- Email: [email protected]
Genetic selection of rats with high and low body temperatures
Journal of Thermal Biology 26 (2001) 223–229
Genetic selection of rats with high and low body temperatures$ Christopher J. Gordon*, Amir H. Rezvani1, With the technical assistance of Beth Padnos and Peggy Becker Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA Received 27 August 2000; received in revised form 16 October 2000; accepted 11 November 2000
Abstract Body (core) temperature (Tc ) directly affects all biological processes, including sensitivity to toxic chemicals, development, aging, and drug metabolism. To understand how Tc affects these processes it is necessary to alter Tc independently of other physiological processes. The purpose of this study was to determine whether selective breeding techniques can be used to develop lines of rats with hyperthermic and hypothermic Tc ’s. Tc and motor activity of 24 female and 23 male rats (parental line) of the NIH heterogenous stock were monitored by telemetry for 96 h at a Ta of 228C. The mean 24 h Tc of the male and female rats was 37.38C with a range of 37–38.28C. Tc was not correlated with motor activity or body weight. Pairs with the lowest and highest Tc’s were selected for breeding. The F1 generation consisted of 10 offspring from the hyperthermic group and 20 from the hypothermic group. They were implanted with transmitters at 60 d of age. Tc of rats derived from the hyperthermic parental line had a significantly warmer Tc than the rats derived from the hypothermic parental line. Motor activity was significantly higher in the hyperthermic F1 males and hypothermic F1 females. Breeding of hyperthermic and hypothermic rats has shown that adult offspring of the fourth generation maintain significantly different core temperatures but have similar patterns of motor activity. The results demonstrate that Tc is heritable and that it should be feasible to develop lines of rats that regulate Tc above or below normal. Published by Elsevier Science Ltd. Keywords: Genetic selection; Body temperature; Hyperthermia; Hypothermia
1. Introduction Because of the direct effect of temperature on membrane potentials, chemical reactions, and enzyme
$ This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. *Corresponding author. Tel.: +1-919-541-1509, fax: +1-919541-4849. E-mail address: [email protected] (C.J. Gordon). 1 Current address: Duke University Medical Center, Department of Psychiatry, Durham, NC 27710, USA.
0306-4565/01/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 3 0 6 - 4 5 6 5 ( 0 0 ) 0 0 0 4 6 - 2
activities, body temperature has a direct effect on the function of all molecular, cellular, and organismic processes. There are innumerable studies showing that a change in body temperature will affect a variety of processes, including sensitivity to drugs and toxic chemicals, drug metabolism, development, growth, and aging (Prosser, 1973; Gordon, 1993). One particularly interesting possibility that this area of study has generated is that a lower body temperature (DT¼ 1:08C) will increase life span, possibly by reducing metabolism and the damaging effects of free radicals (Lane et al., 1996; Duffy et al., 1997). In order to study how temperature affects these processes it is essential to alter body (i.e., core) temperature independently of other physiological and
224
C.J. Gordon, A.H. Rezvani / Journal of Thermal Biology 26 (2001) 223–229
behavioral processes. However, to the best of our knowledge, achieving a chronic change in core temperature that is essentially independent of other physiological processes has not been achieved. Body temperature of a homeotherm can be changed with relative ease by altering heat production and/or heat loss. Techniques such as exposure to heat or cold stress, exercise, administration of drugs that alter thermoregulation, lesions in CNS thermoregulatory centers, and surgically implanted heat exchangers have been used in innumerable studies to alter core temperature. However, regardless of the method used, the change in body temperature is accompanied by physiological and behavioral changes that are likely to be unrelated to the change in body temperature. In most instances, body temperature is forced above or below the set-point and physiological responses are activated to counter the change in temperature. Hence, body temperature was essentially a dependent variable in the past studies. To understand how body temperature affects cellular and organismic processes, it is essential to develop an animal model with a permanent change in their regulated core temperature (i.e., independently change body temperature without altering thermoeffector activity). To this end, we have explored the use of selective breeding to develop genetic lines of rats that regulate their core temperature above or below normal. Genetic selection has been used to alter a variety of thermoregulatory related processes such as increased survival to acute heat stress (Furuyama and Ohara, 1993), altered resistance to the hypothermic effects of ethanol (Crabbe et al., 1987) and the anticholinesterase agent DFP (Overstreet et al., 1979), and a high or low rate of heat loss (Nielsen et al., 1997). However, to the best of our knowledge, there has been no successful attempt to develop lines of animals with permanently different body temperatures. A fundamental premise of our hypothesis is that there is an inherent differences in body temperature within a species with a normal distribution that is attributed to genotypic differences in the set-point or regulated body temperature. The purpose of this study was to test the hypothesis that individual differences in the level at which deep body temperature is regulated is genetically determined, meaning that a high or low temperature can be passed on to successive generations with selective breeding in the rat.
