INTRODUCTION

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WITHIN THE HYPOTHALAMUS, 5-hydroxytryptamine (5-HT; serotonin) has been reported to be involved in body temperature regulation (see Ref. 6 for review). However, the picture is clouded by conflicting information obtained even when the same species of animals is used. For example, intracranial administration of 5-HT has produced falls (8, 9, 26) and rises (33) in the colon temperature of rats. Rats with brain 5-HT depletion produced by neurotoxins displayed no change (25, 28) or a lesser rise (32, 49) in colon temperature when exposed to acute heat stress. Furthermore, Sheard and Aghajanian (45) reported that electrical stimulation of rat midbrain raphe nuclei or intrahypothalamic administration of 5-HT produced an increase in colon temperature. On the other hand, Lin et al. (29) showed that either electrical stimulation of rat midbrain raphe nuclei or intrahypothalamic administration of 5-HT produced a decrease in colon temperature.

Experiments that have attempted to assess the effects of systemic administration of different 5-HT receptor subtype agonists on body temperature have also produced conflicting information. The stimulation of 5-HT_1A and 5-HT₂ receptor subtypes has been shown to have opposite effects on body temperature. For example, systemic administration of 5-HT_1A receptor agonists such as 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), gepirone, ipsapirone, and buspirone decreased body temperature, whereas a 5-HT₂ agonist such as (2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) increased body temperature in both rodents and humans (1, 11, 12, 15, 20, 23, 24, 31, 38).

According to the findings of Myers and his colleagues (33, 34, 42), 5-HT microinjected into the hypothalamus ordinarily produces a rise and norepinephrine produces a fall in the body temperature of the cat, rat, monkey, rabbit, and many other species. The increase in temperature caused by intrahypothalamic 5-HT is prevented by methysergide (a 5-HT receptor antagonist; see Ref. 39). Similarly, the fall in temperature evoked by intrahypothalamic norepinephrine is prevented by intrahypothalamic pretreatment with phentolamine (14). The dose of each putative neurotransmitter used by different investigators probably complicates the functional interpretation of the local action of monoamine on the thermosensitive region of the hypothalamus. A high dose of 5-HT given either in the cerebral ventricle or into the hypothalamus can cause a sharp fall in temperature (10, 37). This could be due to activation of the 5-HT₁ receptors in the presynaptic locus, which results in a decrease in 5-HT release in the synaptic cleft. In addition, because a high dose of 5-HT produces a characteristic inactivity, sleep and behavioral incapacitation, the decline in the temperature thus could occur as a secondary side effect of the high dose (5). Moreover, 5-HT in a high dose not only can saturate the 5-HT receptor sites but can also be taken up by catecholamine receptors that mediate the heat loss pathway (42).

To our knowledge, little information is available on the effects of increasing or decreasing the endogenous 5-HT release in the hypothalamus on body temperature in rats. Therefore, in the present study, in vivo dialysis coupled with HPLC with electrochemical detection was used for measuring extracellular 5-HT or its metabolites in the hypothalamus of unanesthetized rats. A series of experiments was carried out to determine whether the hypothalamic 5-HT could be manipulated by agents that act on serotonergic neurons to determine the proportion of hypothalamic 5-HT that is dependent on neuronal depolarization, calcium influx, and body temperature in unanesthetized rats. It is hoped that, with the use of these varied approaches, a clearer picture will emerge about the nature of the contribution that the endogenous 5-HT release in the hypothalamus might make to thermoregulatory control.

	MATERIALS AND METHODS

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Surgery. Male Sprague-Dawley rats weighing 250-300 g were housed singly under a 12:12-h light-dark cycle (light on at 0600) with free access to food and water in a temperature-regulated (22 ± 1°C) room. For surgery, rats were anesthetized with pentobarbital sodium (40 mg/kg ip) and were placed in a Kopf stereotaxic frame (David Kopf Instruments, Tujunga, CA) with a tooth bar setting at the upper incisor. A 10-mm guide tube cannula (21-gauge stainless steel tubing) was planted in the upper corner of the anterior commissure (anterior 8.7 mm, lateral 0.8 mm, and vertical 6.7 mm below the dura; see Ref. 41). After surgery, the guide cannula was plugged with a stylet, and rats were returned to their cages for a minimum recovery period of 1 wk.

