J. Physiol. (1977), 266, pp. 423-433 With 5 text-figurew Printed in Great Britain

423

A DISSOCIATION BETWEEN TEMPERATURE REGULATION AND FEVER IN THE RABBIT

BY D. BORSOOK, HELEN P. LABURN,* C. ROSENDORFF, G. H. WILLIES AND C. J. WOOLF From the Department of Physiology, University of the Witwatersrand Medical School, Johannesburg 2001, South Africa

(Received 23 August 1976) SUMMARY

1. The role of 5-hydroxytryptamine (5-HT) in temperature regulation and in fever in the rabbit has been investigated. 2. Intrahypothalamic microinjections of 5-HT in the conscious rabbit alters body temperature in a dose-dependent manner. 3. Low doses (5.5 nmol) of 5-HT and control saline injections produce a small, non-significant increase in temperature, with a long latency. 4. Doses of 14 nmol 5-HT produce a hyperthermia with a 45 min delay; while microinjections of 28 nmol result in a biphasic response; an initial short hypothermia is followed later by a hyperthermia. 5. Depleting the rabbit's brain of 5-HT by pretreatment with pchlorophenylalanine (PCPA) fails to affect its body temperature at thermoneutral temperatures but significantly impairs the ability to thermoregulate against a cold stress. 6. PCPA pretreatment did not, however, impair the febrile response to bacterial pyrogen and prostaglandin E1. 7. These results reveal a dissociation between the effects of 5-HT depletion on temperature regulation, and on fever. The site of action of 5-HT in temperature regulation must be proximal to the fever input, but distal to the convergence of peripheral and hypothalamic temperature inputs. INTRODUCTION

The interrelation between fever and temperature regulation in homoeothermic animals is complex. Fever is a disturbance of thermoregulation which manifests as an elevation in body temperature, but neither the synaptic chemical events involved in the pathogenesis of fever nor the actual manner in which pyrogenic agents alter normal thermoregulation *

To whom requests for reprints should be addressed.

D. BORSOOK AND OTHERS 424 are known. Whether fever is produced by an alteration in the activity of the neural elements of the temperature regulatory pathways of the hypothalamus, or whether a functionally and anatomically separate fever input converges with the temperature regulatory pathway at some appropriate point, remains to be determined. In the rabbit there have been extensive investigations of the possible role of 5-hydroxytryptamine (5-HT) in temperature regulation and in fever. Microinjections of 5-HT intraventricularly or into the hypothalamus of the rabbit have been reported to produce hyperthermia (Canal & Ornesi, 1961; Ruckebusch, Grivae & Laplace, 1965; Jacob & Robert, 1966), hypothermia (Cooper, Cranston & Honour, 1965; Bligh, Cottle & Maskrey, 1971) and a biphasic response (Jacob, Girault & Peindaries, 1972). Depleting the rabbit's brain of 5-HT by pretreatment with p-chlorophenylalanine (PCPA) alters the response of the rabbit to hypothalamic heating and cooling (Woolf, Laburn, Willies & Rosendorff, 1975) and to an environmental heat stress (Glauber, Woolf, Willies & Rosendorff, 1976). Depletion of brain 5-HT in the rabbit either has no effect on, or slightly potentiates, the fever produced by pyrogen (Giarman, Tanaka, Mooney & Atkins, 1968; Teddy, 1971; Metcalf & Thomson, 1975) or by prostaglandin E1 (PGE1, Sinclair & Chapman, 1974). The present series of experiments has been designed to examine further the role of 5-HT in temperature regulation and in fever in the rabbit, and in particular to determine whether this amine has the same or different roles in the two processes. Microinjections of 5-HT at a variety of doses have been made directly into the hypothalamus, and the responses of 5-HT depleted (PCPA pretreated) rabbits to a cold environmental stress and to the administration of bacterial pyrogen or PGE1 have been

compared. METHODS Experiments were performed on a total of thirty New Zealand White rabbits of both sexes weighing between 2-5 and 3*5 kg. Animals used for intrahypothalamic injections had stainless-steel headplates fixed to their skulls at least 1 week before the experiments began. The method used for stereotaxic alignment of the headplates and the positioning of the injection cannulae was a modification of that used by Monnier & Gangloff (1961). The techniques for intrahypothalamic microinjection into conscious rabbits have been described in a previous paper (Woolf, Willies, Laburn & Rosendorff, 1975). 5-Hydroxytryptamine creatinine sulphate (Sigma) was dissolved in sterile saline and unilateral intrahypothalamic injections were made at doses of 5-5, 14 and 28 nmol. Control unilateral injections of sterile saline were made in several of the ten rabbits used in this experimental series. Fevers were produced by an intrahypothalamic injection of PGE, (Upjohn), or by an injection of bacterial pyrogen. For PGE1 administration a dose of 1-4 nmol in a volume of 5 ,ul sterile saline was injected into one side of the anterior hypothalamus.