2. Materials and methods Animals used in this study were from the heterogenous stock developed and maintained at the National Institutes of Health (N:NIH strain). Inbred and outbred rats from conventional animal suppliers could lack thermoregulatory traits because of inbreeding and genetic isolation. The NIH heterogenous strain is
considered to be ideal for selective breeding studies (Hansen and Spuhler, 1984). The rats were obtained at approximately 21 d of age and maintained at a Ta of 228C, a relative humidity of 50%, and a 12:12 light:dark photoperiod. Core temperature and motor activity were monitored in undisturbed rats using radiotelemetry (Data Sciences International, St. Paul, MN). Details of the telemetry system have been published (Gordon, 1994). Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg; IP) and an abdominal incision was made for the implantation of the transmitter (TA10TA-F40) into the abdominal cavity. The abdominal muscle was sutured and the skin was closed with wound clips. Following surgery, rats were administered a penicillin antibiotic (30,000 units; i.m.) and analgesic (buprenorphine; 0.03 mg/kg; SC). The rats were allowed at least 10 d of recovery before testing. Core temperature and motor activity were monitored at 5 min intervals by receivers placed under the floor of each cage. The rat’s motor activity was measured from the change in position of the transmitter in relation to the antennae in the receiver board. This technique does not give a quantitative measurement of activity. Instead, the technique provides a relative measure of the rat’s activity in dimensions of counts/10 min. The telemetry variables were monitored for 96 h continuously in 90 d-old male (N=23) and female (N=24) rats (parental line) while they were individually housed in the animal facility. The telemetry system was capable of monitoring 24 animals simultaneously. Thus, two cohorts of males and females were measured in separate experiments. Care was taken to not disturb the rats in any way during the monitoring period.
2.1. Data analysis The 96 h of data were ensembled for each rat into a single 24 h cycle. It was decided that 96 h of recording would be sufficient to dampen or eliminate changes in body temperature that would occur from extrinsic (e.g., ambient noise) and intrinsic sources (e.g., infradian oscillations, estrous cycle, sleep–wake patterns, etc.). The mean and standard deviation (SD) of core temperature and motor activity over the 96 h period was calculated for each rat. It was intended to isolate rats whose core temperature was above or below one SD of the mean as based on the definition of hypothermia and hyperthermia (IUPS, 1987). The temperature responses were further evaluated for any unusual characteristics of the nychthemeral temperature rhythm and patterns of motor activity. It was intended to select rats from the parental lines that were hyperthermic or hypothermic but did not have unusually high or low levels of motor activity, abnormal differences in
C.J. Gordon, A.H. Rezvani / Journal of Thermal Biology 26 (2001) 223–229
amplitude or timing of the nychthemeral temperature rhythm, or marked deviations in body weight. Two pairs of rats with core temperatures above and below the mean population were isolated and placed together for breeding. The offspring were weaned at 21 d of age. There was no culling of litters. At 60 d of age the offspring (F1) were implanted with transmitters and their core temperature and motor activity patterns were recorded in the same manner as described above.
3. Results There was a near normal frequency distribution of core temperature in male and female rats (Fig. 1). The mean core temperature of male and female rats was 37.32 and 37.368C, respectively. Core temperature of females ranged from 37.05 to 37.558C; for males, the range was 37.1–38.18C. The outliers with core temperatures greater or less than 1.0 SD from the mean were
Fig. 1. Frequency distribution of mean 24 h core temperatures in male and female rats monitored undisturbed for 96 h. Noted outliers (arrows) were used for breeding hyperthermic and hypothermic lines.
225
selected for the hyperthermic and hypothermic parental lines, respectively. There was one male that had a relatively high core temperature of 38.18C, which was more than 3 SD’s above the mean. There was no relationship between the 24 h mean of motor activity and core temperature in the parental line (Fig. 2). In addition, body weight was not related to core temperature (data not shown). The four breeding pairs from the parental line gave birth to 22 pups from the hyperthermic line and 21 pups from the hypothermic line. There was substantial mortality in the hyperthermic line with 10 pups surviving through weaning whereas 18 hypothermic pups survived through weaning. These deaths occurred within one week of parturition. When tested as adults, core temperature of the F1 rats derived from the hyperthermic parental line was significantly warmer than that of the rats derived from the hypothermic parental line (Fig. 3). Mean core temperature of hyperthermic males and females was 0.20 and 0.298C warmer than the hypothermic line in the daytime and 0.13 and 0.198C warmer at night. Motor activity of hypothermic females was higher than males, whereas activity of hypothermic males was lower than
Fig. 2. Relationship between mean motor activity and core temperature of parental males and females. Data fit with linear regression. Slopes of regression lines for male and female rats not different from zero.
226
C.J. Gordon, A.H. Rezvani / Journal of Thermal Biology 26 (2001) 223–229
that observed in the hyperthermic males. The differences in motor activity were more distinguishable during the dark phase (Fig. 4). The mean core temperature of hyperthermic offspring was cooler than that of the parental line. Similarly, the core temperature of hypothermic offspring was warmer than that of the parental line (Table 1). Body weight of F1 at adulthood was significantly greater in animals derived from the hyperthermic line: 336 13 g for hyperthermic males;
300 10 g for hypothermic males; 205 6 g for hyperthermic females; and 184 4 g for hypothermic females. 3.1. F4 Generation Continued selective breeding of hyperthermic and hypothermic rats through four generations to this point has shown that adult offspring maintain significantly different core temperatures but similar patterns of motor activity (Fig. 5). Overall, the 24 h core temperature of the F4 hyperthermic line was 0.238C warmer than the hypothermic line in males and 0.218C in females. Similar to the F1 generation, body weights of both sexes of the hyperthermic F4 generation were greater than in the hypothermic animals (39 and 20 g heavier in males and females).
4. Discussion
Fig. 3. 24 h core temperature of hyperthermic and hypothermic parents (P line) used for generating F1 of hyperthermic and hypothermic lines. Data are ensembled means of 96 h of continuous data.
