INTRODUCTION

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IT IS WELL KNOWN THAT DEHYDRATION impairs thermoregulation in heat and can lead to heat stroke in extreme conditions. Two factors during dehydration, a reduction in blood volume and an increase in plasma osmolality, are thought to be involved in the mechanism (1, 2, 6-9, 14, 15, 19). These factors suppress autonomic responses necessary for thermoregulation such as panting (1, 2, 9), sweating (6, 7), salivary secretion (8), and skin vasodilation (14, 15, 19). The suppression of these thermoregulatory responses during dehydration would be important to preserve fluid in the body and/or maintain a greater central blood volume. Besides the autonomic responses, all animals use behavioral means to regulate body temperature (3, 16, 20, 21). In general, they first try to escape thermally untenable environments (16). Indeed, when readily available, heat-escape and/or cool-seeking behaviors are the least costly responses in terms of energetics and fluid balance. Thus, for dehydrated animals, alterations in core temperature would be most efficiently accomplished with behavioral responses. Brummermann and Rautenberg (3) first analyzed the effect of osmotic stress on behavioral thermoregulation in the heat: pigeons conditioned to get cold-air rewards in the heat by an instrumental response increased their responding following hypertonic saline infusion. However, it still remains unknown if heat-escape/cold-seeking behavior is activated by osmotic stress per se or if the activation is a compensatory response as a result of attenuated autonomic responses.

In the present study, we investigated heat-escape/cold-seeking behavior in rats following hypertonic saline injection to evaluate the importance of osmotic changes in this behavior. Rats spread saliva on their body surface for the evaporative heat loss. To minimize the influence of this additional evaporative heat loss, salivary secretion was blocked by surgical ligature of the salivary ducts. In addition, a novel operant system that Chen et al. (4) recently reported was used for quantitative assessment of heat-escape/cold-seeking behavior in freely moving rats. We hypothesized that heat-escape/cool-seeking behavior in rats with attenuated salivary secretion would be increased following hypertonic saline injection.

	METHODS

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Male crj-Wistar rats (Charles River Japan, Osaka, Japan) were used for the present experiments. The experimental protocol for this study was approved by the Animal Committee of Osaka University Medical School. Rats were housed individually at the room temperature of 23°C in a 12:12-h light-dark cycle (light on at 7:00 AM) and had free access to food and water.

Surgery. Each rat was anesthetized with an intraperitoneal injection of pentobarbital sodium (5 mg/100 g body wt). The major salivary ducts from the parotid, major sublingual, and submaxillary glands were doubly ligated, then cut in the middle as previously reported (12). The successful procedure was verified by polydipsia for several days following the surgery and postmortem examination of salivary glands that showed atrophy. A biotelemetry device (15 × 30 × 8 mm; Physiotel, Data Science, St. Paul, MN) for the measurement of core body temperature was placed in the peritoneal cavity. The accuracy of the measurement was within 0.1°C. At least 2 wk after the surgery, each rat was anesthetized in the same way as above, and a silicone catheter (diameter 1.0 mm; Fuji Systems, Tokyo, Japan) was placed in the intrathoracic inferior vena cava through the right femoral vein. Its free end was pulled out through the skin of the nape. The catheter was filled with heparinized (50 units/ml) saline, and the end was plugged with a stainless rod. To avoid clogging, the catheter was flushed with the heparinized saline every day.

Experimental operant behavior system. For quantitative assessment of heat-escape/cool-seeking behavior in heat, we used the experimental system previously reported (4). Briefly, a box (50 × 10 × 30 cm) made of Plexiglas was placed in an environmental chamber (80 × 65 × 60 cm, Fig. 1A). The box was well ventilated with numerous 1-cm diameter holes on the sides and a metal mesh lid. The chamber had an air inlet and an outlet that were connected to two air-supply units (CAU-210, Tabai Espec, Osaka, Japan). One of the units supplied hot air ranging from 25 to 40°C, and the other supplied cold air ranging from 0 to 30°C. Computer-controlled electromagnetic valves switched the unit ventilating the chamber. Rats were able to move freely within the box. Movement of the rats was continuously monitored with an infrared video camera. In addition, five pairs of photosensor units located the animals' position to one of five 10 × 10-cm square areas.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   The experimental system is illustrated. A: Plexiglas box was put in a chamber, which was connected to 2 air-supply units. One supplied cold air (0-30°C), and the other supplied warm air (25-40°C). The unit supplying the chamber was switched by computer-controlled valves. B: top view of the box. The position of rats in the box was located with 5 pairs of photosensors, dividing the box into five 10 × 10-cm square areas in a single plane (1).

