activity level of the autonomic heat loss mechanism cannot be evaluated. To eliminate such influences, C was assessed as a function of body movement (Fig. 3). C from 21.00 to 03.00 h, 15.00 to 21.00 h and 03.00 to 09.00 h was consistently greater in the HE rats than in the Cn rats regardless of the intensity of body movement. The results may agree with the report that non-evaporative heat loss capacity is improved by acclimation to intermittent heat exposure (Shido & Nagasaka, 1990b). Between 09.00 and 15.00 h corresponding to the previous heat exposure period, however, C of the HE rats never exceeded C of the Cn rats at any intensity of body movement. The decrease of C may not indicate the facilitation of heat loss responses but the occurrence of activation in the heat conservation mechanism. It is, therefore, unlikely that heat dissipation mechanisms contributed to the fall in body temperature during the period of previous heat exposure after the heat exposure schedule had ended in heat-acclimated rats. Reduction of M is another potent cause of the decrease in body temperature. After the heat exposure schedule in the HE rats, M significantly decreased between 09.00 and 15.00 h, which implies the predominant contribution of metabolic heat production mechanism to the formation of the Thy trough in the dark phase. The change of M obtained in the present system involves changes of muscular work intensity associated with behaviour and of resting metabolic rate. The acclimation to an intermittent heat exposure schedule obviously modulated the distributions of body movement determined by piezoelectric ceramics and of WR in a day. The HE rats showed less body movement during the previous heat exposure time on the first day after heat exposure schedule termination. However, the reduction of activity became unclear on the second day after heat exposure termination where the fall in Thy and the definite reduction of M were constantly observed. Similarly, WR decreased significantly during the last half of the dark phase, but the low locomotor
MODIFICATION OF TEMPERATURE RHYTHM BY HEAT
69
activity lasted only for one day after the heat exposure schedule had ended. Thus the suppression of M in the HE rats cannot be explained by only the redistribution of locomotor activity. When assessed as a function of body movement (Fig. 4), M from 03.00 to 09.00 h, 15.00 to 21.00 h and 21.00 to 03.00 h was consistently greater in the HE rats than in the Cn rats at all intensities of body movement except between 15.00 and 21.00 h at the range of body movement from 401 to 800 counts. From 09.00 to 15.00 h corresponding to the previous heat exposure period, however, M of the HE rats became smaller than that of the Cn rats regardless of body movement magnitude. Furthermore, mean M during this period of the HE rats was significantly lower than that of the Cn rats, although the amount of body movement was not different between the two groups. These results may suggest that resting metabolic rate, heat production without muscular movement, was suppressed during a particular period of a day in the HE rats. The characteristic trough of Thy formed during the previous heat exposure period after termination of the heat exposure schedule was, therefore, attributed to both the reduction of heat production accompanied by behavioural activity and to the suppression of resting metabolic rate. The detailed mechanism of the suppression of resting heat production during the previous heat exposure period is not yet clear. At the same level of locomotor activity, M before meals was markedly lower than that after feeding in rats on restricted feeding regimes (Boyle, Storlien, Harper & Keesey, 1981; Nagasaka & Shido, 1989). The reduction of postprandial thermogenesis was possibly involved in the low M of the previous heat exposure period in the HE rats. In this case, the distributions of feeding activity in a day are expected to be modified by daily heat exposure at a fixed time as observed in the alterations in diurnal variation of locomotor activity. Acclimation to chronic heat exposure brings about several adjustments of thermogenic pathway (Cassuto, 1968) and of endocrine functions (Collins & Weiner, 1968; Cassuto, Chayoth & Rabi, 1970; Arieli & Chinet, 1986). Some reports give evidence of modulations in circadian rhythm of endocrine systems by chronic heat exposure (Cure, 1989; Brandenberger, Follenius, Di Nisi, Libert & Simon, 1989). It is, therefore, possible that the pattern of diurnal cycle of hormones related to metabolism or the activity of thermogenic organs was influenced by the heat exposure schedule adopted in this study and the persistence of re-established rhythm contributed to the reduction of M during the period of previous heat exposure.