Dialysis probe. The dialysis probe was a CMA-12 microdialysis probe purchased from CMA/Microdialysis (RosLagsvägen, Stockholm, Sweden). The working surface was a 4.0-mm length of permeable nitrocellulose membrane of 0.2 mm outer diameter and 6,000-Da cutoff. Fluid entered through 36-gauge stainless steel tubing that extended to the base of the probe and exited through an outer cannula of 26-gauge stainless steel tubing. The length of the dialysis probe was adjusted to place the probe tip at a point 2.0 mm below the guide tube cannula or 0.5 mm above the base of the medial preoptic area of the hypothalamus (anterior 8.7 mm, lateral 0.8 mm, and vertical 8.7 mm below the dura) in animals under brief anesthesia.

Dialysis perfusion. The evening before an experiment, a dialysis probe was slowly lowered through the guide cannula and cemented in place. Rats were restrained in rat stocks. Ringer solution was perfused through the microdialysis probe at a rate of 2 µl/min by a high-precision pump (CMA/Microdialysis; Roslagsvägen) attached to a liquid swivel. Sequential 15-µl perfusates were collected by a microfraction collector (CMA/Microdialysis; Roslagsvägen) into polyethylene microsample tubes containing 5 µl of 0.1 mol/l perchloric acid with 10⁷ mol/l ascorbic acid; 5-µl perfusates were analyzed for 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) on a microbore HPLC system.

Samples were identified and quantified by comparison with a standard solution containing 5-HT and 5-HIAA and by measurement of retention time and peak size. The mobile phase composition was 60 ml acetonitrile, 0.42 g sodium octyl sulfate (2.2 mmol/l), 200 g monosodium dihydrogen othophosphate (14.7 mmol/l), 8.82 g sodium citrate (30 mmol/l), 10 mg EDTA (0.027 mmol/l), and 1 ml diethylamine in double distilled water (21). The solution was adjusted to pH 3.5 by concentrated orthophosphoric acid, and its final volume was adjusted to 1.0 liters. The mixture was filtered with a 0.22-µm nylon filter under reduced pressure and degassed for 20 min.

In vitro recovery. The performance of each microdialysis probe was calibrated by a dialysis of a known amount of the standard mixture, and recoveries of all analytes were then determined. In vitro tests were run to determine the recovery of 5-HT in the dialysis solution at the flow rate used. At room temperature, the dialysis probe was immersed in dialysis solution containing 5-HT. At a flow rate of 2 ml/min, the relative recovery (e.g., 5-HT concentration in the dialysis solution divided by 5-HT concentration in the beaker) was determined.

Drugs. All chemicals were analytical grade and were dissolved in distilled, filter-purified water (Millipore, Bedford, MA). 5-HT and 5-HIAA were obtained from Research Biochemicals (Natick, MA) and were prepared as stock solutions in 0.1 M HCl with 100 mM EDTA; tetrodotoxin (TTX) was obtained from Sigma and was prepared as a stock solution in diluted sodium citrate buffer, pH 4.8; 8-OH-DPAT was obtained from Research Biochemicals; and fluoxetine was provided by Eli Lilly (Indianapolis, IN).

Determination of thermoregulatory parameters. The changes in body temperature and in metabolic, respiratory, and vasomotor activity in the animals were assessed by means of a partitional calorimeter (27). In each case, the metabolic rate was calculated from the animal's oxygen consumption, which was measured using a modified open-flow draw technique. Air was drawn at a constant flow rate (340 ml/min) through a Plexiglas helmet enclosing the animal's head so that all of the animal's expired gas was drawn into the chamber's efferent tube. The deficit in oxygen content of the efferent air was measured downstream by putting a dry sample of this air through a S-3A oxygen analyzer (Applied Electrochemistry, San Francisco, CA). The respiratory quotient, assumed to be 0.83 for the rodent, was used for all calculations. The metabolic rate was then calculated in watts so that 1 liter of oxygen consumed per hour was equivalent to heat production of 5.6 watts. Respiratory evaporative heat loss was calculated by measuring the increase in water vapor content in the efferent air in the helmet over that of the ambient air. Two pairs of wet and dry bulb thermocouples were used. The first pair, placed in the environmental chamber, measured the water content of the inspired air, whereas the second pair was positioned downstream from the helmet and continuously monitored the water content of the expired air of the animals. Evaporative heat loss expressed as watts per kilogram was calculated from evaporative water loss assuming the latent heat of the vaporization of 0.7 W · h¹ · g¹. T_co and foot skin temperature were measured with copper and constantan thermocouples. T_co was also measured with copper and constantan thermocouples enclosed in polyethylene 200 tubing, sealed at one end, and inserted 60 mm into the colon. All measurements were taken one time per minute throughout the experiments.

