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. 2003 Jun 18;551(Pt 2):713–720. doi: 10.1113/jphysiol.2003.040592

Attenuation of metabolic heat production and cold-escape/warm-seeking behaviour during a cold exposure following systemic salt loading in rats

Masahiro Konishi 1, Kei Nagashima 1, Kento Asano 1, Kazuyuki Kanosue 1
PMCID: PMC2343232  PMID: 12815190

Abstract

The reduction of body core temperature (Tcore) after salt loading has been reported. In this study, we tested the hypothesis that, during a cold exposure in rats, (1) salt loading would decrease metabolic rate (MR), reducing Tcore, but (2) Tcore would be maintained when cold-escape/warm-seeking behaviour is available. In the first experiment (n = 7), MR and Tcore were measured by indirect calorimetry and telemetry, respectively, during 26, 20 and 10 °C exposure for 1 h each, in that order. In the second experiment (n = 7), each rat was placed in an operant system during the same exposure protocol as in the first experiment, where it could trigger a 40 °C air reward for 30 s at 20 and 10 °C by moving into specific areas (operant behaviour). In each experiment, rats repeated the same protocol twice with a subcutaneous injection (10 ml kg−1) of either isotonic saline (154 mm) or hypertonic saline (2500 mm). In the first experiment, MR in the isotonic-saline trial increased (P < 0.05) at 20 and 10 °C compared with that at 26 °C by 21 ± 5 and 48 ± 6 %, respectively (means ± s.e.m.), with Tcore unchanged. However, values for MR and Tcore in the hypertonic-saline trial were lower (P < 0.05) than those in the isotonic-saline trial in any ambient temperature. In the second experiment, Tcore was also lower (P < 0.05) in the hypertonic-saline trial than in the isotonic-saline trial. The counts of the operant behaviour in the hypertonic-saline trial remained unchanged in each exposure period, but those in the isotonic-saline trial increased (P < 0.05) at 10 °C. These results may suggest that salt loading attenuates both metabolic and behavioural thermoregulatory responses to the cold.

In homeothermic animals, body core temperature (Tcore) is regulated by both autonomic and behavioural processes. However, it is well known that during a heat exposure in dehydrated animals both processes are modulated by an increase in plasma solute concentration, e.g. Na+, and/or a decrease in blood volume. The two factors attenuate both evaporative and non-evaporative heat loss mechanisms, e.g. saliva secretion and tail blood flow in rats, and panting and skin blood flow in dogs (Baker & Doris, 1982; Horowitz & Nadel, 1984; Baker & Dawson, 1985; Horowitz & Meiri, 1985; Nakajima et al. 1998). This attenuation of heat loss mechanisms would be important in preserving body fluid and preventing an excessive blood distribution to the periphery; however animals may lose their body temperature homeostasis. In contrast, it was reported that operant heat-escape/cold-seeking behaviour increased after subcutaneous (S.C.) hypertonic-saline injection in rats (Nagashima et al. 2001; Konishi et al. 2002) and pigeons (Brummermann & Rautenberg, 1989). These results may indicate that behavioural thermoregulatory processes are activated by an increase in plasma solutes, i.e. elevation of plasma osmolality and/or Na+ concentration, compensating the attenuation of the autonomic heat loss mechanisms.

Dehydration has been reported to decrease the resting metabolic rate (MR) in rats (Horowitz & Samueloff, 1989) and camels (Schroter et al. 1987). We also reported that, in rats, Tcore decreased by 0.9 °C after S.C. hypertonic-saline injection in a thermoneutral condition at 26 °C (Nagashima et al. 2001; Konishi et al. 2002). These responses may prevent Tcore from reaching a critical level in a hot environment. However, animals also become dehydrated in a cold environment due to cold-induced diuresis, blunted thirst, increased respiratory water loss, or low availability of food and water (Fregly, 1982; Allen & Gellai, 1993). Therefore, the reductions of MR and Tcore might be critical for animals during a cold exposure. However, it remains uncertain whether dehydration and/or salt loading (systemic hypertonic-saline injection) also have influences on autonomic and behavioural thermoregulatory responses to the cold.

