thermoregulatory mechanisms proposed by Myers & Yaksh (1969). According to their theory a cholinergically coded heat production pathway

Transcription

1 J. Physiol. (1972), 22, pp With 3 text-figures Printed in Great Britain THERMOREGULATORY EFFECTS OF INTRAHYPOTHALAMIC INJECTIONS OF ADRENERGIC AND CHOLINERGIC SUBSTANCES AT DIFFERENT ENVIRONMENTAL TEMPERATURES BY DAVID D. AVERY From the Department of Psychology, Colorado State University, Ft. Collins, Colorado, U.S.A. (Received 15 March 1971) SUMMARY 1. Rats were unilaterally implanted with chronic indwelling cannulae directed towards the medial preoptic area of the hypothalamus. Carbamylcholine chloride (carbachol), noradrenaline, and saline were microinjected in volumes of 5 psl. at each of three environmental temperatures, 5, 24, and 35 C. 2. At an ambient temperature of 24 C injections of carbachol significantly raised core temperature, whereas injections of noradrenaline to the same sites significantly lowered temperature. 3. Injections of carbachol did not affect core temperature at 35 C, but injections of noradrenaline defended against the normal hyperthermia observed in this environment. 4. Hyperthermia resulted from hypothalamic injections of both substances at an ambient temperature of 5 C. 5. The data generally support a neurochemical model for hypothalamic thermoregulatory mechanisms proposed by Myers & Yaksh (1969). According to their theory a cholinergically coded heat production pathway courses from the anterior to the posterior hypothalamus. This pathway is activated by 5-HT and inhibited by noradrenaline in the anterior hypothalamus. The observation in the present experiments of hyperthermia following adrenergic stimulation at 5 C cannot be accounted for by this model and therefore an addition to the model is proposed. It is suggested that a heat loss mechanism is also located, at least partially, in the anterior hypothalamus and that this system is inhibited by noradrenaline. Thus a thermal balance is established via an interaction of the two systems. 9-2

2 258 DA VID D. A VERY INTRODUCTION Several studies have pointed to the importance of ambient temperature as a factor affecting thermal responsiveness of animals to systemically injected drugs (Shemano & Nickerson, 1958; Buckley, Heading & Taylor, 1969; Paolino & Bernard, 1968). On the basis of such data it could reasonably be expected that the temperature of the environment will affect thermal responses to centrally injected substances. This notion derives support from Paolino & Bernard's (1968) data with respect to the effects on body temperature of injecting morphine in the anterior hypothalamus while the animals were maintained in environments of differing temperatures. At an ambient temperature of 5, hypothermia developed; at 32 C, there was an increase in core temperature; at thermal neutrality, (24 C) body temperature was not altered. More recently, Bligh, Cottle & Maskrey (1971) found that thermal responsiveness to intraventricular injections of monoamines and cholinergically active substances was influenced by ambient temperature. In addition, it has been shown that both brain levels of noradrenaline (Ingenito & Bonnyeastle, 1967; Ingenito, 1968) and the rate of turnover of catecholamines in the brain (Gordon, Spector, Sjoerdsma & Udenfriend, 1966; Simmonds & Iversen, 1968) are affected by both positive and negative deviations in ambient temperature from thermal neutrality, although these results are not unequivocal (Moore, Calvert & Brody, 1961; Gibson, McGeer & McGeer, 1966; Cox & Potkonjak, 1967; Corrodi, Fuxe & Hokfelt, 1967). In the case of central injections into discrete brain loci of neurohumours or their mimetics which have been shown to be involved in thermoregulation (Avery, 197, 1971; Myers & Yaksh, 1968, 1969; Sharpe & Myers, 1969), the effects of ambient temperature have not been investigated, although it has been proposed that this may be a critical factor (Avery, 197; Myers & Yaksh, 1968). The present experiments were therefore conducted to determine whether thermoregulatory responses to intrahypothalamic injections of neurohumours would be influenced by the ambient temperature at the time of the injection. Specifically, changes in core temperature as a result of injecting noradrenaline and carbachol into the anterior hypothalamus of rats maintained at each of three different environmental temperatures were investigated. METHODS Eight male albino rats weighing between 35 and 45 g at the beginning of the experiment were unilaterally implanted with double-wall cannulae directed towards the anterior-preoptic hypothalamic area. The outer cannula was constructed from 22-gauge stainless-steel tubing and the inner cannula from 3-gauge stainless-steel

