Effects of ethanol on thermoregulation

Pharmac. Ther. Vol. 23, pp. 313 to 364, 1984

0163-7258/84 $0.00 + 0.50 Copyright © 1984 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

Specialist Subject Editors: E. SCHONBAUM and P. LOMAX

EFFECTS

OF ETHANOL

ON THERMOREGULATION

H. KALANT*t and A. D. L~t *Department of Pharmacology, University of Toronto, Canada tBiobehavioral Studies Department, Addiction Research Foundation of Ontario, Toronto, Canada

1. INTRODUCTION It is common knowledge that ingestion of alcohol leads, in most people, to a feeling of warmth and frequently to some visible flushing of the face and the extremities. On the other hand, it is also 'common knowledge' among pharmacologists that ethanol typically produces a lowering of body temperature. Careful review of the literature, however, reveals that these two seemingly contradictory statements are considerable oversimplifications and that the effects of ethanol on thermoregulatory processes are complex, incompletely understood, and subject to modification by a number of internal and external variables. In the present review we shall begin with a brief glance at published reports of spontaneous clinical observations of hypothermia following alcohol ingestion. This is followed by experimental observations of the acute effects ofethanoI on body temperature in humans and in experimental animals under varied environmental conditions. A more detailed analytical survey is then presented of the acute effects of ethanol on known and postulated thermoregulatory mechanisms. This is followed by a survey of the changes in thermic effects of ethanol during the development of tolerance and physical dependence. The effects of ethanol are compared with the corresponding effects of a number of other drugs which affect thermoregulation. Finally, the somewhat limited literature on the biological impact of alcohol induced changes in thermoregulation is examined briefly. 2. ACUTE EFFECTS OF ETHANOL ON BODY TEMPERATURE 2.1. CLINICALCASE REPORTS According to Freund (1979), the earliest known report of alcohol induced hypothermia was by Reincke in 1875, and dealt with persons "who fell or jumped into the cold harbor water of Hamburg" in Germany. Most cases described since then have been similar, involving alcohol intoxicated individuals who fell asleep outdoors in winter. A celebrated case (Laufman, 1951) was that of a 23 year old Chicago woman who survived a lowering of body temperature to 18°C, though all four extremities had to be amputated because of intravascular thrombosis and gangrene. Similar, though less dramatic, recent cases have been reported by Fernandez et al. (1970), Tolman and Cohen (1970), Hudson and Conn (1974), Weyman et al. (1974), and Carter (1976). Hirvonen (1976) described a series of 22 fatalities due to hypothermia, in 12 of which blood ethanol levels of 125-249 mg/dl were found at autopsy. Such case reports do not, of course, permit any conclusion about the role of ethanol in the production of hypothermia, even though intoxicated individuals are over-represented among the victims of accidental hypothermia. Ethanol is clearly not unique in this respect. Barbiturates, other hypnotic and anxiolytic agents, neuroleptics and opiates have all been implicated in similar cases. Moreover, the clinical, laboratory, and autopsy findings are similar to those in cases of accidental hypothermia unassociated with alcohol or drugs (Hirvonen, 1976, 1979; Hudson and Conn, 1974). It was conceivable, therefore, that the role of the drugs was merely to increase the risk of being exposed to environmental circumstances which were directly responsible for the hypothermia. In order to ascertain the direct role of ethanol it was necessary to administer ethanol to healthy individuals under controlled conditions; such studies are reviewed in the next section. Jp.T. 23/3- A