In the present study, we evaluated the effects of S.C. hypertonic-saline injection on metabolic heat production and cold-escape/warm-seeking behaviour during consecutive 20 and 10 °C cold exposure in rats. The behavioural response was assessed using the operant behaviour system that our laboratory has previously reported (Chen et al. 1998). We hypothesized that, after the hypertonic-saline injection, (1) MR during the cold exposure would be attenuated; and (2) Tcore would decrease; however (3) when the operant cold-escape/warm-seeking behaviour was available, the rats would be able to maintain Tcore by increasing the behaviour.

METHODS

Fourteen male crj-Wistar rats (320 ± 5 g; Charles River Japan, Osaka, Japan) were used in this study. Rats were individually housed at an ambient temperature (Ta) of 23.0 ± 0.5 °C in a 12:12 h light:dark cycle (lights on at 08.00 h) and had free access to food and water. All procedures used conformed with the UK Animals (Scientific Procedures) Act 1986 and the Institutional Animal Care and Use Committee, School of Allied Health Sciences, Osaka University Faculty of Medicine.

Surgical preparations

Under general anaesthesia induced by an intraperitoneal (I.P.) injection of sodium pentobarbital (nembutal, 50 mg kg−1; Dainippon, Osaka, Japan), a radio transmitter (15 mm × 30 mm × 8 mm; Physiotel, Data Science, St Paul, USA) for the measurement of Tcore was placed in the abdominal cavity of each rat. A silicone catheter (1.0 mm o.d.; Fuji Systems, Tokyo, Japan) for blood sampling was placed in the inferior vena cava through the right femoral vein. The other end of the catheter was exited at the nape and plugged with a stainless steel rod. Lignocaine jelly (Xylocaine jelly, AstraZeneca, Osaka, Japan) was applied to the area of closed incisions to minimise post-surgical discomfort. The catheter was flushed with heparinized saline (50 u ml−1) every day to avoid clogging. The rats were allowed to recover from the surgery for at least 2 weeks before experiments.

In the present study, two different experiments were conducted to evaluate metabolic heat production (experiment 1) and cold-escape/warm-seeking behaviour (experiment 2). Two groups of rats (n = 7 for each group) were used for the experiments. After all the experiments were finished, rats were killed by I.P. injection of sodium pentobarbital (200 mg kg−1).

Experiment 1. Metabolic heat production during cold exposure

A Plexiglas metabolic box (18 cm × 18 cm × 18 cm) with a metal mesh stage in the bottom (height, 1 cm) was placed in an environmental chamber (80 cm × 65 cm × 60 cm; CAU-210, Tabai Espec, Osaka, Japan). The metabolic box had air-inlets (15 holes of 5 mm diameter on its side walls) and an outlet (10 mm diameter), and was ventilated at a constant flow rate of 400 ml min−1. The air from the outlet was collected via tubing at O2 and CO2 analysers (S-103 and S-155 Analyser, Qubit Systems, Kingston, Canada) placed outside of the chamber. The inlet air was also collected at the same analysers at 5 min intervals. The signals of Tcore were collected with a receiver board underneath the metabolic box (CTR86, Data Science), and the ambient temperature (Ta) in the chamber was monitored with thermocouples. O2 consumption and CO2 production rates (Inline graphicand Inline graphic) were calculated as the products of the differences in O2 and CO2 concentrations between the inlet and outlet airs, and the flow rate. The values were divided by the 0.75 power of body weight (Brody-Kleiber formula) and corrected to the standard temperature and pressure, dry (STPD) condition. MR was estimated by Inline graphicand respiratory quotient (RQ, Inline graphic/Inline graphic; indirect calorimetry). All the data were stored on a personal computer at 5 s intervals.

Prior to the experiment, the rats were allowed to acclimatize to the system by placing them in the metabolic box for 2 h on three separate days. At 10.00 h on the experimental day, a rat had a S.C. injection (10 ml kg−1) of either isotonic (154 mm NaCl) or hypertonic saline (2500 mm NaCl) on its flank under local anaesthesia induced by a S.C. injection of lignocaine hydrochloride (0.5 ml; Xylocaine). We injected the saline, loosely wrapping a rat with a towel on our thighs. Thirty minutes after the saline injection, the rat was placed in the metabolic box for 3 h. Ta was changed to 26, 20 and 10 °C for 1 h each, in that order. At least 1 week after the first trial, the rat repeated the same protocol with a S.C. saline injection of the other tonicity on the other side of the back. The order of the two trials was randomized. The rats were deprived food and water during the experiment.