Histology. All animals were killed by an overdose of pentobarbital sodium at the end of the experiment. The brain was removed and fixed in 30% sucrose in a 10% Formalin solution. Frozen 25-µm sections were used for histological verification of the path of probes.

Data calculation and statistics. Basal 5-HT levels and thermoregulatory parameters were considered stable after 45 min of observation with no upward or downward trend. The 5-HT level of samples was expressed as a percent of the mean baseline. Time course data were analyzed by repeated ANOVA measures (general linear model). Duncan's multiple range test (multiple time point experiments) was used for post hoc determination of significant differences (P < 0.05). In all experiments, data collection was begun at least 8 h after probe implantation (2).

	RESULTS

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A total of 49 rats was used. Four rats were withdrawn, either because some became sick midway in the experiment or the HPLC systems malfunctioned. Forty-five rats were used for the entire dialysis experiment. After histological verification of the probe's path, all of the data obtained were included in our results. Figure 1 illustrates the locations of the dialysis probe in the anterior hypothalamus.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Schematic drawing of coronal sections of the rat brain adapted from the atlas of Paxinos and Watson (41) and showing the location of the dialysis surface of probes in the hypothalamus: AC, anterior commissure; MPO, median preoptic area of the anterior hypothalamus; OC, optic chiasma.

Studies with enhancers of 5-HT release. The following three enhancers of 5-HT release in dialysis solution were tested in this study: 5-hydroxytryptophan (5-HTP), fluoxetine, and high potassium concentration in the dialysis solution. The 5-HTP was dissolved in the dialysis solution at a concentration of 20 mM and was administered for 22 min through a dialysis probe (Fig. 2, n = 5). Colon temperature, extracellular 5-HT, and extracellular 5-HIAA were increased by 1.19 ± 0.15°C (Fig. 2A), 1,258 ± 25% of baseline (Fig. 2B), and 308 ± 21% of baseline (Fig. 2C) by 45 min after 5-HTP, respectively. Both the colonic temperature (T_co) and the extracellular levels of 5-HT and 5-HIAA returned to baseline by 75 min after 5-HTP perfusion. In addition, fluoxetine was dissolved in the dialysis solution at a concentration of 5 µM and was administered for 150 min through the dialysis probe (Fig. 3, n = 5). T_co, extracellular 5-HT, and extracellular 5-HIAA were increased to 1.28 ± 0.14°C (Fig. 3A), 690 ± 72% of baseline (Fig. 3B), and 141 ± 7% of baseline (Fig. 3C), respectively, by 150 min after perfusion of fluoxetine through the dialysis probe. Furthermore, potassium in the dialysis solution was increased from 3 to 120 nM to cause local depolarization. As shown in Fig. 4, T_co and extracellular 5-HT increased to 1.24 ± 0.08°C and 700 ± 82% of baseline, respectively, by 150 min after high potassium. In contrast, extracellular 5-HIAA was not altered during the 150-min perfusion of 120 mM potassium solution (Fig. 4C). The data are summarized in Table 1. Table 1 also shows that the hyperthermia produced by any of the three enhancers of 5-HT release is brought about by an increase in metabolic rate and a decrease in foot skin temperature. Intrahypothalamic administration of DOI (0.2 µg), but not Ringer solution, saline, or 2-methylserotonin (0.5 µg), produced hyperthermia, decreased skin temperature, and increased metabolic rate (Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effects of perfusion with dialysis solution containing 5-hydroxytryptophan (; 5-HTP; n = 5) or vehicle (; n = 5) for colonic temperature (Tco; A), serotonin (5-HT) concentrations (B), and 5-hydroxyindoleacetic acid (5-HIAA) concentrations (C). Duration of 5-HTP perfusion is indicated by the horizontal bar. Basal 5-HT level was 1.8 ± 0.6 pg/60 µl. * P < 0.05 for differences from the final predrug value; Duncan's multiple-range test.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effects of perfusion with dialysis solution containing 10 mM fluoxetine (; n = 5) or vehicle (; n = 5) on Tco (A), 5-HT concentrations (B), and 5-HIAA concentrations (C). Duration of fluoxetine perfusion is indicated by the horizontal bar. Basal 5-HT level was 1.5 ± 0.5 pg/60 µl. * P < 0.05 for differences from the final predrug value; Duncan's multiple-range test.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effects of perfusion with dialysis solution containing 120 mM potassium (n = 5) on Tco (A), 5-HT concentrations (B), and 5-HIAA concentrations (C). Basal 5-HT level was 1.7 ± 0.5 pg/60 µl. * P < 0.05 for differences from the final predrug value; Duncan's multiple-range test. Duration of high potassium perfusion is indicated by the horizontal bar.