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2.2. EXPERIMENTAL OBSERVATIONS IN HUMANS 2.2.1. A t Normal Ambient Temperature Although experimental observations of alcohol effects on human body temperature were recorded over 130 years ago (Lichtenfels and Fr6hlich, 1852), there have been suprisingly few reports since then. Moreover, no consistent pattern of alcohol effects emerges from the available publications. Andersen et al. (1963) found no change in rectal temperature in healthy young men, sleeping nude at an ambient temperature (Ta) of 20°C after consuming 1 or 1.5 g of ethanol/kg of body weight. Martin and Cooper (1978) also found no effect in subjects immersed quietly in water at 22°C. Isbell et al. (1955) found a fall in body temperature of normally dressed subjects only when blood alcohol levels reached values of 200-300mg/dl. In contrast, Marbach and Schwertz (1964) reported that rectal temperature was slightly increased in healthy young men during sleep that followed consumption of ethanol (1.2 g/kg). However, the majority of investigators have reported small and variable decreases in oral or rectal temperature in normal subjects at rest. at normal Ta, after consumption or intravenous infusion of small doses (0.3-1 g/kg) of ethanol (Lichtenfels and Fr6hlich, 1852; Mullin et al., 1933; Horwitz et al., 1949; Risbo et al., 1981). One study in a group of alcoholic volunteers, consuming fixed small doses of ethanol every hour during the day (total daily dose, 3.2 g/kg), revealed a fall in temperature relative to their own control values at 22:00 hr every day, but a rise in 06:00 hr temperature (Gross et al., 1975). These diverse observations suggest that ethanol does tend to lower body temperature in healthy humans, but that the effect is strongly influenced by dose, clothing, level of physical activity and other factors modifying the total thermal load on the body. In order to clarify the role of ethanol a number of investigators have studied its effect on body temperature of subjects exposed to abnormally low or abnormally high ambient temperatures.

2.2.2. A t Low Ambient Temperature 2.2.2.1. Studies on non-exercising subjects. If ethanol facilitated the development of hypothermia in patients exposed accidentally to winter conditions (Section 2.1), it might be anticipated that this facilitation would be easily demonstrable experimentally. Surprisingly, the majority of experiments on human volunteers have not borne out this expectation. Small doses of ethanol (0.3-0.4 g/kg) did cause a small but significant fall in rectal temperature in healthy young men immersed for 24 rain in water at 13°C (Graham and Baulk, 1980), or clothed but lying quietly outdoors at - 2 ° C (Gupta, 1960). However, five studies failed to show a significant enhancement of hypothermia by the combination of similar or larger doses of ethanol and exposure to cool air (Andersen et al., 1963; Risbo et al., 1981) or cold water (Keatinge and Evans, 1960; Martin et al., 1977; Fox et al., 1979). Indeed, Hobson and Collis (1977) even reported that ethanol retarded the fall in core temperature of subjects immersed in water at 7.5°C for periods of 38-75 min. These puzzling findings become even more difficult to explain when certain associated observations are considered. It might be reasoned that cutaneous vasoconstriction, in direct local response to the cold air or water, prevented loss of heat to the surroundings and thus masked an ethanol effect on central thermoregulation. Yet several reports indicate that the subjects felt warmer and more comfortable after ethanol, regardless of whether it lowered the body temperature more (Gupta, 1960; Graham and Baulk, 1980) or failed to do so (Keatinge and Evans, 1960; Hobson and Collis, 1977; Martin et al., 1977; Fox et al., 1979). This might appear to indicate that alcohol prevented cutaneous vasoconstriction by the cold, and this should have resulted in greater heat loss; this point is considered further in Section 3. In any case, several researchers reasoned that vigorous exercise, by increasing the metabolic load on thermoregulatory mechanisms, might permit a clearer view of the

