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Retention of Carbon Dioxide during Entrance into Torpor in Dormice

RALF ELVERT1 AND GERHARD HELDMAIER1 Abstract. Metabolic rate (MR), heart rate (HR) and body temperature (Tb) of two edible dormice (Glis glis) were studied during entrance into torpor at two temperature ranges, Ta= 0 to 5°C and 15 to 20°C. MR was reduced rapidly from 275 to 84 ml O2*h-1 within the first hour after onset into torpor at the low temperature range and from 154.5 to 82.2 ml O2*h-1 at the high temperature range. HR decrease paralleled the reduction of MR. Between 0-5°C the HR decreased rapidly from 388 to 171 BPM within the first hour and from 284 to 193 BPM in the range of 15-20°C. After the onset of metabolic reduction Tb decreased within one hour from 36.9°C to 30.8°C at the low temperature range (cooling rate 6.1°C*h-1). At the high temperature range cooling rate within the first hour was at 2.4°C*h-1 with a Tb decrease from 37.2°C to 34.8°C. A decrease of respiratory quotient (RQ) appeared closely correlated to the reduction of Tb at Ta below 5°C. The RQ decreased to 0.25 indicating a retention of CO2. In the temperature range of 15 to 20°C no reduction of RQ could be observed during entry into torpor. The RQ even rose slightly from 0.7 – 0.8 to about 1.0. We suggest that the depression of metabolism and heart activity is an active downregulation, but the decrease of RQ at low Tb point out a temperature dependent relationship caused by cooling of body fluids.

Introduction The entrance into torpor by Djungarian hamsters has been shown to be always initiated by a suppression of metabolic rate (MR) (Heldmaier and Ruf 1992). The authors showed that body temperature (Tb) decrease is caused by the deficit of heat production, or more precisely, of active metabolic suppression (Heldmaier et al 1999). This observation was also made in other species entering torpor or hibernation (Lyman 1958, Wang 1978, Cranford 1983, Nestler 1990, Heldmaier et al 1993, Wilz and Heldmaier 2000). In woodchucks entering hibernation it was observed that heart rate (HR) decreased simultaneously with MR and both reached their minimal values several hours before Tb had approached the low hibernation level (Lyman, 1958). Milsom et al (1999) described that the reduction of HR paralleled the reduction of MR in all hibernators and anticipated the decline in Tb during entrance into hibernation. The authors concluded that the changes in HR and cardiac output mirror the changes in MR. The initial fall in HR is considered as a result of parasympathetic activation and vagal slowing of the heart (Lyman 1982). 1

Dept. of Biology, Philipps-University, Karl von Frisch Str., 35032 Marburg, Germany Life in the Cold G. Heldmaier and M. Klingenspor (eds.) © Springer-Verlag Heidelberg 2000

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R. Elvert and G. Heldmaier

Several investigators have shown that the entrance into torpor is further accompanied by a decrease in the respiratory exchange rate (RQ) (Snapp and Heller 1981, Bickler 1984, Malan 1986, Nestler 1990, Milsom 1993). The reduction could be caused by a retention of CO2 in body fluids instead of its elimination during ventilation (Malan 1982, 1986, 1988, Bharma and Milsom 1993). The CO2 content in arterial blood and other tissues is raised (Malan et al 1973). This is accompanied by an increase in bicarbonate concentration during entry into hibernation or daily torpor (Withers 1977). A retention of CO2 could reflect the greater solubility of CO2 in body fluids at low temperature, as well as contributing to the pH control in hibernation. To analyze the effect of temperature on CO2 balance we measured HR, Tb, O2 consumption and CO2 production simultaneously in edible dormice (Glis glis) and compared the entrance into torpor at high ambient temperature (Ta) 15-20°C and low Ta (0-5°C).

Methods MR, HR and Tb were continuously recorded in two edible dormice (Glis glis) from April 1998 to December 1999. The dormice were housed in wire mesh cages (80 x 50 x 42 cm) inside a climate chamber with food and water ad libitum. For monitoring metabolic and cardiorespiratory changes into torpor food was removed. Ta was maintained between 0 and 5°C or alternatively between 15 and 20°C. Humidity was maintained constant at 80 %. Through a revolving door the dormice had free access to a sleep and nesting box placed outside the cage. HR and Tb data was recorded by a modified physiological implantation system (Data Sciences, DSI, St. Paul, USA) using a temperature sensitive transmitter with biopotential option (ETA-F20) as described elsewhere (Elvert and Heldmaier 2000). Ta was measured with a thermocouple placed inside the nesting box. Data of HR, Tb and Ta were stored on a computer. Oxygen consumption and carbon dioxide production were recorded by pumping air through the sleeping box with a flow rate of 30 to 35 l*h-1. O2 and CO2 content was measured by an O2-analyzer (Ametek S 3a/II, Pittsburgh, USA) and a CO2analyzer (UNOR 6N Maihak, Hamburg, FRG) as described in Wilz and Heldmaier (2000), and data were stored on a second computer. A magnetic valve system allowed switching to reference air every 55 min for automated zero readjustment and calibration checks for 5 min. HR, MR, RQ, Tb and Ta were measured continuously and stored in time intervals of 1 min.