The key observation of this study is the demonstration that small individual variations in the level at which body temperature is regulated is heritable in the rat. Selective breeding of rats with higher than normal core temperature had offspring with significantly warmer core temperatures compared to selective breeding of rats
Fig. 4. Time-course of core temperature and motor activity of F1 hyperthermic and hypothermic lines. Data ensembled from 96 h recordings as in Fig. 3. Repeated ANOVA: Core temperature; male; treatment, F(25, 1)=21.7, p50.0001, treatment-time, F(23, 552)=3.0, p50.0001; female, treatment, F(29, 1)=34, p50.0001; treatment-time, F(23, 644)=6.5, p50.0001; motor activity, male, treatment, F(25.1)=14. P=0.001; treatment-time, F(23, 552)=9.3, p50.0001; female, treatment, F(29, 1)=11, p=0.003; treatment-time, F(2, 644), p50.0001. Numbers in parentheses indicate sample size.
C.J. Gordon, A.H. Rezvani / Journal of Thermal Biology 26 (2001) 223–229
with colder than normal core temperatures. The altered core temperature in the offspring was maintained throughout most of the light and dark phase of a 24 h period. There were significant differences in motor activity between the hypothermic and hyperthermic offspring of F1; however, these differences are probably not the reason behind the differences in core temperatures. Motor activity was higher in the hyperthermic males but lower in the hyperthermic females. Like the parental line, there was no correlation between motor activity and core temperature in the F1 generation. Overall, spontaneous activity has minor effects on resting core temperature of the rat (Gordon and Yang, 1997). Thus, the core temperatures in the hyperthermic and hypothermic lines are likely to be a result of innate differences not related to the heat produced from motor activity. In addition, the separation of core temperature
Table 1 Mean S.E. of core temperature in parental line selected for breeding and in F1 generation Group/sex
Parental line, Tc (8C)
F1, Tc (8C)
Hyperthermic/male Hyperthermic/female Hypothermic/male Hypothermic/female
37.8 0.3 37.7 0.1 37.1 0.004 37.1 0.08
37.4 0.05 37.5 0.02 37.2 0.03 37.3 0.04
227
in the F4 hyperthermic and hypothermic lines but with similar patterns of motor activity is further support that the differences in core temperature are not a result of differences in motor activity. It is essential to use radiotelemetry to monitor core temperature in animals to be used for developing lines of hyperthermic and hypothermic rats. A single measurement of core temperature can be affected by numerous variables, including stress of handling, ultradian oscillations, and estrous cycle (Briese and de Quijada, 1970; Gordon, 1990, 1993). In addition, single or multiple measurements of core temperature with a colonic probe may not necessarily reflect the animal’s core temperature over a 24 h period. Stress from this technique can lead to abnormal measurements of core temperature. Indeed, the presence of personnel in the animal facility can lead to artifactual changes in core temperature (Briese and de Quijada, 1970). On the other hand, telemetric monitoring gives the best estimate of resting core temperature that should reflect the core temperature regulated by thermoregulatory centers in the CNS. By monitoring for 96 h, there should be an adequate number of samples to eliminate the extrinsic and intrinsic factors that would transiently alter the set-point temperature. Since the estrous cycle of the rat is 4–5 d, the 96 h period should also take into account the slight rise in core temperature (0.28C) of the female rat during the night of proestrus (Yang and Gordon, 1996). There have been considerable success in artificial selection of heat-tolerant lines of birds and mammals
Fig. 5. Time-course of core temperature and motor activity of F4 hyperthermic and hypothermic lines. Data ensembled from 96 h recordings. Repeated ANOVA: Core temperature; male; treatment, F(1, 14)=87.9, p50.0001, treatment-time, F(23, 322)=1.9, p=0.006; female, treatment, F(1, 10)=27, p=0.0004; treatment-time, F(23, 230)=2.2, p=0.002; motor activity, male, treatment (NS), treatment-time, (NS) female, treatment, (NS) treatment-time, (NS). Numbers in parentheses indicate sample size.
228
C.J. Gordon, A.H. Rezvani / Journal of Thermal Biology 26 (2001) 223–229
(Furuyama and Ohara, 1993; Wilson et al., 1975). In these studies, animals with improved heat tolerance were selectively bred and the resulting generations showed a marked improvement in their tolerance to acute heat stress. Baseline core temperature was not a factor in the selection process. However, the artificial selection for heat tolerance did lead to changes in the baseline core temperature when the animals were challenged with other stimuli such as cold stress and thermoregulatory drugs (Furuyama and Ohara, 1993). Little is known of the extent to which individual variations in body temperature are heritable. Connolly and Lynch (1981) estimated the heritability of individual variations in the body temperature of mice by comparing the day and night core temperatures in four strains. They concluded that heritability was very low for daytime body temperature but relatively high during the night when motor activity and body temperature are elevated. In other words, body temperature at night may be subject to more selective pressure than during the day because it is associated with expression of complex behaviors (Connolly and Lynch, 1981). We endeavored to artificially select rats with altered day time and night time temperatures because selecting for only a high or low night time temperature would also result in a change in the amplitude of the nychthemeral pattern of core temperature. Moreover, raising or lowering the diurnal temperature of a nocturnal rodent allows one to study pyrogens such as lipopolysaccharide that are more effective to elevate core temperature in the day time as compared to the night (Feng et al., 1989). We expect heritability of individual variations in core temperature of the rat to be relatively small. The core temperature of the hyperthermic F1’s was 0.2–0.48C below that of the parents while the core temperature of the hypothermic F1’s was 0.1–0.28C above that of their parents (Table 1). After four generations we have found the separation of mean 24 h core temperature to have increased by just 0.18C over that of the first generation (cf. Fig. 5). Hence, we expect a slow separation in the mean core temperature of hyperthermic and hypothermic lines with each generation compared to the relative success of selection of other thermoregulatory parameters (Wilson et al., 1975; Furuyama and Ohara, 1993; Nielsen et al., 1997). What are the physiological mechanisms responsible for the differences in core temperature between the hyperthermic and hypothermic lines? We tested the behavioral thermoregulatory responses of a selected group of hyperthermic and hypothermic rats that had a core temperature difference of 0.58C but found essentially no significant difference in selected ambient temperature (Gordon, unpublished observations). In fact, rats of the hypothermic line selected slightly cooler ambient temperatures than that of the hyperthermic line. All together, these preliminary studies suggest that the
selection of the hyperthermic and hypothermic lines did not arise from a thermoregulatory dysfunction such as an over- or underactive thyroid gland. If this was the case, we would have expected to see marked behavioral thermoregulatory responses.