TEMPERATURE REGULATION AND FEVER

425

Bacterial pyrogen (E pyrogen, Proteus vulgari8, Organon Laboratories), 0-02 jug, was dissolved in 1 ml sterile saline and was injected into an ear vein. Control injections of sterile saline in the appropriate volume into either the anterior hypothalamus, or ear vein had no significant effect on body temperature. Depletion of 5-HT was achieved by intraperitoneal (I.r.) injection of 075 mmol/ kg p-chlorophenylalanine (PCPA, Sigma) suspended in 4 ml sterile corn oil, each day for three consecutive days. Control animals received i.P. injections of 4 ml sterile corn oil. The extent of depletion of 5-HT using the above-mentioned method in rabbits has been ascertained previously in this laboratory using a spectrophotofluorometric technique (Woolf et al. 1975) and by Metcalf & Thompson (1975) who used a similar method of PCPA treatment. One week after the course of PCPA or control injections the rabbits were exposed to a cold stress. After 1 hr at room temperature during which the animals' body temperatures were allowed to stabilize, the rabbits were placed in a thermostatically controlled chamber, which had been precooled to 7 ± 10 C. Cold exposure lasted for 5 hr. No rabbit was exposed to cold more than three times and experiments were never performed on the same animal on consecutive days. All experiments were performed on conscious rabbits restrained in conventional stocks. Rectal temperature was measured by a YSI thermistor probe inserted into the rectum. In the cold exposure experiments ear skin temperature was also measured by taping a small, flat YSI thermistor probe on the shaved surface of the rabbit's ear. These body temperatures, as well as the temperature of the chamber were continually scanned by means of a YSI telethermometer (Model 47) and were recorded on a Beckman Dynograph. All solutions other than the pyrogen and PCPA were passed through a 022 /tm millipore filter before injection. Except in the case of the cold exposure series, all experiments took place at an ambient temperature of 21 ± 1 C. All data were subjected to the Student's t test and values of P equal to or less than 002 were considered significant. RESULTS

Microinjections of 5-HT into the anterior hypothalamus of conscious rabbits resulted in alterations in rectal temperature which varied with the different doses used. The lowest dose microinjected (5.5 nmol) did not initially change the rectal temperature, but after a latent period of between 1 and 2 hr the animals' rectal temperatures rose by 025-05° C. A similar pattern of rectal temperature changes occurred when an identical volume of sterile normal saline was microinjected into the same area of the brain. In the cat, non-specific fevers of like amplitude and onset have been shown to be associated with increased levels of prostaglandins of the E series in the cerebrospinal fluid and these fevers are prevented or attenuated by antipyretic agents (Dey, Feldberg, Gupta, Milton & Wendlandt, 1974). Mechanical trauma of brain tissue does stimulate prostaglandin release (Milton & Harvey, 1975) which could be responsible for the fevers, or there may be pyrogenic contamination of the injectate in spite of all precautionary measures. Microinjection of 14 nmol 5-HT into the anterior hypothalamus

426 D. BORSOOK AND OTHERS produced a hyperthermia after a latent period of approximately 45 min (Fig. 1 B). This fever differed from the non-specific fever in its shorter latent period and its greater increase in rectal temperature. The highest dose of 5-HT microinjected (28 nmol) produced a biphasic response (Fig. IA). A

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Fig. 1. Effects of intrahypothalamic injections of (A) 28 nmol and (B) 14 nmol 5-HT in the rabbit. Each curve represents the rectal temperature change from pre-injection levels (zero on temperature scale) for a single rabbit.

After a latency of 5-10 min rectal temperature fell in all cases. The maximal drop in rectal temperature was 0.75° C which occurred 60 min after the injection was made. The hypothermia was terminated by a steady rise in rectal temperature, culminating in varying degrees of hyperthermia. The highest body temperature rise recorded after the initial hypothermia was 20 C above preinjection levels. At all doses tested the quantitative responses were quite variable among different animals but generally followed a consistent trend at each dose.

427 TEMPERATURE REGULATION AND FEVER Exposure of untreated control rabbits to a cold environment of 7 + 10 C demonstrated the ability of the rabbit to thermoregulate adequately against a cold stress. After 5 hr of cold exposure the mean rectal temperature for ten rabbits had decreased by only 0.1 + 0.20 0 (s.E.). This was not significantly different from the base-line temperature measured at room temperature. The skin temperature of these rabbits fell by a mean value of 22+ 10 C (S.E., n = 10) almost immediately on placement in the cold environmental chamber. Fig. 2 shows typical responses of rectal and skin temperatures for a control, and for a PCPA pretreated rabbit when placed in the environmental chamber. Tr control

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Fig. 2. Typical effects on rectal (T1) and skin (T.) temperatures of rabbits ,a when exposed to a cold (7+± 1 C) ambient temperature (T.). control rabbit; ---, a rabbit pretreated with PCPA (0.75 mmol/kg for 3 days). Note different temperature scales for skin and ambient, and for rectal temperatures. Rabbits were exposed to cold after 1 hr at room temperature.

The rectal temperatures of the PCPA pretreated rabbits did not differ from those of the control rabbits when measured at room temperature. On exposure to the cold environment the rectal temperature of the PCPA pretreated rabbits began to fall almost linearly. The mean fall in rectal temperatures for these rabbits was 0-84 + 0.130 C (s.E., n = 10). After 4'5 and 5 hr of cold exposure the rectal temperatures of the depleted and the control rabbits were significantly different (P < 0-02, Fig. 3). The

D. BORSOOK AND OTHERS 428 PCPA pretreated rabbits exhibited a marked degree of cutaneous vasoconstriction when exposed to the cold stress. Ear skin temperature fell by a mean value 25-0 + 0.70 C (S.E., n = 10). This drop in skin temperature was significantly greater than the fall in skin temperature of the control rabbits

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A dissociation between temperature regulation and fever in the rabbit.

J. Physiol. (1977), 266, pp. 423-433 With 5 text-figurew Printed in Great Britain 423 A DISSOCIATION BETWEEN TEMPERATURE REGULATION AND FEVER IN THE...
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