Experiment 2. Operant cold-escape/warm-seeking behaviour during cold exposure

The details of the system were previously reported (Chen et al. 1998). Briefly, a Plexiglas behavioural box (50 cm × 10 cm × 30 cm) with a metal mesh top was placed in the same environmental chamber used in experiment 1. The chamber was ventilated with either a warm (25–40 °C) or a cold (0–30 °C) air-supply unit (CAU-210, Tabai Espec, Osaka, Japan), which was switched by computer-controlled valves. Five pairs of sensor-units comprising a light-emitting diode and a photoelectric cell on the side walls of the box located a rat to one of five 10 cm × 10 cm square areas (areas 1–5 from the left). Areas 4 and 5 were defined as reward areas: when a rat moved into the reward areas during a cold exposure at 20 or 10 °C, air at 40 °C was ventilated into the chamber for 30 s (operant behaviour). To get another 40 °C-air reward, the rat had to move out of the reward areas and move back there again. A re-entry into the reward areas within 30 s of the previous reward did not trigger another reward and did not count as an operant behaviour. There was no temperature gradient among the five areas during the cold or warm air ventilation. Tcore and Ta were monitored in the same manner as in experiment 1. All the data were stored on a personal computer every 5 s.

Before the experiment, the rats had 2 h training sessions in the system on four separate days. Naive rats tended to stay still during cold exposure even when warm-air rewards were available by the operant behaviour. Therefore, in the first two sessions, the setting of the system was altered so the reward temperature was set at 0 °C in a heat exposure of 40 °C. Then, the rats had two sessions of cold exposure (20 °C for 1 h, followed by 10 °C for 1 h) with 40 °C-air rewards. By this training procedure (Yoda et al. 2000), all the rats learned the operant cold-escape/warm-seeking behaviour.

At 10.00 h on the experimental day, a rat had a S.C. isotonic or hypertonic saline injection in the same manner as in experiment 1. Thirty minutes after the injection, the rat was placed in the behavioural box for 3 h. In the first hour, both air-supply units were set at 26 °C. Then, the warm air-supply unit was changed to 40 °C (reward air temperature) and the cold air-supply unit (load air temperature) was consecutively set at 20 and 10 °C for 1 h each. Each rat repeated the same protocol with the S.C. injection of the other tonicity as in experiment 1.

In both experiments, we continuously monitored rats’ behaviour. In addition, to assess the pain-related responses, we also counted scratching, biting and licking behaviours, which have been considered as visible nociceptive reactions (Hwang & Wilcox, 1986). Rats did not show any apparent abnormal behaviour during the experiments.

Blood and urine sampling and measurements

Blood sampling (0.3 ml) and body weight measurement were conducted at three different time points in both experiments: (1) before the S.C. saline injection, (2) 30 min after the injection and (3) at the end of experiment. Haematocrit (Hct, microcentrifugation), plasma protein concentration (PPC, refractometry; Atago, Tokyo, Japan), plasma osmolality (freezing-point depression; One-Ten osmometer, Fiske, Norwood, USA) and plasma Na+ concentration (flame photometry, Corning, Medfield, USA) were determined. In experiment 1, urine samples produced during the cold exposure were collected from the bottom of the experimental box, and their volume, osmolality and Na+ concentrations were also measured (n = 3). The relative change in blood volume was estimated by changes in Hct and PPC (Miki et al. 1987).

Statistics

Differences among means were assessed by ANOVA with repeated measurements. A post hoc test to identify a significant difference at a specific time point was performed by the Newman-Keuls procedure. A null hypothesis was rejected at the level of P < 0.05. All values are presented as means ± s.e.m.