5. Future perspectives Some outstanding issues also need to be resolved in future studies: (1) How high or low a core temperature can be developed with selective breeding. Are there internal limits to the genetic selection of hyperthermic and hypothermic rats? (2) Does the high or low body temperature selected in adults persist throughout the life of the organism? (3) How early in life can differences in core temperature be detected? (4) Will the differences in core temperature be maintained when the animal is housed at warm or cold ambient temperatures? These questions will be addressed in future investigations.
Acknowledgements We thank Dr. Carl Hansen of NIH for his generosity in supplying us with rats of the NIH:N strain.
References Briese, E., de Quijada, M.G., 1970. Colonic temperature of rats during handling. Acta Physiol. Lat. Am. 20, 97–102. Connolly, M.S., Lynch, C.B., 1981. Circadian variation of strain differences in body temperature and activity in mice. Physiol. Behav. 27, 1045–1049. Crabbe, J.C., Kosobud, A., Tam, B.R., Young, E.R., Deutsch, C.M., 1987. Genetic selection of mouse lines sensitive (cold) and resistant (hot) to acute ethanol hypothermia. Alcohol Drug Res. 7, 163–174. Duffy, P.H., Leakey, J.E., Pipkin, J.L., Turturro, A., Hart, R.W., 1997. The physiologic, neurologic, and behavioral effects of caloric restriction related to aging, disease, and environmental factors. Environ. Res. 73, 242–248. Feng, J.D., Price, M., Cohen, J., Satinoff, E., 1989. Prostaglandin fevers in rats: regulated change in body temperature or change in regulated body temperature? Am. J. Physiol. 257, R695–R699. Furuyama, F., Ohara, K., 1993. Genetic development of an inbred rat strain with increased resistance adaptation to a hot environment. Am. J. Physiol. 265, R957–R962. Gordon, C.J., 1990. Thermal biology of the laboratory rat. Physiol. Behav. 47, 963–991. Gordon, C.J., 1993. Temperature Regulation in Laboratory Rodents. Cambridge University Press, New York. Gordon, C.J., 1994. 24-hour control of body temperature in the rat: I. Integration of behavioral and autonomic effectors. Am. J. Physiol. 267, R71–R77.
C.J. Gordon, A.H. Rezvani / Journal of Thermal Biology 26 (2001) 223–229 Gordon, C.J., Yang, Y., 1997. Contribution of spontaneous motor activity to the 24 hour control of body temperature in male and female rats. J. Thermal Biol. 22, 59–68. Hansen, C., Spuhler, K., 1984. Development of the National Institutes of Health genetically heterogeneous rat stock. Alcohol Clin. Exp. Res. 8, 477–479. IUPS, 1987. Glossary of Terms for Thermal Physiology. Revised by Commission for Thermal Physiology of the International Union of Physiological Sciences. Pflugers Archives, Vol. 410, pp. 567–587. Lane, M.A., Baer, D.J., Rumpler, W.V., Weindruch, R., Ingram, D.K., Tilmont, E.M., Cutler, R.G., Roth, G.S., 1996. Calorie restriction lowers body temperature in rhesus monkeys, consistent with a postulated antiaging mechanism in rodents. Proc. Natl. Acad. Sci. 93, 4159–4164.
229
Nielsen, M.K., Jones, L.D., Freking, B.A., DeShazer, J.A., 1997. Divergent selection for heat loss in mice: I. Selection applied and direct response through fifteen generations. J. Anim. Sci. 75, 1461–1468. Overstreet, D.H., Russell, R.W., Helps, S.C., Messenger, M., 1979. Selective breeding for sensitivity to the anticholinesterase DFP. Psychopharmacology 65, 15–20. Prosser, C.L., 1973. Temperature. In: Prosser, C.L. (Ed.), Comparative Animal Physiology. W.B. Saunders, Philadelphia, pp. 362–428. Wilson, H.R., Wilcox, C.J., Voitle, R.A., Baird, C.D., Dorminey, R.W., 1975. Characteristics of White Leghorn chickens selected for heat tolerance. Poult. Sci. 54, 126–130. Yang, Y., Gordon, C.J., 1996. Ambient temperature limits and accuracy of temperature regulation in unrestrained male and female rats. J. Thermal. Biol. 21, 353–363.