Studies with inhibitors of 5-HT release. The following three inhibitors of synaptic 5-HT release were tested: TTX, 8-OH-DPAT, and zero calcium dialysis solution. Their effects on extracellular 5-HT, extracellular 5-HIAA, and thermoregulatory parameters are summarized in Table 1. During the 150-min perfusion of the hypothalamus with 500 nM TTX, the colon temperature and the extracellular 5-HT fell to 1.25 ± 0.08°C and 50 ± 8% of baseline, respectively (Fig. 5). This manipulation had no significant effect on 5-HIAA concentrations. When 8-OH-DPAT (500 µg/kg sc) was administered, colon temperature, the extracellular 5-HT, or the extracellular 5-HIAA was decreased significantly (Fig. 6). Finally, removal of calcium plus addition of 0.2 mM EDTA reduced T_co and extracellular 5-HT (Fig. 7). Again, this manipulation had no significant effect on 5-HIAA concentration. It can be seen from Table 1 that the hypothermia induced by any of the three inhibitors of 5-HT release was brought about by an increase in foot skin temperature and a decrease in metabolic rate. Furthermore, local administration of 8-OH-DPAT (0.5 µg) into the hypothalamus produced hypothermia, increased skin temperature, and decreased metabolic rate.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effects of perfusion with dialysis solution containing 500 nM tetrodotoxin (TTX; n = 5) on Tco (A), 5-HT concentrations (B), and 5-HIAA concentrations (C). Duration of TTX perfusion is indicated by the horizontal bar. Basal 5-HT level was 2.1 ± 0.6 pg/60 µl. * P < 0.05 for differences from the final predrug value; Duncan's multiple-range test.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effects of subcutaneous injection of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; n = 5) on Tco (A), 5-HT concentrations (B), and 5-HIAA concentrations (C). Basal 5-HT level was 1.9 ± 0.7 pg/60 µl. * P < 0.05 for differences from the final predrug value; Duncan's multiple-range test.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effects of perfusion with dialysis solution containing zero Ca2+ (n = 5) on Tco (A), 5-HT concentrations (B), and 5-HIAA concentrations (C). Duration of zero Ca2+ perfusion is indicated by the horizontal bar. Basal 5-HT level was 2.0 ± 0.7 pg/60 µl. * P < 0.05 for differences from the final predrug value; Duncan's multiple-range test. Arrow, start of perfusion.

	DISCUSSION

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Both present and previous results observed that local perfusion of the hypothalamus with fluoxetine (16, 18, 43, 44), 5-HTP (4, 47), or high potassium solution was associated with elevated levels of hypothalamic 5-HT in rats. In the present results, we further showed that elevated levels of hypothalamic 5-HT were associated with hyperthermia. The hyperthermia was brought about by an increase in metabolic heat production and a decrease in dry heat loss. The increase in 5-HT produced by fluoxetine is in accord with the hypothesis that reuptake is an important physiological mechanism for clearance of synaptically released 5-HT. Our results reaffirm the concept that the hydroxylation of tryptophan is the rate-limiting step of 5-HT synthesis (47). In addition, the increase in hypothalamic 5-HT during perfusion with 120 mM potassium was probably derived from hypothalamic neurons since 5-HT release from platelets and mast cells is not depolarization dependent (30, 40).