RESULTS

Experiment 1. Metabolic response to the cold exposure

Figure 1 shows examples of the changes in Tcore (A), Inline graphic(B) and RQ (C) of one rat in the isotonic- and hypertonic-saline trials in experiment 1. In the hypertonic-saline trial, Tcore and Inline graphicwere lower than those in the isotonic-saline trial throughout the experiment. In both trials, rats stayed still in the box during the cold exposure. In addition, the counts of scratching, biting and licking behaviours did not differ between the isotonic-saline (2.5 ± 0.8) and hypertonic-saline trials (2.2 ± 0.8); these behaviours were mostly seen within 30 min after the injection.

Figure 1. Examples of metabolic response during the cold exposure.

Figure 1

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Changes in body core temperature (Tcore; A), oxygen consumption rate (Inline graphic; B) and respiratory quotient (RQ; C) of one rat in the isotonic- and hypertonic-saline trials in experiment 1. Each result was the average for 5 min.

Figure 2 illustrates the averaged Tcore (A) and MR (B) during the last 30 min of the 26, 20 and 10 °C exposure. In the hypertonic-saline trial, Tcore and MR were lower (P < 0.05) than those in the isotonic-saline trial in the three different Ta conditions. In the isotonic-saline trial, Tcore at 20 and 10 °C was not different from that at 26 °C. MR at both 20 and 10 °C increased (P < 0.05) from the level at 26 °C (337 ± 10 J min−1 kg−0.75) by 21 ± 5 and 48 ± 6 %, respectively. In the hypertonic-saline trial, Tcore at both 20 and 10 °C was lower (P < 0.05) than that at 26 °C. In addition, MR at 20 °C remained unchanged from that at 26 °C; however that at 10 °C increased (P < 0.05) from that at 26 °C by 35 ± 5 %.

Figure 2. Changes in Tcore (A) and metabolic rate (MR; B) in experiment 1.

Figure 2

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Tcore and MR were averaged during the last 30 min of each 1 h exposure period at 26, 20 and 10 °C. Values are means ± s.e.m. (n = 7). * Significantly different between the two trials, P < 0.05. † Significantly different from the value at 26 °C in each trial, P < 0.05.

Experiment 2. Behavioural response to the cold exposure

Figure 3 illustrates examples of the operant behaviour of one rat in the isotonic- and hypertonic-saline trials in experiment 2. In both trials, rats stayed still in the operant box during the last 30 min of 26 °C exposure. The operant behaviour at 20 °C was similar to that at 26 °C; however it increased at 10 °C in the isotonic-saline trial only. Moreover, during the cold exposure, rats did not move much except for the operant behaviour. The counts of scratching, biting and licking behaviours were at the same levels in experiment 1 with no significant difference between the two trials.

Figure 3. Examples of operant behaviour during the cold exposure.

Figure 3

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The operant cold-escape/warm-seeking behaviour of one rat in the isotonic- (A) and hypertonic-saline (B) trials in experiment 2. The position of the rat in the behavioural box (POS), body core temperature (Tcore) and ambient temperature (Ta) were measured. A 40 °C-air reward was given for 30 s when a rat entered the reward areas (area 4 or 5, shown in shaded area) in the behavioural box during 20 and 10 °C exposure. At 26 °C, reward temperature was set at 26 °C.

Figure 4 shows the averaged Tcore (A) and the counts of operant behaviour (B) during the last 30 min of the 26, 20 and 10 °C exposure. Similar to the result in experiment 1, Tcore was lower (P < 0.05) in the hypertonic-saline trial than in the isotonic-saline trial in the three Ta conditions. The counts of operant behaviour at 26 and 20 °C were not different between the two trials. In the isotonic-saline trial, the counts at 10 °C were greater (P < 0.05) than those at 26 °C (7.0 ± 1.2 and 1.5 ± 0.2, respectively). However, in the hypertonic-saline trial, the counts at 10 °C (2.5 ± 0.9) remained unchanged from those at 26 °C (2.8 ± 0.8).

Figure 4. Changes in Tcore (A) and counts of operant behaviour (B) in experiment 2.

Figure 4

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Tcore was averaged, and the operant behaviour was counted during the last 30 min of each 1 h exposure period at 26, 20 and 10 °C. Values are means ± s.e.m. (n = 7). * Significantly different between the two trials, P < 0.05. † Significantly different from the value at 26 °C in each trial, P < 0.05.