Genetic selection of rats with high and low body temperatures$ Christopher J. Gordon*, Amir H. Rezvani1, With the technical assistance of Beth Padnos and Peggy Becker Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA Received 27 August 2000; received in revised form 16 October 2000; accepted 11 November 2000
Abstract Body (core) temperature (Tc ) directly affects all biological processes, including sensitivity to toxic chemicals, development, aging, and drug metabolism. To understand how Tc affects these processes it is necessary to alter Tc independently of other physiological processes. The purpose of this study was to determine whether selective breeding techniques can be used to develop lines of rats with hyperthermic and hypothermic Tc ’s. Tc and motor activity of 24 female and 23 male rats (parental line) of the NIH heterogenous stock were monitored by telemetry for 96 h at a Ta of 228C. The mean 24 h Tc of the male and female rats was 37.38C with a range of 37–38.28C. Tc was not correlated with motor activity or body weight. Pairs with the lowest and highest Tc’s were selected for breeding. The F1 generation consisted of 10 offspring from the hyperthermic group and 20 from the hypothermic group. They were implanted with transmitters at 60 d of age. Tc of rats derived from the hyperthermic parental line had a significantly warmer Tc than the rats derived from the hypothermic parental line. Motor activity was significantly higher in the hyperthermic F1 males and hypothermic F1 females. Breeding of hyperthermic and hypothermic rats has shown that adult offspring of the fourth generation maintain significantly different core temperatures but have similar patterns of motor activity. The results demonstrate that Tc is heritable and that it should be feasible to develop lines of rats that regulate Tc above or below normal. Published by Elsevier Science Ltd. Keywords: Genetic selection; Body temperature; Hyperthermia; Hypothermia
1. Introduction Because of the direct effect of temperature on membrane potentials, chemical reactions, and enzyme
$ This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. *Corresponding author. Tel.: +1-919-541-1509, fax: +1-919541-4849. E-mail address: [email protected] (C.J. Gordon). 1 Current address: Duke University Medical Center, Department of Psychiatry, Durham, NC 27710, USA.
0306-4565/01/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 3 0 6 - 4 5 6 5 ( 0 0 ) 0 0 0 4 6 - 2
activities, body temperature has a direct effect on the function of all molecular, cellular, and organismic processes. There are innumerable studies showing that a change in body temperature will affect a variety of processes, including sensitivity to drugs and toxic chemicals, drug metabolism, development, growth, and aging (Prosser, 1973; Gordon, 1993). One particularly interesting possibility that this area of study has generated is that a lower body temperature (DT¼ 1:08C) will increase life span, possibly by reducing metabolism and the damaging effects of free radicals (Lane et al., 1996; Duffy et al., 1997). In order to study how temperature affects these processes it is essential to alter body (i.e., core) temperature independently of other physiological and
224
C.J. Gordon, A.H. Rezvani / Journal of Thermal Biology 26 (2001) 223–229
behavioral processes. However, to the best of our knowledge, achieving a chronic change in core temperature that is essentially independent of other physiological processes has not been achieved. Body temperature of a homeotherm can be changed with relative ease by altering heat production and/or heat loss. Techniques such as exposure to heat or cold stress, exercise, administration of drugs that alter thermoregulation, lesions in CNS thermoregulatory centers, and surgically implanted heat exchangers have been used in innumerable studies to alter core temperature. However, regardless of the method used, the change in body temperature is accompanied by physiological and behavioral changes that are likely to be unrelated to the change in body temperature. In most instances, body temperature is forced above or below the set-point and physiological responses are activated to counter the change in temperature. Hence, body temperature was essentially a dependent variable in the past studies. To understand how body temperature affects cellular and organismic processes, it is essential to develop an animal model with a permanent change in their regulated core temperature (i.e., independently change body temperature without altering thermoeffector activity). To this end, we have explored the use of selective breeding to develop genetic lines of rats that regulate their core temperature above or below normal. Genetic selection has been used to alter a variety of thermoregulatory related processes such as increased survival to acute heat stress (Furuyama and Ohara, 1993), altered resistance to the hypothermic effects of ethanol (Crabbe et al., 1987) and the anticholinesterase agent DFP (Overstreet et al., 1979), and a high or low rate of heat loss (Nielsen et al., 1997). However, to the best of our knowledge, there has been no successful attempt to develop lines of animals with permanently different body temperatures. A fundamental premise of our hypothesis is that there is an inherent differences in body temperature within a species with a normal distribution that is attributed to genotypic differences in the set-point or regulated body temperature. The purpose of this study was to test the hypothesis that individual differences in the level at which deep body temperature is regulated is genetically determined, meaning that a high or low temperature can be passed on to successive generations with selective breeding in the rat.
