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J. Physiol. (1979), 288, pp. 393-399 393 With 3 text-ftgures Printed in Great Britain THE EFFECT

1 J. Physiol. (1979), 288, pp With 3 text-ftgures Printed in Great Britain THE EFFECT OF DOPAMINE ON SHIVERING IN THE RAT BY HAZEL W. MARSHALL AND H. B. STONER From the M.R.C. Trauma Unit, Stopford Building, Manchester University Medical School, Oxford Road, Manchester M13 9PT (Received 25 August 1978) SUMMARY 1. Dopamine ( Etmole) injected into a lateral cerebral ventricle inhibited shivering and lowered core temperature in rats at an ambient temperature of 0-5 0C. 2. These effects were inhibited by the dopamine antagonist, pimozide, and imitated by the dopamine agonists, piribedil and apomorphine. 3. Pimozide itself had no effect on shivering. 4. It is concluded that while there are inhibitory dopamine receptors on the cold sensor-shivering pathway in the rat they are not concerned in the normal function of this pathway. INTRODUCTION While there is general agreement that acetylcholine, noradrenaline and serotonin are concerned as neurotransmitters in the central control of body temperature, the role of dopamine, if it has one, is obscure. Nevertheless, centrally administered dopamine can affect the body temperature of several species (see Cox, 1978) and recently Cox, Kerwin & Lee (1978) have produced evidence suggesting that it has a role in the heat sensor-heat loss pathway. In our experiments we have studied the effects of dopamine on the cold sensor-shivering pathway in the rat. The results showed that dopamine can affect this pathway but its physiological role is uncertain. METHODS Male albino Wistar rats of the Porton strain fed on either M.R.C. diet 41B (Bruce & Parkes, 1956) or Porton Mouse Diet (Oakes Millers, Congleton, Cheshire) were used. They had been kept from weaning at the age of 3-4 weeks in an 'C environment with controlled lighting giving 12 h light per day ( h). The guide tubes devised by Goodrich, Greehey, Miller & Pappenheimer (1969) were inserted in the skull as previously described (Stoner, 1977). The experiments were performed 7 days later (average; range 3-13 days) when the rat weighed 232 g (average; range g). On the day of the experiment the ventral caudal artery was cannulated, under ether anaesthesia, with polyethylene tubing (PE 10; Clay Adams, Parsippany, N.J., U.S.A.) and the pressure recorded with a transducer (Type 4-422; Bell & Howell, Basingstoke) and Devices (Ormed Engineering Ltd, Welwyn Garden City) equipment. The electromyogram (e.m.g.) and core (Tc) and ambient (T.) temperatures were recorded as before (Stoner, 1971). At least an hour elapsed between the end of the anaesthesia and the start of the experiment. The rat was placed in an environmental cabinet (Stoner, 1971) and shivering was induced by reducing T. to 0-5 IC. Injections into a lateral cerebral ventricle were made through the guide as described previously (Stoner, 1977). This route was chosen in preference to injecting into the hypothalamus itself because of the damage which can be produced when probes are inserted into it (unpublished observations) and because materials introduced into a lateral ventricle are known to reach the parts of the brain concerned in thermoregulation.

394 H. WV. MARSHALL AND H. B. STONER At the end of the experiment the rat was anaesthetized with ether, injected with 1o 1Ul. Indian ink through the guide tube and then killed.

