El (PGE,) and E2 (PGE2) have been injected into a lateral cerebral

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1 J. Phygsiol. (1974), 236, pp With 1 text-figure Printed in Great Britain THERMOREGULATORY RESPONSES TO THE INJECTION OF MONOAMINES, ACETYLCHOLINE AND PROSTAGLANDINS INTO A LATERAL CEREBRAL VENTRICLE OF THE ECHIDNA By J. A. BAIRD,* J. R. S. HALES AND W. J. LANG* From C.S.I.R.O., Division of Animal Physiologvy, Ian Clunies Ross Animal Research Laboratory, Prospect, N.S. W. 21, Australia (Received 2 April 1973) SUMMIARY 1. The putative neurotransmitter substances 5-hydroxytryptamine (5-HT), noradrenaline (NA), acetylcholine (ACh) and prostaglandins El (PGE,) and E2 (PGE2) have been injected into a lateral cerebral ventricle of the conscious echidna (Tackhyglossus aculeatus); thermoregulatory responses in thermoneutral (ambient dry bulb temperature, Tdb, of approximately 22 C, cool (Tdb of approximately 14C) and warm (Tdb of approximately 26 C) environments were determined. 2. Under all conditions, all of the drugs tested either caused deep body temperature to fall or else had no effect; the fall was brought about by peripheral vasodilatation and/or reduced metabolic rate due to a decrease in shivering or to general relaxation. 3. Responses of the many placental mammals to 5-HT, NA and ACh vary widely, and the echidna, a monotreme, appears to exhibit responses most like those of the rat. 4. Placental mammals tested to date invariably exhibit a hyperthermic response to prostaglandins, and the hypothermic responses of this monotreme is therefore unique. The present study included the confirmation of a hyperthermic response to PGE, and PGE2 in cats and rats. 5. It is concluded that the concept of thermoregulation by amines and other substances in the hypothalamus of placental mammals may also be applicable to the monotremes which have evolved separately from the marsupials and placental mammals for about 18 million years. * Present address: Department of Pharmacology, University of Melbourne, Parkville, Victoria, 352, Australia.

2 544J. A. BAIRD, J. R. S. HALES AND W. J. LANG INTRODUCTION There is now considerable evidence supporting the proposal by Feldberg & Myers (1964) that the amines 5-fiT and NA act as transmitters in the hypothalamus to mediate thermoregulatory responses. More recently, cholinomimetic substances and some of the prostaglandins have also been implicated. To date, experiments have been carried out on common laboratory animals which belong to the so-called 'higher' or placental mammal group. The monotremes have evolved separately from the marsupials and placental mammals for about 18 million years, and depending on the extent to which thermoregulation had already evolved before this divergence, and the extent of subsequent changes in the separate evolutionary lines, the central nervous regulation of body temperature in the monotrematous-, marsupial- and placental-mammals could be similar or quite different. In an attempt to determine whether putative transmitter substances which influence the central nervous regulation of temperature in placental mammals have similar effects on the monotremata, experiments have been performed on the echidna (Tachygloosw aculeatu~s). As thermoregulatory responses to the putative transmitter substances injected into a lateral cerebral ventricle can vary with ambient temperature (Findlay & Thompson, 1968; Bligh, Cottle & Maskrey, 1971), the echidnas were exposed to thermoneutral, cool, and warm environments in different experiments in which the thermoregulatory responses produced by injections of 5-HT, NA, ACh and prostaglandins El (PGE), and E2 (PGE2) into a lateral cerebral ventricle were determined. A preliminary report of this work has been previously published (Hales & Baird, 1972). METHODS Animals. Six echidnas were obtained from Kangaroo Island (South Australia) and from New South Wales. Body weights were 2-4 kg, and all animals appeared to be healthy, maintaining or slightly' increasing body weights. Because other workers have experienced difficulty in keeping echidnas, in captivity the following details are given: Groups of two to four animals were housed in concrete pens (6 ft. x 4 ft.), the floors of which were covered with several inches of clean wood shavings. During the winter, heaters were installed to prevent environmental temperatures falling to low levels that might have induced torpor. The daily feeding ration per'six animals consisted of: two hard boiled egg yolks, 25 g baby cereal with vitamins ('Farex'), 25 g synthetic replacement for bitch's milk ('Animalac'), -25 ml. infant multivitamin drops ('Pentavite'), 35 g lean mince steak and 6 g fine soil. These ingredients were mixed to a soft, almost fluid consistency with water, and were presented each afternoon.