Changes in body weight and blood and urine measurements

Percentage changes in body weight (A) and blood volume (B), and changes in plasma osmolality (C) and Na+ concentration (D) in experiment 2 are shown in Fig. 5. These changes were similar to those in experiment 1, and thus the data in experiment 1 are not presented. Body weight decreased (P < 0.05) only in the hypertonic-saline trial at 30 min after the injection. Although body weight deceased (P < 0.05) in both trials at the end of experiment, the reduction in the hypertonic-saline trial was greater (P < 0.05) than that in the isotonic-saline trial. In addition, urine volume accounts for about 70 % of the body weight reduction (3.2–4.6 and 1.1–1.7 ml (100 g body weight)−1 in the hypertonic- and isotonic-saline trials, respectively), and the rest was mostly from faeces. In both trials, blood volume 30 min after the saline injection and at the end of experiment was not different from the level before the saline injection. Plasma osmolality and Na+ concentration in the hypertonic-saline trial increased (P < 0.05) after the injection by 34 ± 1 mosmol (kg H2O)−1 and 15.2 ± 1.0 mmol l−1, respectively; however, plasma osmolality and Na+ concentration in the isotonic-saline trial remained unchanged. Urine solutes excretion during the 3 h exposure period were greater in the hypertonic-saline trial than in the isotonic-saline trial (1.11–1.72 and 0.02–0.04 mmol (100 g body weight)−1 in Na+ and 3.22–4.63 and 0.62–0.84 mosmol (100 g body weight)−1, respectively). Rats recovered from the influences of the hypertonic-saline injection within 1 week, i.e. there were no differences from baseline values. Moreover, the body weight gain between the baseline periods of first and second trials was similar regardless of the type of the first trial (18 ± 2 and 16 ± 2 % after the hypertonic- and isotonic-saline trials, respectively).

Figure 5. Percentage changes in body weight (%Δ body weight; A) and blood volume (%Δ blood volume; B), and changes in plasma osmolality (C) and plasma Na+ concentration (D) in experiment 2.

Figure 5

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The data were obtained before the S.C. saline injection, 30 min after the injection and at the end of experiment. Values are means ± s.e.m. (n = 7). * Significantly different between the two trials, P < 0.05. † Significantly different from the value before the S.C. saline injection in each trial, P < 0.05.

DISCUSSION

In the present study, we evaluated the effects of S.C. hypertonic-saline injection on metabolic heat production and the operant cold-escape/warm-seeking behaviour during the cold exposure at 20 and 10 °C in rats. The two thermoregulatory responses to the cold were attenuated after the S.C. hypertonic-saline injection.

Metabolic response to the cold exposure during salt loading

In the hypertonic-saline trial, Tcore decreased not only at 26 °C but also during the cold exposure at 20 and 10 °C. Our previous studies also showed a decrease in Tcore at 26 °C following the same hypertonic-saline injection in rats (Nagashima et al. 2001; Konishi et al. 2002). In addition, we showed here that MR in the hypertonic-saline trial decreased from the level in the isotonic-saline trial at 26 °C and during the cold exposure at 20 and 10 °C (Fig. 1 and Fig. 2).

Autonomic heat loss processes such as panting, sweating, saliva secretion and skin blood flow are suppressed in a cold environment. In addition, Hosono et al. (2001) reported that the tail blood flow of rats decreased at Ta of 19 and 13 °C from the level at 25 °C, which facilitated heat insulation. Dehydration and/or salt loading are also known to attenuate the autonomic heat loss processes by an increase in concentration of plasma solutes such as Na+ and a decrease in blood volume (Harrison et al. 1978; Doris & Baker, 1981; Baker & Doris, 1982; Horowitz & Nadel, 1984; Baker & Dawson, 1985; Horowitz & Meiri, 1985; Nakajima et al. 1998). The present study showed that the S.C. hypertonic-saline injection increased plasma osmolality and Na+ concentration by 34 mosmol (kg H2O)−1 and 15 mmol l−1. However, in the isotonic-saline trial, these values remained unchanged during the experiment (Fig. 5C and D). Schoorlenmer et al. (2000) reported that a cell-volume sensor (not a Na+ sensor) located outside the blood-brain barrier in the brain was involved in the mechanism of renal Na+ excretion and thirst. Thus we speculate that the increase in plasma osmolality during the salt loading, rather than that in plasma Na+ concentration itself, is the stimulus inducing the metabolic and behavioural thermoregulatory changes. The greater reduction in body weight in the hypertonic-saline trial indicates that the rats were dehydrated, probably due to osmotic diuresis (Fig. 5A): greater urine volume and solutes excretion than those in the isotonic-saline trial. However, blood volume did not change in the two trials during the experiment (Fig. 5B). Therefore, the least likely mechanism is that the decrease in Tcore in the hypertonic-saline trial was caused by a facilitation of heat loss. Supporting this speculation, heat conductance estimated as MR/(Tcore–Ta) was lower (P < 0.05) in the hypertonic-saline trial than in the isotonic-saline trial (e.g. 26.4 ± 0.1 and 29.5 ± 0.4 J min−1 kg−0.75°C−1 at 26 °C, respectively). Thus it is thought that MR was decreased by the effect of the increase in plasma osmolality, and this reduction in MR was a primary mechanism involved in the decrease in Tcore in the hypertonic-saline trial.