Protocol 1 (operant behavior in heat). Six rats (200-300 g body wt) with ligated salivary ducts were individually put in the experimental system for 2 h three times. A 2-day interval separated the training sessions. The following parameters were used. The hot unit was set at 40°C, and the cold unit was set at 0°C. The chamber was normally ventilated by the hot unit. Once rats moved into the reward area (areas 4 and 5 in Fig. 1B), ventilation via the cold unit was given by switching the valves for 30 s. Rats had to move out of the area and then move into the area again to get another 30 s of cold air. The rats easily learned this procedure during the training sessions. At least 3 days after the training sessions, either normal (154 mM NaCl) or hypertonic (2,500 mM NaCl) saline was injected (1 ml/100 g body wt) into the subcutaneous tissue of the lower back under local anesthesia (0.5% lidocaine). After 1.5 h, each rat was put in the experimental box with temperatures of both air-supply units set at 26°C. The rat was kept under these conditions for >1 h until the core temperature became stable (within 0.2°C for >10 min). The air-source temperatures were then altered to 40 and 0°C for another 2 h. After a 5- or 6-day interval, the same experimental protocol was repeated with a saline injection of the other concentration, e.g., normal saline injection in case of hypertonic saline injection in the first trial. The order of the trials was randomly chosen. Food and water were not given to the rats during the measurement period.

Protocol 2 (periodical passive cooling in heat). Five rats (200-300 g) with ligated salivary ducts were used for this experiment. Each rat was placed in the box for one 2-h session to familiarize them with the apparatus. The system was programmed to switch intermittently the ventilating air from 40°C to 0°C regardless of the rats' movements in the box. The switch for the cold unit was maintained for 30 s. The number of cold-air ventilations (35 times during the session) was determined based on the mean number of cold-air rewards in the normal saline trial of protocol 1. Two days after the session, normal or hypertonic saline was injected in the same way as in protocol 1. Each rat was first placed in the box for 1 h with the air temperature set at 26°C. For another 2 h, the air temperature was set at 40°C with intermittent 0°C air ventilation as in the training session. The same experimental protocol was repeated after the saline injection with the other concentration following a 5- or 6-day interval.

Protocol 3 (passive heating). Three rats (250-300 g) with ligated salivary ducts were used. In the initial session, each rat was put in the box with 26°C ventilating air for 2 h. Two days after this session, either normal or hypertonic saline was injected in the same way as in protocols 1 and 2. The rat was placed in the box for 1 h with 26°C ventilating air followed by 40°C for 2 h. No cold air was given in this protocol. We stopped the experiment when the body core temperature exceeded 40°C. The same heat exposure was repeated in the second saline injection following a 5- or 6-day interval.

Measurements. Measurements of body weight and blood sampling (0.5 ml) were done before and 1.5 h after the saline injection and at the end of the experiment for protocols 1 and 2. Hematocrit (Hct, microcentrifuge), total plasma protein concentration (TP, refractometry; Atago, Tokyo, Japan), and plasma osmolality (freezing-point depression; One-Ten osmometer, Fiske, MA) were determined. Percent changes in blood volume (%BV) were estimated by changes in Hct and TP, assuming plasma content of total protein was constant throughout the measurement period. The following formula was used (13):
where Hct₀ and Hct_t represent Hct at baseline and measurement point, respectively; TP_o and TP_t represent TP at baseline and measurement point, respectively.

Statistics. Differences between variables were determined by analysis of variance for repeated measurements (18). Significant differences at specific time points were identified by Newman-Keuls post hoc test. Confidence level for statistical significance was set at P < 0.05. All variables are expressed as means ± SE.

	RESULTS

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Protocol 1. Figure 2 illustrates data for one rat in the normal-saline trial. The rat first moved restlessly in the box (Fig. 2A). However, the rat moved periodically in and out of the reward area (areas 4 and 5 in Fig. 1) during the heat exposure. The core body temperature was basically stable during the heat exposure (Fig. 2B). The ambient temperature measured in the box reached ~10°C within 30 s after the rat moved in the reward area, then returned immediately to 40°C (Fig. 2C).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Position (A) and core temperature (Tcore; B) of 1 rat as well as the ambient temperature (Ta; C) in the box in the normal-saline trial of protocol 1 are illustrated. Air at 0°C was given for 30 s when the rat entered the reward area (areas 4 and 5) in the box.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   A: changes in body Tcore averaged for each 30 min at the Ta of 26°C (baseline) and during operant behavior at 40°C for the normal- and hypertonic-saline trials of protocol 1. The rats received the saline injections 1.5 h before being placed in the box. B: counts of cold-air rewards in the normal- and hypertonic-saline trials of protocol 1. Values are means ± SE (n = 6). *Significantly different between normal- and hypertonic-saline trials (P < 0.05). Significantly different from the baseline in each trial (P < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   %Changes in body wt (A) and blood volume (B) and plasma osmolality (C) before and 1.5 h following saline injections (pre-heat exposure) and at the end of the experiment (post-heat exposure) in the normal- and hypertonic-saline trials of protocol 1. Values are means ± SE (n = 6). *Significantly different between normal- and hypertonic-saline trials (P < 0.05). Significantly different from the baseline in each trial (P < 0.05). , Change.