3 THERMOREGULATOR Y TRANSMITTERS IN ECHIDNA 541 Four animals were used for each drug treatment under thermoneutral conditions and two to four animals were used for experiments in warm and cool environments. Surgical preparation. Anaesthesia was induced and maintained with a halothane/2 mixture using an open-circuit apparatus (it might be noted that respiratory frequency was only 1-3 breaths mini-' during anaesthesia compared with 7-14 breaths min-' when conscious). A Collison-type cannula (Feldberg & Sherwood, 1954) modified to contain interchangeable injection shanks (18 s.w.g.) which fitted into a fixed head, was used for cannulation of a lateral cerebral ventricle. Shanks of two lengths were available to allow for variations in ventricular position between animals. During implantation the animal was held so that the snout was horizontal. The skull was exposed by mid-line incision, and the site of cannulation located at 3 mmn anterior to the highest point of the skull, and 2-3 mmn lateral to the sagittal suture. The final depth of implantation varied slightly and was determined by recording pressure changes (via a Statham transducer) within the cannula while it was lowered into the brain. As the tip of the cannula entered the ventricle (at an average depth of 9.5 mm into the brain) a fall in pressure was observed, and a weak pulse coinciding with respiratory changes was then detected. The implanted cannula was held in position with dental acrylic anchored by a stainless-steel screw inserted into the skull. During the same operation a polyethylene re-entrant tube, for subsequent use in monitoring deep body temperature, was implanted into the muscles of the mid-back, so that the tip lay approximately 1 mm below the outer surface of the body. This method was adopted because insertion of a cloacal probe invariably upset the animal and caused changes in its body temperature. Oxytetracycline was injected i.m. for 5 days post-operatively, and all animals were allowed at least one week to recover from surgery before commencement of experiments. Experimental procedure. One or two hr before each experimental session the animal was placed in a climatic room with ambient dry bulb temperature controlled to within +.5 C, and relative humidity between 1 and 2 % Temperatures were measured using 38 s.w.g. copper/constantan thermocouples. At the commencement of the session thermocouples were glued to the mid dorsal surface of a foot and between two toes to provide an index of peripheral vascular activity, the deep body thermocouple was inserted into the guide tube, and a 26 S.W.G. needle, with attached polyethylene tubing containing drug-solutions for injection, was inserted into the ventricular cannula. The animal was then placed in a metal box (28 x 23 x 19 mm) with Perspex lid, and inlet and outlet tubes. Room air was drawn through the box at a measured rate of approximately 41 min-', and a portion of the mixed expired and room air was continually sampled and the 2 and CO2 content analysed for subsequent calculation of metabolic rate, as described by Hales & Hutchinson (1971). Shivering was assessed as being absent, faint, moderate or vigorous. Respiratory frequency was determined by counting external body movements. Environmental temperatures. For the thermoneutral environment ambient dry. bulb temperatures were adjusted so that the temperatures of the extremity skin lay approximately midway between deep body and environmental temperatures. The cool environment was controlled so that the animal showed continual faint to moderate shivering and peripheral vasoconstriction. The warm environment was such that deep body temperature was raised by approximately.5 C, and there was peripheral vasodilatation. The approximate values for the three ambient dry bulb temperatures were cool: 14' C, thermoneutral: 22' C and warm: 26' C.