2. Materials and methods Animals used in this study were from the heterogenous stock developed and maintained at the National Institutes of Health (N:NIH strain). Inbred and outbred rats from conventional animal suppliers could lack thermoregulatory traits because of inbreeding and genetic isolation. The NIH heterogenous strain is
considered to be ideal for selective breeding studies (Hansen and Spuhler, 1984). The rats were obtained at approximately 21 d of age and maintained at a Ta of 228C, a relative humidity of 50%, and a 12:12 light:dark photoperiod. Core temperature and motor activity were monitored in undisturbed rats using radiotelemetry (Data Sciences International, St. Paul, MN). Details of the telemetry system have been published (Gordon, 1994). Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg; IP) and an abdominal incision was made for the implantation of the transmitter (TA10TA-F40) into the abdominal cavity. The abdominal muscle was sutured and the skin was closed with wound clips. Following surgery, rats were administered a penicillin antibiotic (30,000 units; i.m.) and analgesic (buprenorphine; 0.03 mg/kg; SC). The rats were allowed at least 10 d of recovery before testing. Core temperature and motor activity were monitored at 5 min intervals by receivers placed under the floor of each cage. The rat’s motor activity was measured from the change in position of the transmitter in relation to the antennae in the receiver board. This technique does not give a quantitative measurement of activity. Instead, the technique provides a relative measure of the rat’s activity in dimensions of counts/10 min. The telemetry variables were monitored for 96 h continuously in 90 d-old male (N=23) and female (N=24) rats (parental line) while they were individually housed in the animal facility. The telemetry system was capable of monitoring 24 animals simultaneously. Thus, two cohorts of males and females were measured in separate experiments. Care was taken to not disturb the rats in any way during the monitoring period.
2.1. Data analysis The 96 h of data were ensembled for each rat into a single 24 h cycle. It was decided that 96 h of recording would be sufficient to dampen or eliminate changes in body temperature that would occur from extrinsic (e.g., ambient noise) and intrinsic sources (e.g., infradian oscillations, estrous cycle, sleep–wake patterns, etc.). The mean and standard deviation (SD) of core temperature and motor activity over the 96 h period was calculated for each rat. It was intended to isolate rats whose core temperature was above or below one SD of the mean as based on the definition of hypothermia and hyperthermia (IUPS, 1987). The temperature responses were further evaluated for any unusual characteristics of the nychthemeral temperature rhythm and patterns of motor activity. It was intended to select rats from the parental lines that were hyperthermic or hypothermic but did not have unusually high or low levels of motor activity, abnormal differences in
C.J. Gordon, A.H. Rezvani / Journal of Thermal Biology 26 (2001) 223–229
amplitude or timing of the nychthemeral temperature rhythm, or marked deviations in body weight. Two pairs of rats with core temperatures above and below the mean population were isolated and placed together for breeding. The offspring were weaned at 21 d of age. There was no culling of litters. At 60 d of age the offspring (F1) were implanted with transmitters and their core temperature and motor activity patterns were recorded in the same manner as described above.
3. Results There was a near normal frequency distribution of core temperature in male and female rats (Fig. 1). The mean core temperature of male and female rats was 37.32 and 37.368C, respectively. Core temperature of females ranged from 37.05 to 37.558C; for males, the range was 37.1–38.18C. The outliers with core temperatures greater or less than 1.0 SD from the mean were
Fig. 1. Frequency distribution of mean 24 h core temperatures in male and female rats monitored undisturbed for 96 h. Noted outliers (arrows) were used for breeding hyperthermic and hypothermic lines.
225
selected for the hyperthermic and hypothermic parental lines, respectively. There was one male that had a relatively high core temperature of 38.18C, which was more than 3 SD’s above the mean. There was no relationship between the 24 h mean of motor activity and core temperature in the parental line (Fig. 2). In addition, body weight was not related to core temperature (data not shown). The four breeding pairs from the parental line gave birth to 22 pups from the hyperthermic line and 21 pups from the hypothermic line. There was substantial mortality in the hyperthermic line with 10 pups surviving through weaning whereas 18 hypothermic pups survived through weaning. These deaths occurred within one week of parturition. When tested as adults, core temperature of the F1 rats derived from the hyperthermic parental line was significantly warmer than that of the rats derived from the hypothermic parental line (Fig. 3). Mean core temperature of hyperthermic males and females was 0.20 and 0.298C warmer than the hypothermic line in the daytime and 0.13 and 0.198C warmer at night. Motor activity of hypothermic females was higher than males, whereas activity of hypothermic males was lower than
Fig. 2. Relationship between mean motor activity and core temperature of parental males and females. Data fit with linear regression. Slopes of regression lines for male and female rats not different from zero.
226
C.J. Gordon, A.H. Rezvani / Journal of Thermal Biology 26 (2001) 223–229
that observed in the hyperthermic males. The differences in motor activity were more distinguishable during the dark phase (Fig. 4). The mean core temperature of hyperthermic offspring was cooler than that of the parental line. Similarly, the core temperature of hypothermic offspring was warmer than that of the parental line (Table 1). Body weight of F1 at adulthood was significantly greater in animals derived from the hyperthermic line: 336 13 g for hyperthermic males;
300 10 g for hypothermic males; 205 6 g for hyperthermic females; and 184 4 g for hypothermic females. 3.1. F4 Generation Continued selective breeding of hyperthermic and hypothermic rats through four generations to this point has shown that adult offspring maintain significantly different core temperatures but similar patterns of motor activity (Fig. 5). Overall, the 24 h core temperature of the F4 hyperthermic line was 0.238C warmer than the hypothermic line in males and 0.218C in females. Similar to the F1 generation, body weights of both sexes of the hyperthermic F4 generation were greater than in the hypothermic animals (39 and 20 g heavier in males and females).
4. Discussion
Fig. 3. 24 h core temperature of hyperthermic and hypothermic parents (P line) used for generating F1 of hyperthermic and hypothermic lines. Data are ensembled means of 96 h of continuous data.