3 NEUROCHEMICAL SYSTEMS IN THERMOREGULATION 259 tubing. The inner cannula extended 1 mm below the outer cannula. A microlitre syringe (Hamilton-Stoelting, Inc.) was used for all injections. Subjects were individually housed in a constantly illuminated room, the temperature of which was controlled at 24 C +.5 C, with food and H2 available at all times. Each animal was allowed at least 1 days post-operative recovery time before the chemical injections. Each subject received separate applications of two substances and control injections of normal saline at each of three different ambient temperatures, 24 C +. 8 C, 35 C + -5 C, and 5 C +.5 C. The substances injected were noradrenaline bitartrate 15 /sg (an adrenergic substance), and carbamylcholine chloride 3 /zg (carbachol, a cholinergic substance). These doses were chosen on the basis of doseresponse data from previous experiments (Avery, 197, 1971). Each substance was dissolved in normal saline and each injection was -5 jed. At least 72 hr elapsed between injections. The experiments at 24 C were conducted in the animals' home cages. At 5 and 35 C the animals were injected and immediately placed in individual cages in an environmental chamber (Hot Pack, Shearer Gillette Inc.) The relative humidity under all conditions was 2 + 5%. Core temperature was measured using a telethermometer (model no. 46 TUC., Yellow Springs Instruments Inc.). The probe was inserted 6 cm beyond the anal orifice and a period of 45-6 sec was allowed for thermal equilibrium before the temperature was recorded. Measures were made just before the injection, and at 5, 1, 2, 3, 4 and 5 hr after injection. At ambient temperatures of 5 and 35 C the animals were returned to their home cages 5 hr after stimulation. RESULTS Histology At the end of the experiments each animal was killed via standard 1% formol-saline procedures. The brain was removed and sections were cut on a freezing microtome. The sections were stained using the luxol-fast-blue procedure. The locations of the cannula tips for each animal are depicted in Fig. 1. In four of the animals, the cannula tips were located in the medial preoptic area of the hypothalamus. The remaining four animals had placements mm anterior to the medial preoptic area. Of these animals one had a cannula in the diagonal band of Broca -4 mm medial to the preoptic area. The data for these animals were included because their thermal responses in each condition were not appreciably different from those of animals with medial preoptic placements. In addition, on the basis of Myers's (1966) data with respect to spread of centrally injected solutions, it can be reasonably concluded that liquids injected in the more anterior loci did diffuse to the medial preoptic area, and vice versa. Temperature The effects on colonic temperature during the first 5 hr after injecting noradrenaline 15 /ug, carbachol 3,sg, and control solutions of saline at each of the three ambient temperatures, 5, 24 and 35 C, are in Fig. 2. Differences in mean maximum thermal responsiveness to the three injection conditions in each ambient thermal condition, as presented in

4 26 DAVID D. AVERY Fig. 3, were tested using the Matched Pairs Signed Ranks Test (Siegel, 1956). In the 24C environment injections ofnoradrenaline significantly reduced core temperature below those levels observed after saline injections (P <.5), whereas cholinergic stimulation caused a significant hyperthermia (P <.5). With both neurochemical substances deep body temperature was altered during the first 3 min after injection. However, the Fig. 1. SJX~~~~~ Cairmula tip locations for each of the animals used in the experiments (after Pellegrino ; Cushman, 1967). length of time the induced temperature changes persisted was different for the two substances. Cholinergically induced hyperthermia. persisted throughout the 5 hr of the experiments, but adrenergically induced hypothermia, was not observed after 2 hr. At an ambient temperature of 5eC, both noradrenaline and carbachol produced an increase in colonic temperature as compared with the control injection (P < h5)ụndoftherimets butedimental conditions the hyperthermia persisted throughout the experimental period. Injections of noradrenaline significantly depressed the increases in body temperature usually observed in the 35 C environment as compared with injections of carbachol (P <.5) and with saline (P <.5). The effects associated with these latter two substances were not significantly different (P >.1). Under all three injection conditions, the mean maximum increases in colonic temperature occurred at 2-3 hr after injection. During the remaining 3 hr of the experiment, body temperatures were maintained

5 NEUROCHEMICAL SYSTEMS IN THERMOREGULATION 261 c +3- E +2 LI g +1.'ao lx I. V E +2 LI._ o +1- '5.E C co ' -1- IX Inj. Inj. --- Saline Ambient temp.=5 C - Carbachol -.Noradrenaline... I I I I Time (hr) 4- Iur --- Saline Ambient temp.=24 C -Carbachol Noradrenaline O_ -.1 u,.. _ I I I I I Time (hr) 5 E +2-1/... U._ o +1-._ C 6 bo C -C Ix ---Saline Ambient temp.=35 C -Carbachol -. Noradrenaline 1 I I I I I Inj. Time (hr) Ffg. 2. Mean thermal responses of eight animals during the first 5 hr after they were intrahypothalamically injected with carbamylcholine chloride (carbachol), noradrenaline, and saline and placed in environments of 5, 24 and 35 C.