Protocol 2. Figure 5A illustrates averaged core temperature. Thirty-second ventilation of 0°C air was automatically and intermittently given during the heat exposure regardless of the rats' movement. The core temperature in the normal-saline trial remained stable throughout the experiment. Despite a lower (P < 0.05) baseline core temperature, core temperature in the hypertonic-saline trial increased (P < 0.05) during the heat such that there was no significant difference from the normal-saline trial. The increase in core temperature from the baseline value in the hypertonic-saline trial was 1.3 ± 0.2°C at 90-120 min, which was greater (P < 0.05) than that of the normal-saline trial (0.3 ± 0.3°C). Although the cold air was automatically given regardless of the rats' position in protocol 2, we counted movements of the rats into the area corresponding to the "reward area" in protocol 1 to evaluate nonspecific movements of rats in the heat (Fig. 5B). The counts of movements during the heat were greater (P < 0.05) than the baseline at 0-120 min in the normal-saline trial and at 0-60 min in the hypertonic-saline trial. However, the counts of nonspecific movements were significantly (P < 0.05) lower than the counts of cold-air rewards in protocol 1 at 30-120 min in both trials. The percent changes in body weight and blood volume and plasma osmolality were similar to the values in protocol 1.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   A: changes in body Tcore at an ambient temperature of 26°C (baseline) and during heat exposure of 40°C in the rats for the normal- and hypertonic-saline trials of protocol 2. Cold air of 0°C intermittently and automatically ventilated the chamber during the heat exposure. B: movements into areas 4 or 5 of the experimental box. The counts estimate nonspecific movements. Values are means ± SE (n = 5). *Significantly different between normal- and hypertonic-saline trials (P < 0.05). Significantly different from baseline in each trial (P < 0.05).

Protocol 3. The rats were exposed to 40°C without either operant or automatic cold-air ventilation. The baseline core temperature before the heat exposure was 37.6 ± 0.2 and 36.8 ± 0.2°C in the normal- and hypertonic-saline trial, respectively, with a significant difference (P < 0.05). We had to cease the protocol for all the rats within 30 min after the onset of heat exposure because the core temperature exceeded 40°C. The mean duration of heat exposure was 20 ± 2 and 24 ± 3 min in the normal- and hypertonic-saline trial, respectively.

	DISCUSSION

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In the present study, we evaluated the effect of hypertonic saline injection on heat-escape/cool-seeking behavior in the rats with ligated salivary ducts. The results strongly suggest that hypertonic saline injection increases heat-escape/cool-seeking behavior.

During constant exposure to environmental temperature of 40°C without a cold-air reward in protocol 3, the rats with ligated salivary ducts could not regulate body core temperature following either normal or hypertonic saline injections. In addition, the latency to reach the body temperature of 40°C was not different between the normal- and the hypertonic-saline trials. Thus the results would indicate that the contribution of skin vasodilation to heat dissipation was similar in the two trials. However, core temperature was maintained within the normal range if 0°C air was available following an operant behavior (Fig. 3) or when administered automatically (Fig. 5). Therefore, getting cold air would be the only viable mechanism for rats with blocked salivary secretion to regulate core temperature in the heat.

The rats using the operant behavior (moving to 1 part of the box for cold-air rewards in protocol 1) increased the counts of cold-air rewards during the heat exposure both in the normal- and hypertonic-saline trials (Fig. 3). In addition, the counts in the hypertonic-saline trial were greater than those of the normal-saline trial in the first one-half hour and the last hour of heat exposure. In this operant system, rats learned the operant behavior (Fig. 2) much more rapidly than when it involves a lever press (12). We judged that the rats had learned the operant behavior when they showed the periodical movements into the reward area as shown in Fig. 2A. However, we have no precise criteria to separate the operant behavior from nonspecific movements, because nonspecific movements increased during heat exposure (Fig. 5). Nonspecific movements of the rats in the heat were assessed in protocol 2 by counting movements into areas 4 or 5 (Fig. 1), which corresponded to the reward area in protocol 1 (Fig. 5). The counts in protocol 1 were significantly greater than those in protocol 2 at 30-120 min of 40°C. Therefore, the cold-air rewards in protocol 1 were, partly at least, due to an operant behavior elicited by thermoregulatory demand.