Carbon Dioxide Retention

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Fig. 1. Individual records of entry into torpor at low ambient temperature (#R4S, Ta=4.7°C, top) and at high ambient temperature (#R13G, Ta=19.4°C, bottom). The courses of MR, HR, Tb and RQ are shown. Dormice were exposed to a LD regime of 10:14 with light on at 8am. Tb body temperature, MR metabolic rate, RQ respiratory quotient, HR heart rate.

Results Metabolic rate Individual dormice showed entries into torpor at low and at high Ta´s (Fig. 1 and 2). The entrance into torpor was always indicated by a final peak of MR followed by rapid decline (Fig. 1). Several entries of two individuals into torpor were summarized and 10min mean values were calculated to smooth short term fluctuations in MR, HR, Tb and RQ during entrance into torpor (Fig. 2, N=2, n=7).

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time (h) Fig. 2. 10min mean values of metabolic rate (MR), heart rate (HR), body temperature (Tb) and the respiratory quotient (RQ) during entry into torpor of two dormice at low Ta (0-5°C) with N=2, n=7 and at high Ta (15-20°C) with N=2, n=8.

10min mean values of VO2 within the low temperature range of 0-5°C showed a rapid decrease from 275 ml O2*h-1 during normothermia to 84 ml O2*h-1 within the first hour and reached 40.6 ml O2*h-1 after two hours entry into torpor. Within the high temperature range of 15 to 20°C dormice also entered torpor frequently (Fig. 1 and 2). In this case 10min mean value of MR during normothermia was 154.5 ml O2*h-1 and was reduced to 82.2 ml O2*h-1 within one hour and reached 41.5 ml O2*h-1 after two hours (Fig. 2, N=2, n=8).


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Heart rate The onset of torpor was indicated by a final peak of HR in both temperature ranges, as observed in MR. Also the reduction in HR parallels the reduction of MR (Fig. 1 and 2). At Ta= 0-5°C the HR of normothermic dormice was about 388 BPM and was reduced after one hour to 171 BPM. After two hours HR was about 91 BPM (Fig. 2, N=2, n=7). At Ta=15-20°C we observed a HR of 284 BPM during normothermia. It was reduced to 193 BPM after one hour and to 108 BPM after two hours (Fig. 2, N=2, n=8). Body temperature The decrease of Tb followed the reduction of MR and HR, but decreased with a much slower rate (Fig. 1 and 2). At the low temperature range mean normothermic Tb was 36.9°C. 10min mean values declined to 30.8°C after one hour, resulting in a cooling rate of 6.1°C*h-1 after onset into torpor. After two hours Tb was 23°C with a cooling rate of 7.8°C*h-1 (Fig. 2, N=2, n=7). At the temperature range of 15 to 20°C the mean Tb of normothermic dormice was 37.2°C. Within one hour 10min mean values of Tb decreased to 34.8°C, resulting in a cooling rate of 2.4°C*h-1 after onset into torpor. Tb reached 30.9°C after two hours, revealing a cooling rate of 3.9°C*h-1 (Fig. 2, N=2, n=8). Respiratory exchange rate (RQ): The mean RQ of normothermic dormouse was 0.7 at Ta= 0 to 5°C and decreased during entry into torpor. Its time course closely paralleled the decrease of Tb (Fig. 1 and 2). A minimum of 10min mean values of RQ = 0.25 was reached about 9.5 hours after the beginning of metabolic depression (Fig. 2, N=2, n=7). At Ta=15-20°C the mean RQ during normothermia was at 0.77 (Fig. 1 and 2). During entrance into torpor no reduction of RQ was observed (Fig. 1 and 2). The RQ varied between of 0.75 to 1.0, in spite of the reduction of metabolism and HR and the decrease of Tb (Fig. 2, N=2, n=8).

Discussion Our results revealed that entrance into torpor at low Ta, and thus low Tb, is accompanied by a decrease in RQ, whereas high levels of RQ were maintained when torpor occurred at high Ta and Tb. The reduction of RQ during torpor in the cold was closely correlated with the reduction of Tb but not with the reduction of HR (Fig. 2). Bickler (1984) and Malan (1988) observed that the entrance into daily torpor or hibernation is accompanied by a falling respiratory exchange rate and suggested that this contributes to a CO2 retention. Malan (1986) noticed that a decrease in

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[HCO3-] PCO2 pH

normothermia Tb= 37°C

hibernation Tb= 5-6°C

Ref.