4 54252J. A. BAIRD, J. R. S. HALES AND W. J. LANG Drug injections. After being prepared for an experiment animals were left undisturbed for 1-2 hr so that stable base line recordings were obtained for all parameters, before any drug treatment. Responses were recorded until after peak effects had been observed, or until no changes had been observed for at least 1 hr. All drugs were injected as pyrogen-free solutions warmed to body temperature just before administration. Doses, expressed in terms of the salt, were administered in -1 ml. volume, and were flushed in with a further -1 ml. sterile -9 % NaCl solution. Noradrenaline acid tartrate (May & Baker Ltd), 5-HT creatinine sulphate (May & Baker Ltd) and acetylcholine chloride (Sigma Chemical Co.) were dissolved in sterile -9 % NaCl solution. Prostaglandins E, and E2 (Ono Pharmaceutical Co. and Upjohn Co.) were prepared and stored as stock solutions of 1 jtg/ml. in -9 % NaCl containing 18,ug Na2CO3 and -1,t~i of radioactive PG per ml1. The latter was included so that the stability of the compounds could subsequently be checked (by means of thin layer chromatography) to show that the particular PG prepared had not changed to another member of the group. Stock solutions were stored at -I1 C, and were thawed and diluted with -9 % NaCl solution immediately before use. The thermoregulatory action of prostaglandin solutions was also checked by injection of appropriate doses, prepared from stock solutions described above, into a lateral cerebral ventricle of conscious cats and iats. In cats, drugs were injected via an indwelling modified Collison cannula, as described by Banerjee, Feldberg & Lotti (1968), while in rats, the indwelling cannula was of the type described by Hayden, Johnson & Maickel (1966). In each species experiments were conducted at a thermoneutral temperature (Tdb~ 24" C). Body temperatures were monitored using thermocouples inserted approximately 1 mm into the rectum in cats and approximately 5 mm into the rectum in rats. At least 2 days were allowed to elapse between drug injections. RESULTS Responses to the various drugs showed some individual variability between animals. For this reason the general response patterns to each drug are shown as group data in Table 1. Fig. 1 shows representative results from one animal, and indicates the variability from the norm which occurred. The brain weight of the echidna was found to be g, and was thus comparable to that of the cat brain. On this basis the doses of drugs used in the present experiments were similar to those previously used in experiments in the cat. Doses of 5-HT and NA lower than those routinely used (125 and 1 jag respectively), were tested in one animal: 1#jg 5-HT produced a response similar to that reported after 125/fig, but of smaller magnitude; 75 jag NA failed to produce any effect, and doses greater than 1 jag were not tested because of restlessness which followed this dose level. 5-Hydroxytryptamine. Injection of 5-HT (125 jag) into the cerebral ventricle failed to produce any effect on deep body temperature under warm or cool conditions, while under thermoneutral conditions deep body temperature fell (Table 1). Vasodilatation occurred after injection

5 T21HERMOREGULATORY TRANSMITTERS IN ECHIDNA 543 of 5-HT at neutral or warm environmental temperatures. Moderate shivering, which was present only in the cool environment, was decreased transiently (Fig. 1), as was metabolic rate. In both thermoneutral and warm environments metabolic rate was unaltered and respiratory frequencies remained unaltered throughout. TAiBLE 1. Effects on deep body and peripheral skin temperatures and on metabolic rate and shivering of injection of 5-hydroxytryptamine, noradrenaline, acetylcholine and prostaglandins E1 and E2 into a lateral cerebral ventricle of the echidna at various ambient temperatures. The direction of responses are indicated by the arrows, a broken arrow representing a small or irregular response, a complete arrow a regular response, and a thick arrow a much stronger response. Parameters that did not change are indicated as being absent (). or at low (i"), medium (-) or high (in) levels Drug and dose 5-Hydroxytryptamine (125 4ug) Noradrenaline (125 gg) Acetylcholine (2 fig) Prostaglandin El (2 fig) Prostaglandin E2 (2 /zg) Function Body temp. Foot temp. Metabolic rate Shivering Body temp. Foot temp. Metabolic rate Shivering Body temp. Foot temp. Metabolic rate Shivering Body temp. Foot temp. Metabolic rate Shivering Body temp. Foot temp. Metabolic rate Shivering Cool Neutral Warm Noradrenaline. Responses following the injection of NA (1 fig) into the cerebral ventricle, were much less consistent than those following 5-HT. On average, there was a small fall in deep body temperature under both warm and cool conditions, and a larger fall under thermoneutral conditions. Associated with the small fall in deep body temperature under cool conditions was either a small vasoconstriction or no change in peripheral vasomotor tone, a decrease from moderate to faint shivering 4' ' I9 I9 I9 t 4t 4 4t I 1 I LiJ I I in