Rats increase heat production with shivering and/or non-shivering thermogenesis (Foster & Frydman, 1979). In the isotonic-saline trial, Tcore was well maintained during the cold exposure at both 20 and 10 °C, during which period MR increased by 21 ± 5 and 48 ± 6 %, respectively. However, MR at 20 °C in the saline trial remained unchanged from the level at 26 °C with a reduction of Tcore by 0.9 °C (Fig. 2). At 10 °C, MR in the hypertonic-saline trial increased from the level at 26 °C by 35 ± 5 %; however the increase maintained Tcore only at the 20 °C level. It was reported that thermal inputs from both the body core and the surface were important for autonomic and behavioural thermoregulatory responses (Roberts, 1988). Therefore, the results may suggest that the threshold Tcore and/or skin temperature for shivering and/or non-shivering thermogenesis decreased after the S.C. hypertonic-saline injection.

Dehydration and/or salt loading increase arginine vasopressin (AVP) and/or oxytocin levels in both the cerebrospinal fluid (CSF) and the plasma (Szczepanska-Sadowska et al. 1983; Negoro et al. 1988). It was also reported that dehydration increased arginine vasotocin (AVT) levels in CSF and plasma in ducks (Gray & Simon, 1987). Moreover, Hassinen et al. (1994) reported that both intravenous and intrahypothalamic injections of AVT decreased MR and Tcore in pigeons, and also suppressed shivering at 2 °C. These results may indicate that shivering thermogenesis was suppressed in the hypertonic-saline trial in the present study and AVP and/or oxytocin are the key mediators in rats. However, it remains unclear whether non-shivering thermogenesis was also attenuated in the hypertonic-saline trial and was involved in the decrease in MR.

Behavioural response to the cold exposure during salt loading

In the isotonic-saline trial, Tcore remained unchanged during the experiment (Fig. 3A and Fig. 4A). Although the counts of operant behaviour at 20 °C did not differ from those at 26 °C, the counts at 10 °C were greater than the values at 26 and 20 °C (Fig. 4B). As a result, the averaged Ta was 21.2 ± 0.5 and 15.0 ± 0.6 °C during the 20 and 10 °C exposure, respectively, which were far below the reported thermoneutral range in rats (22–34 °C; Gordon, 1993). Yoda et al. (2001) also reported, using the same system, that the operant cold-escape/warm-seeking behaviour during 15 and 5 °C exposure was sporadic in well-fed rats. These results may indicate that the rats utilized metabolic heat production to maintain Tcore even when 40 °C-air rewards were available by the operant behaviour. Moreover, the metabolic heat production was a dominant process maintaining Tcore in the cold slightly below the thermoneutral condition (at 20 °C). However, it is likely that the rats effectively utilize the operant behaviour during the 10 °C cold exposure, although we did not assess how much energy consumption for heat production the rats saved by the increase in the operant behaviour. In contrast to our hypothesis, the counts of operant behaviour did not increase in the hypertonic-saline trial during the cold exposure despite the decrease in Tcore (Fig. 3B and Fig. 4). The movement at 26 °C, i.e. non-specific movements, was not different between the isotonic- and hypertonic-saline trials. Moreover, our previous study showed that the heat-escape/cold-seeking behaviour at 40 °C increased following the same hypertonic-saline injection (Nagashima et al. 2001; Konishi at al. 2002). The injection of hypertonic saline in the skin, muscle and intraperitoneal cavity has been reported to induce several nociceptive responses (Hylden & Wilcox, 1983; Hwang & Wilcox, 1986; Kawakita et al. 1993). However, to minimise the pain, we injected the saline in the subcutaneous fat tissue of the flank at which the fat layer was thicker, under local lignocaine anaesthesia (Bukhari, 1999). In addition, estimated from the licking, biting and scratching behaviours (Hwang & Wilcox, 1986), non-specific behaviour due to the pain was small enough and not different from that in the normal-saline trial. These results indicate that the reduction of the operant behaviour was not induced by discomfort, circulatory shock, and/or pain after the salt loading.