16 meq*l-1 38.5 Torr 7.24

34 meq*l-1 27.4 Torr 7.44

1

Estimated torpor values of present study, Ta=0-5°C, N=2, n=7 41.7 meq*l-1

1 1

7.42 - 7.56

Table 1. α(37°C) = 0.0301 mmol*l-1*Torr-1, α(5-6°C) = 0.073 mmol*l-1*Torr-1 (2, 3), pK´(37°C) = 6.1, pK´(5-6°C) = 6.25 (2). Blood gas values obtained from the literature: 1Kreienbühl et al, 1976; 2Kent and Peirce II, 1967; 3Malan, 1982.

RQ does not reflect a change in substrate utilization, but only a temporary imbalance between production and elimination of CO2. Inhibitory effects of CO2retention and respiratory acidosis on thermoregulatory structures, glycolysis, neural activity and brown fat thermogenesis have already been demonstrated (Malan et al 1973, 1985, 1988, Malan et al 1988). At the beginning of entries, significant drops in RQ have been oberved in Spermophilus lateralis and S. tereticaudus (Snapp and Heller 1981, Bickler 1984). Nestler (1990) reported in deer mice (Peromyscus maniculatus) that several hours prior to torpor entrance, RQ began to decline slightly until reaching 0.74. A further decrease to 0.63 occurred simultaneously with a rapid drop in MR, lasting only 9-12min. Our measurements in dormice showed no change in RQ prior to the entrance into torpor but a continuous decrease in RQ with Tb lasting several hours. During normothermia and under food restriction the RQ was about 0.7 indicating a preferential combustion of lipids. If we assume that the combustion of lipids continues during torpor the RQ should remain unchanged. Instead we observed a decrease of RQ in the cold indicating a retention of CO2. During the first 9.5 hours this calculates a retention of 2.34mmol (Fig. 2, N=2, n=7). If we assume that the amount of stored CO2 is converted into bicarbonate and evenly distributed in body water (70% of body mass = 91ml) an increase of 25.7meq*l-1 of bicarbonate concentration could be calculated (N=2, n=7, mean body weight 130g). This increase would raise the total bicarbonate concentration from 16meq*l-1 to 41.7meq*l-1 (Kreienbühl et al 1976, Table 1). Kreienbühl et al (1976) calculated a bicarbonate concentration for dormice of 16meq*l-1 at 37°C and 34meq*l-1 at 6°C. The authors concluded that the actual bicarbonate concentration is not held constant in hibernating mammals. While the solubility of CO2 increases with falling temperature (Kreienbühl et al 1976) the dissociation constant of carbonic acid also increases (Kent and Peirce 1967, Reeves 1976). Studies of blood in closed system conditions have shown that when the temperature of the blood decreased, its pH increased and PCO2 decreased (Musacchia and Volkert 1971, Kreienbühl et al 1976, Rodeau and Malan 1979, Malan 1982). Keeping PCO2 and pH almost constant would require additional storage of large quantities of CO2 (Malan 1982). Hibernating squirrels (Citellus tridecemlineatus) are able to maintain acid-base balance similar to that in normothermic animals (Musacchia and Volkert 1971). They succeeded by increasing total CO2 content in body fluids from 26meq*l-1 at 37°C to 38meq*l-1 at 5°C (Kent and Peirce 1967). Squirrels maintain essentially


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the same arterial pH in normothermic state as in the hibernating state: 7.39 (37°C) and 7.39 (5°C) (Kent and Peirce 1967) and 7.40 (37°C) and 7.44 (6°C) (Musacchia and Volkert 1971). Some authors have found no change in pH, but other data in the literature showed a slight increase in pH during hibernation (see Kreienbühl et al 1976 for overview). Edible dormice showed a retention of CO2 and an increase of bicarbonate content (see Table 1). If we use Kreienbühls´ data for PCO2 and calculated amount of total bicarbonate content, we can estimate a pH of 7.42 following the Henderson-Hasselbalch equation. Assuming that the PCO2 decreased during hibernation to 27.4 Torr a further increase in pH to 7.56 can be calculated (Table 1). Since the neutral pH will increase with decreasing temperature, this represents a relative respiratory acidosis (Bharma and Milsom 1993). Our data confirm previous findings about change of acid-base state during entrance into torpor, but no definitive conclusion about the regulation of acid-base state can be reached from the change in bicarbonate concentration at low Ta.

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