6 544 J. A. BAIRD, J. R. S. HALES AND W. J. LANG and a small fall or no apparent change in metabolic rate. The fall in deep body temperature under thermoneutral conditions was accompanied by vasodilatation and a small decrease in metabolic rate. No changes in parameters other than deep body temperature were detected at warm environmental temperatures (Table 1). No changes in respiratory frequency occurred under any of the conditions used. Acetylcholine. Intraventricular injection of ACh (2,ag) had little effect at warm environmental temperatures. Under cool conditions deep body temperature fell, with associated marked vasodilatation, abolition 5-HT NA ACh PG El PGE2 ( ) (A) (o) Warm muimin 3C -C (26 C) (26 C) (26 C) (2 (25 C) t)29-31,,,,,e",,e_, Thermo- _ neutral h-28-3 (23 C) (22 C) (21 C) (19 C) (21 C; cool (14 C) (14 C) (14 C) (14 C) (14 C) '22-28 hed>s l1b Time after injection (min) Fig. 1. Effects of injecting 125 jug 5-HT, 1 g#g NA, 2 Iug ACh, 2 jg PGEI or 2 fig PGE2 into a lateral cerebral ventricle of an echidna in cool, thermoneutral, and warm environments. Dry bulb ambient temperature for each experiment is shown in brackets. Deep body temperature (), extremity skin temperature (A), oxygen consumption (EL), and shivering (U); height of black bar indicates visually assessed intensity of shivering. Representative results from one animal. of shivering for a short time and decrease in metabolic rate (Fig. 1). In the thermoneutral environment deep body temperature fell, with an associated short-lasting vasodilatation. No shivering was seen and metabolic rate showed no significant change (Table 1). Respiratory frequency was unchanged under all conditions.

7 THERMOREGULATORY TRANSMITTERS IN ECHIDNA 545 Prostaglandin E1. PGE, (2 /tg) injected into the cerebral ventricle decreased deep body temperature over a prolonged period under all conditions tested, but the response in the warm environment was small. Under both cool and thermoneutral conditions foot temperatures rose initially then fell, while in the warm environment little or no change occurred (Fig. 1). Shivering, which was present only in the cool environment, was abolished for a prolonged period; metabolic rate decreased at both cool and thermoneutral environmental temperatures, this response being more marked in the cool (Fig. 1 and Table 1). Respiratory frequency was again unchanged throughout. Prostaglandin E2. Injection of PGE2 (2 jag) into the cerebral ventricle produced marked decreases in deep body temperature in both cool and thermoneutral environments, but failed to produce a consistent effect under warm conditions. Decreases in deep body temperature were accompanied by marked vasodilatation, which was rapid in onset, but changed to vasoconstriction during recovery (Fig. 1). Metabolic rate also decreased during these responses, and in the cool environment shivering was abolished for a considerable time after injection (Table 1). These responses to PGE2 resembled those seen after PGE1 injection, but were generally of shorter duration (Fig. 1). No significant changes in respiratory frequency occurred after injection of PGE2 under any of the conditions tested. Control responses to injection vehicles. Each animal was injected with 2 ml. sterile pyrogen-free 9 % NaCi solution, and with a similar solution containing 3-6 Itg Na2 C3 per ml. These injections consistently failed to produce significant changes in any of the parameters monitored. Responses to prostaglandins E1 and E2 in cats and rats. In each of three cats injection of 1 /tg PGE1 or E2 (in 1 ml. solvent) into a lateral cerebral ventricle produced a hyperthermia of 1P5-2-5 C; the rises in temperature were rapid in onset, beginning within 5 min of injection, and were maintained over 6 hr or longer. During the period in which temperature was increasing, shivering and piloerection were observed, but no vasoconstriction occurred. In three rats, injection of 1 ng PGE1 or E2 (in 1 jll. solvent) into a lateral cerebral ventricle produced a rise in deep body temperature of 1P-2-5 C. These rises were rapid in onset, beginning 2-3 min after injection and were maintained for 3-6 min. Shivering, vasoconstriction and piloerection accompanied the rises in temperature.