Repeated temperature stimuli have been well known to shift the thermoneutral zone and set-point for temperature regulation in mammals (Fox et al. 1963; Shido et al. 1989; Shido & Nagasaka, 1990 a, b). Tokura & Aschoff (1983) have demonstrated that a free-running period of circadian activity rhythm was elongated by a rise in
ambient temperature from 17 to 33 °C in pig-tailed macaques. A similar observation in the effect of ambient temperature on a free-running period of spontaneous activity was made in house finch (Enright, 1966). The results clearly suggest that the internal time-keeping system can also be modified by external temperature stimuli. On the other hand, circadian rhythms of heat production, heat loss, body temperature and activity are considered to be driven by potentially independent oscillators (Aschoff & Pohl, 1970; Aschoff, Biebach, Heise & Schmidt, 1974; Wever, 1975) and can
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desynchronize either spontaneously (Aschoff, Gerecke & Wever, 1967; Sulzman, Fuller & Moore-Ede, 1977) or when forced by manipulations of zeitgeber period (Wever, 1975). To date, there was no available information with regard to the effect of repeated heat exposure on oscillation of heat balance, body temperature or spontaneous activity. However, if heat stress could cause the internal desynchronization between heat gain and loss, the pattern of diurnal changes in body temperature should be altered, which might result in the formation of the characteristic trough in the dark phase in the present rats acclimated to repeated daily heat exposure. In summary, heat exposure for about 5 h at a fixed time once a day modified diurnal changes of body temperature after the heat exposure schedule termination, which had been characterized by a fall in body temperature during the period of previous heat exposure. Since this distinct trough of body temperature persisted for at least 2 days after heat exposure termination, it is suggested that a time memory for heat exposure or for thermoregulation was formed in rats through repeated daily heat exposure at a fixed time. The predominant contributor to the fall in body temperature was the reduction of metabolic heat production associated with and without locomotor activity. The authors gratefully acknowledge the contribution made by Naotoshi Sugimoto in performing the experiments. The authors are also grateful to Akio Yachi and Shigeyuki Tabata for their technical assistance in building the calorimeter system. REFERENCES
ARIELI, A. & CHINET, A. (1986). Thyroid status and noradrenaline-induced regulatory thermogenesis in heat acclimated rats. Hormonal Metabolic Research 18, 103-106. ASCHOFF, J., BIEBACH, H., HEISE, A. & SCHMIDT, T. (1974). Day-night variation in heat balance. In Heat Loss from Animals and Man, ed. MONTEITH, J. C. & MOUNT, L. E., pp. 147-172. Butterworths, London. ASCHOFF, J., GERECKE, V. & WEVER, R. (1967). Desynchronization of human circadian rhythms. Japanese Journal of Physiology 17, 450-457. ASCHOFF, J. & POHL, H. (1970). Rhythmic variations in energy metabolism. Federation Proceedings 29, 1541-1552. BOLLES, R. C. & STOKES, L. W. (1965). Rat's anticipation of diurnal and a-diurnal feeding. Journal of Comparative and Physiological Psychology 60, 290-294. BoULOS, Z., ROSENWASSER, A. M. & TERMAN, M. (1980). Feeding schedules and the circadian organization of behavior in the rat. Behavioral Brain Research 1, 39-65. BOYLE, P. C., STORLIEN, L. H., HARPER, A. E. & KEESEY, R. E. (1981). Oxygen consumption and locomotor activity during restricted feeding and realimentation. American Journal of Physiology 241, R392-397. BRANDENBERGER, G., FOLLENIUS, M., Di Nisi, J., LIBERT, J. P. & SIMON, C. (1989). Amplification of nocturnal oscillation in PRA and aldosterone during continuous heat exposure. Journal of Applied Physiology 66, 1280-1286. CASSUTO, Y. (1968). Metabolic adaptations to chronic heat exposure in the golden hamster. American Journal of Physiology 214, 1147-1151. CASSUTO, Y., CHAYOTH, R. & RABI, T. (1970). Thyroid hormone in heat-acclimated hamsters. American Journal of Physiology 218, 1287-1290. COLLINS, K. J. & WEINER, J. S. (1968). Endocrinological aspects of exposure to high environmental temperatures. Physiologial Reviews 48, 785-839. CURE, M. (1989). Plasma corticosterone response in continuous versus discontinuous chronic heat exposure in rat. Physiology and Behavior 45, 1117-1122.
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ENRIGHT, J. T. (1966). Temperature and free-running circadian rhythm of the house finch. Comparative Biochemistry and Physiology 18, 463-475. Fox, R. H., GOLDSMITH, R., KIDD, D. J. & LEWIS, H. E. (1963). Blood flow and other thermoregulatory changes with acclimatization to heat. Journal of Physiology 166, 548-562. FULLER, R. W. & DILLER, E. R. (1970). Diurnal variations of liver glycogen and plasma free fatty acids in rats fed ad libitum or single daily meal. Metabolism 19, 226-229. KRIEGER, D. T. (1974). Food and water restriction shifts corticosterone, temperature, activity and brain amine periodicity. Endocrinology 95, 1195-1201. MOBERG, G. P., BELLINGER, L. L. & MENDEL, V. E. (1975). Effect of meal feeding on daily rhythm of plasma corticosterone and growth hormone in the rat. Neuroendocrinology 19, 160-169. MORI, T., NAGAI, K. & NAKAGAWA, H. (1983). Dependence of memory of meal time upon circadian biological clock in rats. Physiology and Behavior 30, 259-265. NAGASAKA, T., HIRATA, K., SUGANO, Y. & SHIBATA, H. (1979). Heat balance during physical restraint in rats. Japanese Journal of Physiology 29, 383-392. NAGASAKA, T. & SHIDO, 0. (1989). Locomotor activity and heat production of rats on restricted two-hour feeding regimes. Japanese Journal of Biometeorology 26, 85-90. SHIDO, 0. & NAGASAKA, T. (1990a). Thermoregulatory responses to acute body heating in rats acclimated to continuous heat exposure. Journal of Applied Physiology 68, 59-65. SHIDO, 0. & NAGASAKA, T. (1990b). Heat loss responses in rats acclimated to heat loaded intermittently. Journal of Applied Physiology 68, 66-70. SHIDO, O., YONEDA, Y. & NAGASAKA, T. (1989). Changes in body temperature of rats acclimated to heat with different acclimation schedule. Journal of Applied Physiology 67, 2154-2157. SUGANO, Y. (1983). Heat balance of rats acclimated to diurnal 2-hour feeding. Physiology and Behavior 30, 289-293. SULZMAN, F. M., FULLER, C. A. & MOORE-EDE, M. C. (1977). Spontaneous internal desynchronization of circadian rhythms in the squirrel monkey. Comparative Biochemistry and Physiology 58A, 6367. TOKURA, H. & ASCHOFF, J. (1983). Effects of temperature on the circadian rhythm of pig-tailed macaques Macaca nemestrina. American Journal of Physiology 245, R800-808. WEVER, R. (1975). The circadian multi-oscillator system of man. International Journal of Chronobiology 3, 19-55.