2 394 H. WV. MARSHALL AND H. B. STONER At the end of the experiment the rat was anaesthetized with ether, injected with 1o 1Ul. Indian ink through the guide tube and then killed. The brain was fixed by perfusion with formalin: glacial acetic acid: methanol (1: 1: 8 v/v) mixture and the position of the injection probe verified by dissection and histological examination. In experiments to test the effects of pimozide alone, the Ta threshold for the onset of shivering was determined by the method described previously (Stoner, 1971) in rats weighing g, except that the reduction in Ta began from an ambient temperature of 25 'C. Dopamine hydrochloride (Sigma Chemical Co.) was dissolved in Krebs-Henseleit Ringer Solution or M-NaCl shortly before use. Piribedil methane sulphonate (Servier Laboratories) and apomorphine hydrochloride (Maefarlan-Smith Ltd) were dissolved shortly before injection in M-NaCl and deionized water respectively. The three solutes were injected at a concentration of 1O flmole/ml. The three solvents, tested at maximum volume, had no effect on shivering. Phentolamine mesylate (Rogitine; Ciba) was dissolved in M-NaCl to give a concentration of 15 #umole/ml. Solutions injected into a lateral ventricle were filtered through Millipore filter GSWP shortly before use. Pimozide (Janssen Pharmaceuticals) was prepared as described by Cox et al. (1978) and used at a concentration of 108 #smole/ml. The statistical methods which were used are described by Snedecor & Cochran (1967). RESULTS Effect of dopamine. The injection of dopamine into a lateral cerebral ventricle of a conscious rat in a 0-5 'C environment inhibited shivering, often completely, and caused a fall in TP. Rats varied in their sensitivity but an effect was often seen with 0-05 molee (Fig. 1) and larger doses produced greater responses. A dose of 0-2 molee was always effective. The inhibition usually occurred within 15 s of the injection but longer delays of up to 60 s were sometimes seen. The duration of the response varied but did not exceed 9 min even after the injection of 0-20 #mole, the largest dose used. RT fell between 0-4 and 1-0 'C after O-10 #mole and the nadir was usually at the end of the effect on shivering. The blood pressure did not usually change but in three out of seventeen rats it rose 8-20 % after doses of mole and above. (It is important to monitor the blood pressure since shivering can be inhibited in the rat by both rises and falls in pressure; unpublished results.) Effect of dopamnine agonists. Both piribedil (Corrodi, Farnebo, Fuxe, Hamburger & Ungerstedt, 1972) (Fig. 2) and apomorphine (Ernst, 1967) reproduced the effects of dopamine. Shivering was inhibited, TcJ fell and there was no change in blood pressure. For both compounds 0-10 #mole was an effective dose. These agonists often produced more striking inhibition than equimolecular amounts of dopamine. Effect of antagonists. After first determining the effect of centrally administered dopamine in a shivering rat, the antagonist pimozide (Anden, Butcher, Corrodi, Fuxe & Ungerstedt, 1970) was injected intraperitoneally, 1-08 /Zmole/kg body wt. After 2 h at 20 'C to allow any a blockade action of the drug to disappear, T. again reduced was to 0-5 'C and when the rat was shivering the doses of dopamine were retested. This dose of pimozide completely antagonized the effect of 0-05 /Zmole dopamine (Fig. 1) and greatly reduced the effects of higher doses (Fig. 3). The depression of Tc by dopamine was prevented or reduced. An effect of pimozide itself was sought by determining the threshold Ta for the onset of shivering before and 2 hr aftc the i.p. injection of 1-08,umole pimozide/kg. No significant effect was observed on either the threshold or the slope of the regression line relating the intensity of shivering to T. (before: threshold *62 'C (mean +

DOPAMINE AND SHIVERING 395 S.E. of mean), slope - 0049 + 0 007 (mean + S.E. of mean) after: threshold 17*4 + 1*08 TC, slope - 0*043 + 0006; differences not significant in paired t test, n = 6).

3 DOPAMINE AND SHIVERING 395 S.E. of mean), slope (mean + S.E. of mean) after: threshold 17*4 + 1*08 TC, slope - 0* ; differences not significant in paired t test, n = 6). Pimozide had a mild sedative effect and Tc did not rise on exposure to cold so well after pimozide as before. These differences were about the same as those seen when a control rat was tested with this cycle of Ta changes twice in the same day. E Ui 200 Xi 100_ o 3' ul V.mọk 200- Fig. 1. Effect of 0-05 molee dopamine injected at the arrow into a lateral cerebral ventricle of a rat on shivering (showni as the integrated electromyogram volts2 in arbitrary units, time constant 1-7 sec) and blood pressure (B.P., mmhg); A, before and B, 2 h after the i-p injection of 1 08 flmole pimozide/kg. T. 3 'C. V~m voluntary movement. The effect of molee phentolamine injected into a lateral cerebral ventricle on the response to dopamine was also examined. The interval between the injections of phentolamine and dopamine was about 30 min (range mmn). Phentolamfine

constant 1-7 sec) and B.P. (mmhg). T, = 4 'C. * M~~~~~s.0 - - e E f i AM,. N ~ w 2~~~~00; E 200~ 0 VṂ._ 200r 100 Fig. 3.

4 s_ 396 H. WV. MARSHALL AND H. B. STON,ER > r, W,M. X.. >W >~ ~ ~ ~ ~_ M i S Ui 0 - C_ adi Fig. 2. Effect of 0 lo 1umole piribedil injected at the arrow into a, lateral cerebral ventricle of a rat on shivering (shown as the integrated electromyogram vot2in arbitrary units: time constant 1-7 sec) and B.P. (mmhg). T, = 4 'C. * M~~~~~s e E f i AM,. N ~ w 2~~~~00; E 200~ 0 VṂ._ 200r 100 Fig. 3. Effect of 010,mole dopamine injected at the arrow into a lateral cerebral ventricle of a rat on shivering (shown as the integrated electromyogram volts2 in arbitrary units; time constant 1-7 sec) and B.P. (mmhg); A, before and B, 2 hi after the i.p. pimozide/kg. Ta = 3 'C. Vin. = voluntary movement. injection of 1-08 Itinole