8 546 J. A. BAIRD, J. R. S. HALES AND W. J. LANG DISCUSSION The concept that amines and related substances in the hypothalamus in placental mammals may control normal body temperature was proposed by Feldberg & Myers (1964) and elaborated by Bligh et al. (1971) into a neuronal model postulating sites of action of the various putative transmitters in heat loss and heat production and conservation pathways in the C.N.s. This proposal was based on evidence by Vogt (1954) that the amines occur in the hypothalamus in relatively high concentrations, and on the finding that the action of one of the amines appears to be antagonistic to that of the other, when injected into the cerebral ventricles or hypothalamus. Whereas these and other various points of evidence indicate that the amines NA and 5-HT, together with ACh, may act as thermoregulatory transmitters in the placental mammals, little is known of the C.N.S. regulation of normal body temperature in other animal groups. Studies of thermoregulation in the echidna have indicated that this animal does not sweat or pant, but regulates its body temperature chiefly by changes in behaviour and in metabolic rate and peripheral blood flow (Robinson, 1954; Schmidt-Nielsen, Dawson & Crawford, 1966). In the present experiments it has been shown that 5-HT and ACh, in doses similar to those evoking changes in rectal temperature when injected into the cerebral ventricles of placental mammals, produce changes in deep body temperature together with appropriate thermoregulatory responses in the echidna. However, NA fails to produce similar consistent responses. These responses may indicate a thermoregulatory role of endogenous 5-HT and ACh in the echidna, but further evidence is required concerning naturally occurring levels of these putative transmitters in the hypothalamus and the effects of lower doses of the drugs on body temperature when injected directly into the anterior hypothalamus. The hypothermic response to intraventricular injection of prostaglandins of the E series in echidnas contrasts markedly with the hyperthermia seen in placental mammals. This appears to be a true difference, since the same solutions of PGE1 or PGE2 evoked prolonged hyperthermia and shivering in the cat and rat, and therefore confirmed the findings of Milton & Wendlandt (197, 1971), Feldberg & Saxena (1971) and Potts & East (1972). Also, the inhibited heat production and decreased peripheral vasomotor tone in the echidna is the converse to the inhibited heat loss or stimulated heat production seen in a placental mammal (Hales, Bennett, Baird & Fawcett, 1973). A hyperthermic response to prostaglandins of the E series also occurs in the rabbit (Milton & Wendlandt, 1971; Feldberg & Saxena, 1971) sheep (Hales et al. 1973), and monkey (R. D. Myers &

9 THERMOREGULATORY TRANSMITTERS IN ECHIDNA 547 M. B. Waller, personal communication). Thus there is a uniform finding of hyperthermia evoked by PG's in placental mammals even though the responses to NA, 5-HT and ACh in these species are not uniform. The preliminary finding of an increase in body temperature following PGE2 in one echidna (Hales & Baird, 1972), could not be reproduced, all three subsequent animals invariably exhibiting a decrease in body temperature. The levels of PGE-like substances in the c.s.f. increase during pyrogeninduced fever (Feldberg & Gupta, 1973), and Feldberg & Milton (1973) have proposed that prostaglandins are released by the action of pyrogen and are concerned in the genesis of fever. Consistent with this theory is the observation that anti-pyretics which prevent the release of prostaglandins modify a bacterial pyrogen-induced fever, but are without effect on prostaglandin-induced fever (Milton & Wendlant, 1971). The echidna also develops fever in response to a bacterial pyrogen (i.v. E. typhi), and this can be prevented by the anti-pyretic drug paracetamol (unpublished observation). Thus the hypothermic response of the echidna to PGE1 and PGE2 is in conflict with the proposed roles of the prostaglandins in the genesis of fever in the placental mammals. No explanation can yet be offered for this apparent difference. From the present experiments it is clear that the echidna exhibits thermoregulatory responses to injection into the cerebral ventricle of substances postulated to be neurotransmitters involved in hypothalamic control of body temperature in placental mammals. However, further investigation, e.g. of endogenous levels of the putative neurotransmitters in the hypothalamus of the echidna, is necessary before any conclusions can be reached concerning whether this 'primitive' species complies with theories of hypothalamic regulation of body temperature in placental mammals. The authors are grateful to the Ono Pharmaceutical Co. (Osaka) and the Upjohn Co. (Kalamazoo) for the supply of prostaglandins, and to Drs R. I. Cox and G. D. Thorburn for assistance in preparation and checking the identity of these compounds; also to Messrs A. A. Fawcett and J. W. Bennett for their technical assistance, and to Mr E. Worrell of the Australian Reptile Park for assistance in obtaining echidnas. REFERENCES (1968). Effect on body temperature BANERJEE, U., FELDBERG, W. & LOTTI, V. J. of morphine and ergotamine injected into the cerebral ventricles of cats. Br. J. Pharmac. Chemother. 32, BLIGE, J., COTTLE, W. H. & MASKREY, M. (1971). Influence of ambient temperature on the thermoregulatory responses to 5-hydroxytryptamine, noradrenaline and acetylcholine injected into the lateral cerebral ventricles of sheep, goats and rabbits. J. Physiol. 212,

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