6 262 DA VID D. A VER Y at a fairly constant level under cholinergic and control conditions, whereas during this same period under noradrenergic conditions core temperatures decreased still further. L c I+2 c +3 E * Saline Noradrenaline Carbachol E e C 4, UO E +1 E -c -1 E-~ Ambient temperature (C) Fig. 3. Mean (± s.e.) maximum changes in colonic temperature of eight animals after intrahypothalamic injections of carbamylcholine chloride (carbachol), noradrenaline, and saline at each of three environmental temperatures. DISCUSSION Observations of adrenergically induced hypothermia and increased core temperatures after injections of carbachol at an ambient temperature which is thermally neutral for the rat replicate earlier findings (Avery, 197, 1971). As discussed in previous papers these results support the view that there are both noradrenergic and cholinergic mechanisms in the anterior hypothalamus of the rat which are, at least in part, responsible for the maintenance of a stable core temperature. Recently, Myers & Yaksh (1969) have proposed a neurochemical model of thermoregulatory mechanisms in the hypothalamus. This model, though initially developed on the basis of chemical mapping of the primate hypothalamus, would seem to be applicable to the rat as well, although it can not account for all of the data from the present experiments. These investigators suggest that a cholinergically coded heat production pathway extends from the anterior to the posterior hypothalamus. This pathway is activated by 5-hydroxytryptamine (5-HT) and is inhibited via a blocking action of noradrenaline. They hypothesize that 5-HT is released from

7 NEUROCHEMICAL SYSTEMS IN THERMOREGULATION 263 neurons in the anterior hypothalamus as a function of either lowered brain temperature or pyrogen activity. Noradrenaline, on the other hand, is released when brain temperature goes up, or as a function of antipyretics, and heat production is inhibited via either an interference with the release of 5-HT or a direct blocking of the cholinergic heat production pathway. Data gathered at various ambient temperatures in the present experiments preponderantly support the model even though there has not been as wide a mapping of the rat diencephalon as was done by Myers-Yaksh in the monkey. As has already been pointed out, injections of carbachol in a thermally neutral environment produce hyperthermia. In addition, at an ambient temperature of 5 C cholinergic injections were found to lead to a rise in core temperature above control levels. Thus the notion of a cholinergically mediated heat production pathway which is at least in part located in the anterior hypothalamus is well supported after cholinergic stimulation at both 5 and 24 C. Even though colonic temperatures after cholinergic stimulation were not elevated above those observed after injections of saline at an ambient temperature of 35 C, this is not inconsistent with the model. It could be that, at extremely high ambient temperatures, the normal inhibitory influence of noradrenaline on heat production overrides any effect associated with an artificial increase in the concentration of cholinergic substances in the anterior hypothalamus. These data would then support the notion that noradrenaline interferes directly with cholinergic transmission, rather than having an effect on the release of 5-HT. If this latter notion was true one would expect that hyperthermia above control levels would result from cholinergic injections even at high ambient temperatures. In the ambient environment above thermoneutrality for the rat, noradrenaline effects again support the Myers-Yaksh model. Adrenergic injections significantly reduced the rises in body temperature as compared with the levels reached in the control condition at 35 C as was the case with similar injections at thermoneutrality. This effect could be associated with inhibition of heat production as stated in the model. However, when we consider the fact that in a 5 C environment noradrenergic injections resulted in core temperatures significantly higher than those observed after saline, the Myers-Yaksh model provides no suggestion as to how this phenomenon might be occurring. In light of this latter result the following additions to the Myers-Yaksh model are proposed. Assuming that the model is in fact correct, but incomplete, then the only possible way for central applications of noradrenaline to produce hyperthermia in the cold environment is via an interaction with a heat loss mechanism which is at least partially located in the anterior hypothalamus. It is suggested that this heat loss mechanism is also