5 DOPAMINE AND SHIVERING 397 modified or inhibited shivering, making it difficult to assess changes in the response to dopamine. Out of seven rats tested, the inhibitory effect of dopamine was decreased in two, increased in two and unchanged in the remainder. DISCUSSION Our results confirm those of Stone & Mendlinger (1974) and show that centrally administered dopamine inhibits shivering in the rat. With similar inhibition reported in the cat (Hatton & Wolf, 1977) and goat (De Roij, Frens, Bakker & N6meth, 1977), this may be a general property of dopamine. The effect is not secondary to an action on the blood pressure and it is unlikely that acetylcholinergic neurones are concerned in the response (Cox & Lee, 1978). Dopamine is less active than noradrenaline (unpublished results) but it is unlikely that it acts on central a-receptors or is taken up by central noradrenergic terminals and converted to noradrenaline. Noradrenaline inhibits shivering by an action on a-receptors (Stoner, 1978) which is prevented by phentolamine. In the present experiments the results with phentolamine were obscured by its hypothermic action (Feldberg & Saxena, 1971) and by its effects on dopaminergic receptors (Kennedy & Burks, 1974) but a much more consistent interference would have been expected if dopamine had been acting on a-receptor sites. The inhibition of shivering by piribedil and apomorphine and the antagonism of dopamine by pimozide point to an action on dopamine receptors. Dopamine, therefore, like noradrenaline, can inhibit shivering, and the existence of central dopamine receptors with this effect suggests that there may be dopaminergic neurones with access to the cold sensor-shivering pathway. Does dopamine play a part in the normal control of shivering, e.g. in the threshold mechanism (Bligh, 1972)? As pimozide did not alter the Ta threshold for the onset of shivering or the gain of the response, this seems unlikely. Such a view is supported by previous work with 6-hydroxydopamine by ourselves (Stoner, 1977; Stoner & Marshall, 1977) and others (Bloom, Algeri, Groppetti, Revuelta & Costa, 1969; Simmonds & Uretsky, 1970; Van Zoeren & Stricker, 1976). We used the results to exclude noradrenergic nerve terminals from a role in the shivering threshold mechanism but they can also be used in the same way against dopamine since 6-hydroxydopamine destroys both noradrenergic and dopaminergic terminals (Ungerstedt, 1973). Pre-treatment with 6-hydroxydopamine injected into a lateral cerebral ventricle or into the hypothalamus does not alter the Ta threshold for the onset of shivering and only decreases the gain of the response if it is injected bilaterally into the posterior hypothalamus or dorsal noradrenergic bundle. However, intraventricular 6-hydroxydopamine does increase the T, threshold for the opening of the A-V anastomoses in the tail (Stoner, 1977) which could support the views of Bligh, Tollerton & Smith (1978) and Cox et al. (1978) that dopamine plays a part in the heat sensor-heat loss pathway but would equally well support a role for noradrenaline in this pathway. Is the inhibition of shivering by dopamine a purely pharmacological effect without physiological or pathophysiological counterpart? Shivering is affected by other factors than Ta and T. It is inhibited by changes in blood pressure (Mott, 1963; Little & Stoner, 1977), by injuries such as limb ischaemia (Stoner, 1971) and by 'arousal' (Stitt, 1976). The effects of the last two factors are thought to be mediated by noradrenergic neurones (Stoner, 1977; Stoner & Marshall, 1977; Stitt, 1976) but