The key observation of this study is the demonstration that small individual variations in the level at which body temperature is regulated is heritable in the rat. Selective breeding of rats with higher than normal core temperature had offspring with significantly warmer core temperatures compared to selective breeding of rats
Fig. 4. Time-course of core temperature and motor activity of F1 hyperthermic and hypothermic lines. Data ensembled from 96 h recordings as in Fig. 3. Repeated ANOVA: Core temperature; male; treatment, F(25, 1)=21.7, p50.0001, treatment-time, F(23, 552)=3.0, p50.0001; female, treatment, F(29, 1)=34, p50.0001; treatment-time, F(23, 644)=6.5, p50.0001; motor activity, male, treatment, F(25.1)=14. P=0.001; treatment-time, F(23, 552)=9.3, p50.0001; female, treatment, F(29, 1)=11, p=0.003; treatment-time, F(2, 644), p50.0001. Numbers in parentheses indicate sample size.
C.J. Gordon, A.H. Rezvani / Journal of Thermal Biology 26 (2001) 223–229
with colder than normal core temperatures. The altered core temperature in the offspring was maintained throughout most of the light and dark phase of a 24 h period. There were significant differences in motor activity between the hypothermic and hyperthermic offspring of F1; however, these differences are probably not the reason behind the differences in core temperatures. Motor activity was higher in the hyperthermic males but lower in the hyperthermic females. Like the parental line, there was no correlation between motor activity and core temperature in the F1 generation. Overall, spontaneous activity has minor effects on resting core temperature of the rat (Gordon and Yang, 1997). Thus, the core temperatures in the hyperthermic and hypothermic lines are likely to be a result of innate differences not related to the heat produced from motor activity. In addition, the separation of core temperature
Table 1 Mean S.E. of core temperature in parental line selected for breeding and in F1 generation Group/sex
Parental line, Tc (8C)
F1, Tc (8C)
Hyperthermic/male Hyperthermic/female Hypothermic/male Hypothermic/female
37.8 0.3 37.7 0.1 37.1 0.004 37.1 0.08
37.4 0.05 37.5 0.02 37.2 0.03 37.3 0.04
227
in the F4 hyperthermic and hypothermic lines but with similar patterns of motor activity is further support that the differences in core temperature are not a result of differences in motor activity. It is essential to use radiotelemetry to monitor core temperature in animals to be used for developing lines of hyperthermic and hypothermic rats. A single measurement of core temperature can be affected by numerous variables, including stress of handling, ultradian oscillations, and estrous cycle (Briese and de Quijada, 1970; Gordon, 1990, 1993). In addition, single or multiple measurements of core temperature with a colonic probe may not necessarily reflect the animal’s core temperature over a 24 h period. Stress from this technique can lead to abnormal measurements of core temperature. Indeed, the presence of personnel in the animal facility can lead to artifactual changes in core temperature (Briese and de Quijada, 1970). On the other hand, telemetric monitoring gives the best estimate of resting core temperature that should reflect the core temperature regulated by thermoregulatory centers in the CNS. By monitoring for 96 h, there should be an adequate number of samples to eliminate the extrinsic and intrinsic factors that would transiently alter the set-point temperature. Since the estrous cycle of the rat is 4–5 d, the 96 h period should also take into account the slight rise in core temperature (0.28C) of the female rat during the night of proestrus (Yang and Gordon, 1996). There have been considerable success in artificial selection of heat-tolerant lines of birds and mammals
Fig. 5. Time-course of core temperature and motor activity of F4 hyperthermic and hypothermic lines. Data ensembled from 96 h recordings. Repeated ANOVA: Core temperature; male; treatment, F(1, 14)=87.9, p50.0001, treatment-time, F(23, 322)=1.9, p=0.006; female, treatment, F(1, 10)=27, p=0.0004; treatment-time, F(23, 230)=2.2, p=0.002; motor activity, male, treatment (NS), treatment-time, (NS) female, treatment, (NS) treatment-time, (NS). Numbers in parentheses indicate sample size.
228
C.J. Gordon, A.H. Rezvani / Journal of Thermal Biology 26 (2001) 223–229
(Furuyama and Ohara, 1993; Wilson et al., 1975). In these studies, animals with improved heat tolerance were selectively bred and the resulting generations showed a marked improvement in their tolerance to acute heat stress. Baseline core temperature was not a factor in the selection process. However, the artificial selection for heat tolerance did lead to changes in the baseline core temperature when the animals were challenged with other stimuli such as cold stress and thermoregulatory drugs (Furuyama and Ohara, 1993). Little is known of the extent to which individual variations in body temperature are heritable. Connolly and Lynch (1981) estimated the heritability of individual variations in the body temperature of mice by comparing the day and night core temperatures in four strains. They concluded that heritability was very low for daytime body temperature but relatively high during the night when motor activity and body temperature are elevated. In other words, body temperature at night may be subject to more selective pressure than during the day because it is associated with expression of complex behaviors (Connolly and Lynch, 1981). We endeavored to artificially select rats with altered day time and night time temperatures because selecting for only a high or low night time temperature would also result in a change in the amplitude of the nychthemeral pattern of core temperature. Moreover, raising or lowering the diurnal temperature of a nocturnal rodent allows one to study pyrogens such as lipopolysaccharide that are more effective to elevate core temperature in the day time as compared to the night (Feng et al., 1989). We expect heritability of individual variations in core temperature of the rat to be relatively small. The core temperature of the hyperthermic F1’s was 0.2–0.48C below that of the parents while the core temperature of the hypothermic F1’s was 0.1–0.28C above that of their parents (Table 1). After four generations we have found the separation of mean 24 h core temperature to have increased by just 0.18C over that of the first generation (cf. Fig. 5). Hence, we expect a slow separation in the mean core temperature of hyperthermic and hypothermic lines with each generation compared to the relative success of selection of other thermoregulatory parameters (Wilson et al., 1975; Furuyama and Ohara, 1993; Nielsen et al., 1997). What are the physiological mechanisms responsible for the differences in core temperature between the hyperthermic and hypothermic lines? We tested the behavioral thermoregulatory responses of a selected group of hyperthermic and hypothermic rats that had a core temperature difference of 0.58C but found essentially no significant difference in selected ambient temperature (Gordon, unpublished observations). In fact, rats of the hypothermic line selected slightly cooler ambient temperatures than that of the hyperthermic line. All together, these preliminary studies suggest that the
selection of the hyperthermic and hypothermic lines did not arise from a thermoregulatory dysfunction such as an over- or underactive thyroid gland. If this was the case, we would have expected to see marked behavioral thermoregulatory responses.