8 264 DA VID D. A VER Y inhibited by a release of noradrenaline. Myers and Yaksh's data would suggest that such a heat loss mechanism is probably not coded with acetylcholine, 5-HT, or noradrenaline. The following is a statement of possible ways in which hypothalamic neurohumoral manipulations and ambient temperature in the present experiments may have been interacting in the regulation of body temperature. At both thermoneutrality and lower ambient temperatures heat production may have been incompletely inhibited by noradrenaline; thus central applications of carbachol activated the heat production pathway. At high ambient temperatures the heat production pathway was inhibited sufficiently so that increases in cholinergic levels could not 'override' this normal inhibition. On the other hand, increases in noradrenaline levels at both thermoneutrality and higher ambient temperatures could have inhibited both heat production and heat loss, but differentially; that is, lowered colonic temperatures were observed because the reduction in heat production was greater than the reduction in heat loss. At low ambient temperatures, the reverse may have been true. Central applications of noradrenaline again inhibited both heat production and heat loss. The effect on colonic temperature, however, was an increase. This occurred because heat loss inhibition was greater than heat production inhibition - probably because 5-HT release was high enough to 'override'the inhibitory effects of noradrenaline on the cholinergically mediated heat production pathway. It is obvious that this proposition is very tentative; however, it should provide the impetus for future investigation and a framework for analysis of subsequent data. Specifically, the experiment needs replication which includes measures of metabolic rate, peripheral vasomotor activity, shivering, etc., to verify the heat production and heat loss mechanisms postulated in the preceding discussion. Bligh and his co-workers (1971) came to a similar conclusion that heat loss mechanisms as well as heat production are located in the hypothalamus. In an extensive series of experiments on the effects on body temperature of intraventricular injections of neurohumours at different ambient temperatures, they concluded that noradrenaline had an inhibitory influence on both heat production and heat loss pathways. Even though the site of injection and species were different in Bligh's experiments than in the present investigation, evidence is accumulating that heat production mechanisms alone are not sufficient for explaining the hypothalamic control of thermoregulation. In conclusion, the importance of considering ambient temperature levels in investigations concerned with neurohumoral mechanisms in thermoregulation cannot be denied. This effect has previously been shown to

9 NEUROCHEMICAL SYSTEMS IN THERMOREGULATION 265 be a factor with systemic and intraventricular injections of pharmacological agents, levels and turnover rate of brain catecholamines, and intrahypothalamic injections of morphine. In light of the present experiments, intrahypothalamic injections of neurohumours must be added to the list of manipulations leading to thermal changes which cause differential effects depending on thermal ambient conditions. The data in these experiments were collected during the author's tenure as a postdoctoral fellow in the Division of Psychology, University of Texas Southwestern Medical School at Dallas, Dallas, Texas, U.S.A. The investigations were supported in part by NASA Research Grant NGR awarded to Dr James M. Lipton. Preparation of this manuscript was supported in part by a BSSG Grant No. 1-S5-FR REFERENCES AVERY, D. D. (197). Hyperthermia induced by direct injections of carbachol in the anterior hypothalamus. Neuropharmacology 9, AVERY, D. D. (1971). Intrahypothalamic adrenergic and cholinergic injection effects on temperature and ingestive behaviour in the rat. Neuropharmacology (in the Press). BLIGH, 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. Phy8iol. 212, BucxLuY, G. A., HEADING, C. E. & TAYLOR, K. (1969). Ambient temperature and thermal responses to hexamethonium in the mouse. Br. J. Pharmac. 37, 59. CORRODI, H., FUXE, K. & HOKFELT, T. (1967). A possible role played by central monoamine neurones in thermo-regulation. Acta physiol. scand. 71, Cox, B. & POTRONJAK, D. (1967). The effect of ambient temperature on the actions of tremorine on body temperature and on the concentration of noradrenaline, dopamine, 5-hydroxytryptamine, and acetylcholine in rat brain. Br. J. Pharmac. 31, GIBsoN, S., MCGEER, E. G. & MCGEER, P. L. (1969). Metabolism of catecholamines in cold-exposed rats. J. Neurochem. 16, GORDON, R., SPECTOR, S., SJOERDSMA, A. & UDENFRIEND, S. (1966). Increased synthesis of norepinephrine and epinephrine in the intact rat during exercise and exposure to cold. J. Pharmac. exp. Ther. 153, INGENITO, A. J. (1968). Norepinephrine levels in various levels of rat brain during cold acclimation. Proc. Soc. exp. Biol. Med. 127, INGENITO, A. J. & BONNYCASTLE, D. D. (1967). The effect of exposure to heat and cold upon rat brain catecholamine and 5-hydroxytryptamine levels. Can. J. Physiol. Pharmac. 45, MOORE, K. E., CALVERT, D. N. & BRODY, T. M. (1961). Tissue catecholamine content of cold-acclimated rats. Proc. Soc. exp. Biol. Med. 16, MYERS, R. D. (1966). Injections of solutions into cerebral tissue: Relation between volume and diffusion. Physiol. & Behav. 1, MYERS, R. D. & YAxSH, T. L. (1968). Feeding and temperature responses in the unrestrained rat after injections of cholinergic and aminergic substances into the cerebral ventricles. Physiol. & Behav. 3, MYERS, R. D. & YAKSH, T. L. (1969). Control of body temperature in the unanaesthetized monkey by cholinergic and aminergic systems in the hypothalamus. J. Physiol. 22,

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