6 398 H. ITV. MARSHALL AND H. B. STONER dopamine could be concerned in the effects of these non-thermal inputs. This, and the identification of any dopaminergic neurones involved, remains to be investigated. Our thanks are due to Dr B. Cox for his advice. We also wish to thank Servier Laboratories Ltd, Greenford, Middlesex, Janssen Pharmaceuticals Ltd, Marlow, Bucks and Maefarlan Smith Ltd, Edinburgh for gifts of piribedil, pimozide and apomorphine respectively. REFERENCES ANDEN, N. E., BUTCHER, S. G., CORRODI, H., FUXE, K. & UNGERSTEDT, U. (1970). Receptor activity and turnover of dopamine and noradrenaline after neuroleptics. Eur. J. Pharmacol. 11, BLIGH, J. (1972). Neuronal models of mammalian temperature regulation. In Essays on Temperature Regulation, ed. BLIGH, J. & MOORE, R. E., pp , Amsterdam: North Holland. BLIGH, J., TOLLERTON, A. J. & SMITH, C. A. (1978). The effect of intracerebroventricularly injected noradrenaline on ear skin vasomotor tone of sheep. In New Trends in Thermal Physiology. ed. HOUDAS, Y. & GUIEU, J. D., pp Paris: Masson. BLOOM, F. E., ALGERI, S., GROPPETTI, A., REVUELTA, A. & COSTA, E. (1969). Lesions of central norepinephrine terminals with 6-OH-dopamine: biochemistry and fine structure. Science, N. Y. 166, BRUCE, H. M. & PARKES, A. S. (1956). In 'Correspondence'. J. Anim. Techns Ass. 7, 54. CORRODI, H., FARNEBO, L.-O., FUXE, K., HAMBURGER. B. & UNGERSTEDT, U. (1972). ET495 and brain catecholamine mechanisms: evidence for stimulation of dopamine receptors. Eur. J. Pharmacol. 20, Cox, B. (1978). Dopamine. In Drugs and Body Temperature, ed. LOMAX, P. and SCH6NBAUM, E. New York: Marcel Dekker. (In the Press.) Cox, B., KERWIN, R. & LEE, T. F. (1978). Dopamine receptors in the central thermoregulatory pathways of the rat. J. Physiol. 282, Cox, B. & LEE, T. F. (1978). Is acetylcholine involved in a dopamine receptor mediated hypothermia in mice and rats? Br. J. Pharmacol. 62, DE RoIJ, T. A. J. M., FRENS, J., BAKKER, J. & NEMETH, F. (1977). Thermoregulatory effects of intraventricularly injected dopamine in the goat. Eur. J. Pharmacol. 43, 1-7. ERNST, A. M. (1967). Mode of action of apomorphine and dexamphetamine on gnawing compulsion in rats. Psychopharmacologia. 10, FELDBERG, WV. & SAXENA, P. N. (1971). Effects of adrenoceptor blocking agents on body temperature. Br. J. Pharmacol. 43, GOODRICH, C. A., GREEHEY, B., MILLER, T. B. & PAPPENHEIMER, J. R. (1969). Cerebral ventricular infusions in unrestrained rats. J. appl. Physiol. 26, HATTON, D. G. & WVOLF, H. H. (1977). Effects of intra hypothalamic 6-hydroxydopamine on central thermoregulatory responses to (+) and (-)-norepinephrine and dopamine. Neuropharmacol. 16, KENNEDY, M. S. & BURKS, T. F. (1974). Dopamine receptors in the central thermoregulatory mechanisms of the cat. Neuropharmacology. 13, LITTLE, R. A. & STONER, H. B. (1977). Effect of baroreceptor input to the central nervous system on shivering in the cat. Proc. XXTVII Internat. Congress Physiol. Sciences p Paris: Comite National Frangais des Sciences Physiologiques. MOTT, J. (1963). The effects of baroreceptor and chemoreceptor stimulation on shivering. J. PhysiOl. 166, SIMMONDS, M. A. & URETSKY, N. J. (1970). Central effects of 6-hydroxydopamine on the body temperature of the rat. Br. J. Pharmacol. 40, SNEDECOR, G. WV. & COCHRAN, WV. G. (1967). Statistical Methods. 6th ed. Ames, Iowa: Iowva University Press. STITT, J. T. (1976). Inhibition of thermoregulatory outflow in conscious rabbits during periods of sustained arousal. J. Physiol. 260, 31-32P. STONE, E. A. & MENDLINGER, S. (1974). Effect of intraventricular amines on motor activity in hypothermic rats. Res. Comm. chem. Path. Pharmacol. 7,

DOPAMINE AND SHIVERING 399 STONER, H. B. (1971). Effect of injury on shivering therinogenesis in the rat. J. Physiol. 214, 599-615. STONER, H. B. (1977).

7 DOPAMINE AND SHIVERING 399 STONER, H. B. (1971). Effect of injury on shivering therinogenesis in the rat. J. Physiol. 214, STONER, H. B. (1977). The role of catecholamine in the effects of trauma on thermoregulation, studied in rats treated with 6-hydroxydopamine. Br. J. exp. Path. 58, STONER, H. B. (1978). The effect of ascending noradrenergic nerve fibres on shivering in the rat. In New Trend8 in Thermal Phy8iology, ed. HOUDAS, Y. and GuIEu, J. D., pp Paris: Masson. STONER, H. B. & MARSHALL, H. W. (1977). Localization of the brain regions concerned in the inhibition of shivering by trauma. Br. J. exp. Path. 58, UNGERSTEDT, U. (1973). Selective lesions of central catecholamine pathways: application in functional studies. Neurosci. Re8. 5, VAN ZOEREN, J. G. & STRICKER, E. M. (1976). Thermal homeostasis in rats after intrahypothalamic injections of 6-hydroxydopamine. Am. J. Physiol. 230,

Hypothermia produced in mice by histamine

Br. J. Pharmac. (1971), 42, 205-214. Hypothermia produced in mice by histamine acting on the central nervous system GRAHAM G. SHAW Pharmacology Laboratories, Department of Pharmacy, University of Nottingham,