5. Future perspectives Some outstanding issues also need to be resolved in future studies: (1) How high or low a core temperature can be developed with selective breeding. Are there internal limits to the genetic selection of hyperthermic and hypothermic rats? (2) Does the high or low body temperature selected in adults persist throughout the life of the organism? (3) How early in life can differences in core temperature be detected? (4) Will the differences in core temperature be maintained when the animal is housed at warm or cold ambient temperatures? These questions will be addressed in future investigations.
Acknowledgements We thank Dr. Carl Hansen of NIH for his generosity in supplying us with rats of the NIH:N strain.
References Briese, E., de Quijada, M.G., 1970. Colonic temperature of rats during handling. Acta Physiol. Lat. Am. 20, 97–102. Connolly, M.S., Lynch, C.B., 1981. Circadian variation of strain differences in body temperature and activity in mice. Physiol. Behav. 27, 1045–1049. Crabbe, J.C., Kosobud, A., Tam, B.R., Young, E.R., Deutsch, C.M., 1987. Genetic selection of mouse lines sensitive (cold) and resistant (hot) to acute ethanol hypothermia. Alcohol Drug Res. 7, 163–174. Duffy, P.H., Leakey, J.E., Pipkin, J.L., Turturro, A., Hart, R.W., 1997. The physiologic, neurologic, and behavioral effects of caloric restriction related to aging, disease, and environmental factors. Environ. Res. 73, 242–248. Feng, J.D., Price, M., Cohen, J., Satinoff, E., 1989. Prostaglandin fevers in rats: regulated change in body temperature or change in regulated body temperature? Am. J. Physiol. 257, R695–R699. Furuyama, F., Ohara, K., 1993. Genetic development of an inbred rat strain with increased resistance adaptation to a hot environment. Am. J. Physiol. 265, R957–R962. Gordon, C.J., 1990. Thermal biology of the laboratory rat. Physiol. Behav. 47, 963–991. Gordon, C.J., 1993. Temperature Regulation in Laboratory Rodents. Cambridge University Press, New York. Gordon, C.J., 1994. 24-hour control of body temperature in the rat: I. Integration of behavioral and autonomic effectors. Am. J. Physiol. 267, R71–R77.
C.J. Gordon, A.H. Rezvani / Journal of Thermal Biology 26 (2001) 223–229 Gordon, C.J., Yang, Y., 1997. Contribution of spontaneous motor activity to the 24 hour control of body temperature in male and female rats. J. Thermal Biol. 22, 59–68. Hansen, C., Spuhler, K., 1984. Development of the National Institutes of Health genetically heterogeneous rat stock. Alcohol Clin. Exp. Res. 8, 477–479. IUPS, 1987. Glossary of Terms for Thermal Physiology. Revised by Commission for Thermal Physiology of the International Union of Physiological Sciences. Pflugers Archives, Vol. 410, pp. 567–587. Lane, M.A., Baer, D.J., Rumpler, W.V., Weindruch, R., Ingram, D.K., Tilmont, E.M., Cutler, R.G., Roth, G.S., 1996. Calorie restriction lowers body temperature in rhesus monkeys, consistent with a postulated antiaging mechanism in rodents. Proc. Natl. Acad. Sci. 93, 4159–4164.
229
Nielsen, M.K., Jones, L.D., Freking, B.A., DeShazer, J.A., 1997. Divergent selection for heat loss in mice: I. Selection applied and direct response through fifteen generations. J. Anim. Sci. 75, 1461–1468. Overstreet, D.H., Russell, R.W., Helps, S.C., Messenger, M., 1979. Selective breeding for sensitivity to the anticholinesterase DFP. Psychopharmacology 65, 15–20. Prosser, C.L., 1973. Temperature. In: Prosser, C.L. (Ed.), Comparative Animal Physiology. W.B. Saunders, Philadelphia, pp. 362–428. Wilson, H.R., Wilcox, C.J., Voitle, R.A., Baird, C.D., Dorminey, R.W., 1975. Characteristics of White Leghorn chickens selected for heat tolerance. Poult. Sci. 54, 126–130. Yang, Y., Gordon, C.J., 1996. Ambient temperature limits and accuracy of temperature regulation in unrestrained male and female rats. J. Thermal. Biol. 21, 353–363.