On the other hand, several experiments produced a decrease in extracellular 5-HT. Removal of calcium from the dialysis solution significantly reduced 5-HT levels since 5-HT release from terminals depends on influx of extracellular calcium. Release of 5-HT in the hypothalamus was also inhibited when 500 nM TTX was included in the dialysis solution. TTX blocks voltage-dependent sodium channels and thus voltage-dependent calcium influx into nerve terminals (13). Systemic administration of 8-OH-DPAT significantly reduced extracellular 5-HT (2). Both previous (2) and present results showed that this dose of 8-OH-DPAT reduced 5-HT to 40% or less of basal levels in the rat hypothalamus. We observed lowered levels of hypothalamic 5-HT following local perfusion of the hypothalamus with zero calcium, TTX, or systemic administration of 8-OH-DPAT were associated with decreases of colon temperature in rats. The hypothermia was brought about by a decrease in metabolic rate and an increase in skin temperature at this ambient temperature. Again, the time course of the decrease in hypothalamic 5-HT occurred parallel with, although somewhat in advance of, the decrease in colon temperature.

Although basal levels of 5-HIAA in the hypothalamus were higher than 5-HT levels, they were not a reliable indicator of 5-HT neurotransmission. In the present results, local perfusion of the hypothalamus with dialysis solution containing either fluoxetine or 5-HTP increased 5-HIAA levels in the hypothalamus, whereas local perfusion with high potassium solution did not affect 5-HIAA levels. In addition, systemic injection of 8-OH-DPAT decreased 5-HIAA levels in the hypothalamus, whereas local perfusion of the hypothalamus with either TTX or zero calcium solution did not affect the 5-HIAA levels in the hypothalamus. The present observation confirms previous suggestions that methods for assessing serotonergic neurotransmission based on measurement of 5-HIAA should be interpreted with caution (19, 22). The only effective route of continued metabolism for 5-HT is deamination by monamine oxidase. The product of this reaction can be further oxidized to 5-HIAA or reduced to 5-hydroxytryptophol, depending on the NAD⁺-to-NADH ratio in the tissue. Thus, in the present results, high potassium, TTX, or zero calcium solution have no effect on 5-HIAA but may have an effect on 5-hydroxytryptophol, which has not been assessed yet.

As described early in the introduction, activation of 5-HT₁ and 5-HT₂ receptors appears to mediate the hypothermic and the hyperthermic effects, respectively (1, 11, 12, 15, 20, 23, 24, 31, 38). In the present results, we confirmed that activation of 5-HT₁ and 5-HT₂ receptors in the hypothalamus via an intrahypothalamic cannula injection of 8-OH-DPAT and DOI also produced hypothermia (due to a decrease in metabolic rate and an increase in skin temperature) and hyperthermia (due to an increase in metabolic rate and a decrease in skin temperature), respectively, in rats.

In light of the above findings, nearly all data obtained with direct injections of 5-HT agonists to the hypothalamus confirm the concept of thermogenic action of endogenous 5-HT itself. Within the context of the volume of literature on this topic, any discrepancies in the literature have been clearly related to the injection of an overdose of 5-HT, thus swamping receptor activation by 5-HT. Activation of catecholamine receptors by 5-HT in the hypothalamus can also cause hypothermia (42). In addition, many investigators lucidly showed that 5-HT in the hypothalamus is released by cold environmental temperature (35), alcohol-induced change in temperature in the hypothalamus (17), and pyrogen or prostaglandin E in the hypothalamus (7, 34, 36, 46). Indeed, the present results strengthen the view point that the elevated levels of hypothalamic 5-HT mediate the thermogenic function in thermoregulation. Stimulation of the postsynaptic 5-HT₂ receptors in hypothalamic neurons mediates hyperthermia effects, whereas stimulation of the presynaptic 5-HT₁ receptors in hypothalamic neurons inhibit the endogenous release of 5-HT and leads to hypothermic effects.

Perspectives