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effects of ethanol on thermoregulation during cold exposure. Such studies are reviewed in the next section. 2.2.2.2. Studies on exercising subjects. Only a few groups appear to have undertaken systematic studies of the effects of alcohol on body temperature of subjects exercising actively in the cold. However, the results are more consistent than those obtained in resting subjects. Healthy young male volunteers performed either mild or strenuous exercise (40~o or 70~, respectively, of maximum ~o2) in air at temperatures ranging from - 5 ° C to + 15°C. In all cases, doses of ethanol ranging from 0.3 to 0.8 g/kg produced a significantly greater fall in rectal, esophageal, or mean body temperature than was found in the same or other subjects drinking water or glucose solution (Haight and Keatinge, 1973; Graham, 1981a,b; Graham and Dalton, 1980). Similar but more marked effects were observed in females (Graham, 1983). Subjects receiving a similar dose of ethanol in orange juice and subjected to a much shorter period of exercise (20 min) during cold water immersion also showed a greater fall in body temperature than did controls, although the difference was not statistically significant (Fox et al., 1979). Cross-country skiers, resting at a Ta o f - 2 ° C to +5°C after 5.5 hr of skiing, showed a gradual fall of rectal temperature to pre-skiing levels under control conditions; however, if they consumed ethanol (0.6 g/kg) during the resting period, rectal temperature fell significantly below baseline (Simper et al., 1983). These results suggest that the duration and intensity of the exercise, and possibly the consumption or non-consumption of other food together with the ethanol, are critical determinants of the ethanol effect under these conditions. 2.2.3. A t Raised Ambient Temperature In contrast to the rather rigorous cold exposures mentioned above, only minor ambient heat loads have been used in combination with ethanol in experiments on human subjects. Perhaps it is not surprising, therefore, that subjects receiving 0.7-1 g/kg showed no change in body temperature during a 30 min immersion in water at 30°C (Martin and Cooper, 1978). In another study (Kuznetsov, 1963) immersion of the right arm in water at 45°C resulted in greater sweating in subjects receiving ethanol than in controls, but core temperature does not appear to have been recorded. It is surprising that so little research has been done on the interaction of alcohol and raised Ta. The explanation does not lie in any ethical consideration of intolerable heat load for human subjects since the thermal effects of marijuana have been investigated in humans at an air temperature of 40°C (Jones et al., 1980). The question is important because differentiation between the effects of alcohol on thermoregulatory set point and on the efficacy of thermoregulatory mechanisms (see Section 3) requires a knowledge of the effects at both raised and lowered temperatures. For this, we must rely_mainly_ on observations on experimental animals. 2.3. EXPERIMENTALOBSERVATIONSIN LABORATORYANIMALS 2.3.1. A t Normal Ambient Temperature The hypothermic effect of ethanol on laboratory animals, in contrast to humans, is clearly evident even at normal T,. More than 70 years ago, Pilcher (1912) observed graded intensity and duration of hypothermia in cats after intragastric doses of ethanol ranging from 0.5 to 10 ml/kg. Similar effects were observed many years ago in rabbits (Werner, 1941) and mice (Flacke et al., 1953; Madden and Hiestand, 1954). Since then, numerous confirmations of these early findings have been obtained in experiments with mice (Freund, 1973; Tabakoff et al., 1975; Ritzmann and Tabakoff, 1976a,b,c; Kakihana and Moore, 1977; Moore and Kakihana, 1978; Dinh and Gailis, 1979; Deimling and Schnell, 1980; Papanicolaou and Fennessy, 1980; Rigter et al., 1980; Crabbe et al., 1979, 1981), rats (Altland et al., 1970; Nikki et al., 1971; Pohorecky et al., 1974; Ferko and Bobyock, 1979; Linakis and Cunningham, 1979; Lomax et aL, 1980, 1981; Mullin and Ferko, 1981; Myers,

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1981a), guinea-pigs (Huttunen, 1982) and dogs (Sargent et al., 1980). The degree of effect of the same dose exhibits diurnal variation, being greatest during the dark phase of the daily light cycle, when the body temperature is normally highest, and least during the light phase when body temperature is at its minimum (Kakihana and Moore, 1977; Deimling and Schnell, 1980). The route of ethanol administration was not critical with respect to this effect, since hypothermia was seen after gavage, intraperitoneal injection, inhalation, or injection of much smaller amounts directly into the cerebral ventricles. For example, 8 mg of ethanol injected into the lateral ventricle in the mouse caused as great a fall in temperature as 3 g/kg i.p. (Ritzmann and Tabakoff, 1976c), and even 2 mg i.c.v, produced a significant drop (Ritzmann and Tabakoff, 1976a). The concentration of the administered ethanol is important. For the same total dose, the hypothermic response of rats to i.p. injection of ethanol was proportional to the concentration of the injected solution (Linakis and Cunningham, 1979). Concentration had been shown previously to affect the intensity of behavioral and EEG changes produced by a given dose of ethanol in cats (Perrin et al., 1974), in proportion to the maximum blood alcohol concentration attained. Therefore it is noteworthy that i.c.v, injection of ethanol (5-10/~1 of 29~o ethanol) also caused a small but significant fall in body temperature in Maeaca cyelopis (Chai et al., 1971) at normal room temperature. This finding, in another primate, is consistent with the conclusion that ethanol does tend to lower body temperature in human subjects, even at normal Ta (Section 2.2.1). One recent study (Vuorinen et al., 1976) revealed no hypothermic response to ethanol in rats kept at 21-25°C; indeed, a 3 g/kg dose by stomach tube failed even to intensify the hypothermia produced by a concurrent dose of lidocaine, although it prolonged the narcosis. No explanation for this discrepancy is readily apparent. However, it is obvious that the great preponderance of evidence in animal experiments indicates a very reliable hypothermic effect. Since the rat is much smaller than the human, its surface:mass ratio is much higher, and its potential heat exchange area is greater, despite the presence of fur insulation. It is therefore conceivable that the same T, (20-25°C) that the human can easily cope with, despite alcohol, is able to evoke hypothermia in the rat when thermoregulation is even slightly impaired by alcohol or other drugs. 2.3.2. At Low Ambient Temperature According to Freund (1979), Walther reported in 1865 that a rabbit, given a large dose of ethanol (35 ml of brandy) by gavage and then exposed to an ambient temperature of 12.5°C, died of profound hypothermia while a control animal at the same temperature showed only a minor drop in temperature. That observation has been confirmed repeatedly in our own era, in guinea-pigs (Mufioz, 1937a; Hirvonen and Huttunen, 1977; Huttunen et al., 1980; Huttunen, 1982), rats (Lozinski et al., 1969; Myers, 1981a; Lomax and Lee, 1982), mice (Madden and Hiestand, 1954; Malcolm and Alkana, 1981) and dogs (Mufioz, 1937a). In general, the doses of ethanol used have been relatively large, ranging between 1-7 g/kg by gavage, and 1-3.6 g/kg i.p. In addition, these experiments involved immersion in ice-water at 0°C or exposure to air at - 20°C. These facts probably explain the high degree of concordance of results, in contrast to the variability of findings in human subjects (Section 2.2.2.1). However, even at the lowest dose used (1 g/kg i.p.), the fall in body temperature in the rat was a logarithmic function of the Ta, over the range of 0-18°C (Lomax and Lee, 1982). One must therefore conjecture that part of the difficulty in demonstrating a similar relationship in humans is the much greater genetic and physiological heterogeneity of human subjects, compared to laboratory rats or mice. 2.3.3. A t Raised Ambient Temperatures In contrast to the paucity of experimental data in humans, there is a relative abundance of observations on the interaction of ethanol and high Ta in experimental animals. The

