J. Phygsiol. (1974), 236, pp. 539-548 539 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. 2149, 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); thermo- regulatory responses in thermoneutral (ambient dry bulb temperature, Tdb, of approximately 220 C, cool (Tdb of approximately 140C) and warm (Tdb of approximately 260 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 mono- treme 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 180 million years. * Present address: Department of Pharmacology, University of Melbourne, Parkville, Victoria, 3052, Australia. 54040J. 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 180 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 evo- lutionary 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 trans- mitter substances which influence the central nervous regulation of temperature in placental mammals have similar effects on the mono- tremata, 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 injec- tions 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'), 0-25 ml. infant multivitamin drops ('Pentavite'), 350 g lean mince steak and 60 g fine soil. These ingredients were mixed to a soft, almost fluid consistency with water, and were presented each afternoon. 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/02 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 inter- changeable 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 10 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 + 0.50 C, and relative humidity between 10 and 20 % 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 (280 x 230 x 190 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 02 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 0.50 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. 54252J. A. BAIRD, J. R. S. HALES AND W. J. LANG Drug injections. After being prepared for an experiment animals were left un- disturbed 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 0-1 ml. volume, and were flushed in with a further 0-1 ml. sterile 0-9 % 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 0-9 % NaCl solution. Prostaglandins E, and E2 (Ono Pharmaceutical Co. and Upjohn Co.) were prepared and stored as stock solutions of 100 jtg/ml. in 0-9 % NaCl containing 18 ,ug Na2CO3 and 0-1 ,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 -I100 C, and were thawed and diluted with 0-9 % 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.