Yoda et al. (2000) reported that the counts of cold-escape/warm-seeking behaviour increased greater in food-deprived rats than well-fed rats. In rats, food deprivation is a strong stimulus to decrease Tcore and MR (Munch et al. 1993; Sakurada et al. 2000; Nagashima et al. 2003). Moreover, fasted rats preferred a higher ambient temperature in the thermal gradient compared with normal fed rats (Sakurada et al. 2000). Therefore, to maintain Tcore during fasting, the increase in the cold-escape/warm-seeking behaviour seems to be a dominant thermoregulatory process and compensates the decrease in metabolic heat production. However, in this study, the cold-escape/warm-seeking behaviour was suppressed in the hypertonic-saline trial, despite the decreases in Tcore and MR. These results strongly suggest that salt loading is a strong stimulus to suppress the cold-escape/warm-seeking behaviour as well as metabolic heat production. We previously suggested that central AVP and/or angiotensin II were also involved in the mechanism increasing heat-escape/cold-seeking behaviour after systemic salt loading (Konishi et al. 2002). Thus we speculate that the two hormones also work as mediators attenuating cold-escape/warm-seeking behaviour, although it has not yet been proved.

In contrast to the dehydration due to heat exposure and/or water deprivation, the hypertonic-saline injection protocol did not accompany hypovolaemia. Thus, in considering dehydration in a natural state, it is necessary to investigate the effect of hypovolaemia on behavioural thermoregulation and/or metabolic response in the heat and/or cold. Furthermore, hypovolaemia may have additive or synergistic effects on those thermoregulatory responses.

Functional implications of modulation of thermoregulation during salt loading

As we mentioned, a cold environment is also thought to induce a water-depletion in animals, although the 2 h cold exposure protocol in the present study did not induce an increase in plasma osmolality in the isotonic-saline trial. We speculate that reductions of Tcore and MR are as beneficial for dehydrated and/or salt-loaded animals in a cold environment as in a hot environment. This is because the suppression of metabolic heat production may decrease circulating blood to the heat generating organs such as the muscle and the brown and white adipose tissues, and redistribute the blood to the more critical organs such as the brain or heart. Furthermore, the reduction in body temperature would decrease insensible water loss from the air tract, which increases proportionally to the temperature difference between the body and environment.

In conclusion, systemic salt loading by S.C. hypertonic-saline injection attenuated MR in the thermoneutral condition at 26 °C and during the cold exposure at 20 and 10 °C in rats. Moreover, the reduction in MR induced a decrease in Tcore. In the isotonic-saline injected rats, the operant cold-escape/warm-seeking behaviour during the 10 °C cold exposure was activated, which probably conserved energy for metabolic heat production. However, despite the decreases in MR and Tcore, the operant cold-escape/warm-seeking behaviour was not activated in the hypertonic-saline injected rats, but, in contrast, was completely suppressed. An increase in plasma osmolality may be a strong stimulus suppressing both metabolic and behavioural thermoregulatory responses to the cold, although the mechanism remains unclear.

Acknowledgments

We thank Dr L. I. Crawshaw for helpful comments. The present study was supported partly by a Grant-in-Aid for Science Research from the Ministry of Education, Science Culture of Japan (nos 11557003, 12307001 and 14570058).

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