The baseline core temperature in the hypertonic-saline trial was lower than the normal-saline trial in all protocols. Seven-percent reduction in body weight at the end of the hypertonic-saline trial (Fig. 4A) indicates a loss of body water largely due to osmotic diuresis because the reduction in normal-saline trial was much smaller. Hypohydration induces a decrease in metabolic rate in rats and camels by the effects of either hypovolemia or hyperosmolality (10, 17). In contrast, hypohydration would attenuate the net heat loss because the threshold of core temperature for the tail vasodilation, which is the important heat-loss mechanism, especially in thermoneutral condition, would be elevated by the effect of hyperosmolality (8, 15). Moreover, evaporative heat loss would be minimum in both trials due to the ligation of major salivary ducts, and the core temperatures below 38°C seen in this part of the study are far below the threshold for saliva spreading (20). Thus the lower baseline core temperature in the hypertonic-saline trial may have been a result of attenuated heat production rather than facilitated heat loss.

The greater loss of body weight in the hypertonic-saline trial might have lowered body temperature due to circulatory shock. However, the rats' operant behavior was pursued more vigorously than in the normal-saline trial. In addition, blood volume was preserved even in this condition (Fig. 4B). These findings suggest that circulatory shock would be the least likely mechanism for the lowered body temperature.

Despite lower baseline core temperatures in the hypertonic-saline trials of protocols 1 and 2, the core temperatures increased to the levels of the normal-saline trials within 60 min after the heat exposure (Figs. 3 and 5). As long as the cold air was automatically given, the core temperatures in the normal- and hypertonic-saline trials remained at similar levels (Fig. 5). Therefore, the number of cold-air rewards was sufficient to maintain a balance between heat production and heat loss. In contrast, the rats decreased their core temperature following the hypertonic saline infusion when they could use the operant behavior (Fig. 3), the major mechanism for thermoregulation in the heat. These results strongly suggest that the rats increased the operant behavior in the hypertonic-saline trial to maintain a lower body temperature than in the normal-saline trial.

The operant heat-escape/cold-seeking behavior might have been secondary to a rise in body temperature as a result of attenuated autonomic responses. However, body core temperature is actually lower in the hypertonic-saline trials than in the normal-saline trials (Fig. 3A) as noted above. Furthermore, thermal inputs from the body surface, which would be important both for autonomic and behavioral thermoregulatory responses (16), were lower in the hypertonic-saline trial than in the normal-saline trial because the mean ambient temperature in the hypertonic-saline trial was lower. Therefore, the increase in the operant behavior is not a secondary response to a rise in body temperature but is a direct response to hypertonic saline injections.

The stimulus to increase the operant behavior would be the increase in plasma osmolality and/or the reduction in the intracellular fluid volume (cell dehydration) that occurred subsequently. The hypertonic saline injection induced an increase in plasma osmolality and a reduction in body weight, although the normal saline injection did not change those values except for a slight decrease in body weight at the end of the trial (Fig. 4A). However, blood volume was rather increased in both trials. We surmised that the normal saline injection resulted in an increase in the extracellular volume, including blood volume. In contrast, the hypertonic saline injection led to osmotic forces that likely caused a redistribution of body fluid from the intracellular to the extracellular compartment by osmosis despite a reduction in total body fluid.

Thermally induced dehydration causes not only a reduction in body water, which, when extreme, is accompanied by a decrease in blood volume (11). In this condition, autonomic responses for heat loss are suppressed. The ensuing decrease in evaporative heat loss (1, 2, 6-9) would help maintain body water. The suppression of dry heat loss, i.e., skin vasodilation (14, 15, 19), would augment the central blood volume needed for cerebral circulation. Suppressed autonomic responses for heat loss are generally accompanied by a rise in core temperature, such as during fever. However, the dehydration induced by hypertonic saline injection led to a decreased core temperature under thermoneutral conditions (Figs. 3 and 5). This may function to delay the activation of autonomic heat-loss responses when animals face heat. Facilitation of heat-escape/cold-seeking behavior would prevent or lessen the need to use the autonomic responses, which would result in the preservation of body water. Thus during dehydration, autonomic and behavioral responses for thermoregulation are simultaneously modulated and function to maintain body water homeosatasis.

In summary, we showed that plasma hyperosmolality and/or cell dehydration induced by hypertonic saline injections are stimuli for the direct activation of operant heat-escape/cold-seeking behavior in rats with ligated salivary ducts. The increased operant behavior results in lower body temperature compared with normal-saline controls. Moreover, the hypertonic saline injection also led to a decreased body temperature under thermoneutral conditions, probably by suppressing heat production. These findings indicate that a coordinated set of autonomic and behavioral thermoregulatory adjustments occurs in response to dehydration.

Perspectives