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most common finding has been that a dose of ethanol, sufficient to produce definite hypothermia in rats or mice kept at 20-23°C, fails to reduce body temperature when the animals are kept at 30-38°C (Freund, 1973; Tabakoff et al., 1975; Vuorinen et al., 1976; Ferko and Bobyock, 1978; Grieve and Littleton, 1979b; George et al., 1981; Myers, 1981a). More striking, however, is the finding that the relationship between Ta and post-alcohol body temperature, described by Lomax and Lee (1982) for the Ta range of 0-18°C, applies also in the range of 18-37°C. At a Ta of 28°C, ethanol produced a slight hypothermia in the rat, though significantly less than at 21°C (Oliveira Souza and Masur, 1981); the same was true at 30°C for the mouse (Malcolm and Alkana, 1981). However, at 34°C and 37°C in the mouse (Ritzmann and Tabakoff, 1976a; Malcolm and Alkana, 1981), and at 36°C in the rat (Myers, 1981a), ethanol actually raised the body temperature above that of controls receiving saline. The most elegant demonstration of this effect was by Myers (1981a), who was able to vary the body temperature of alcohol-treated rats (2 to 4 g/kg by gavage) from hypo- to hyperthermic levels at will by simply varying the Ta. At any given dose of ethanol, the effect on body temperature depended solely on Ta, while at any given T~ below thermoneutrality the magnitude of body temperature change depended on the dose. Indirect confirmation of the hyperthermic effect of ethanol at high T~ is provided by several reports that raised ambient temperature increases the toxicity of ethanol in mice of some strains (Freund, 1973; Dinh and Gailis, 1979; Grieve and Littleton, 1979b) and rats (Mufioz, 1937a,b; Thomas and Tr6moli~res, 1968). Conversely, ethanol increases the lethality of high T,, as shown by a reduction in LTs0 (Dinh and Gailis, 1979). While these findings do not, in themselves, prove that alcohol impairs thermoregulation, they are certainly compatible with the idea that it prevents effective coping with an imposed heat load, just as it reduced the ability to cope with cold. 2.4. SUMMARY The literature reviewed to this point has provided references to cases of serious, and even fatal, hypothermia occurring in individuals intoxicated by alcohol and exposed to severe cold. Experimental studies in human volunteers, involving mild intoxication and relatively short exposure to moderately cold environments, have given inconsistent results. However, with the addition of strenuous exercise and longer duration of cold exposure, the same doses of ethanol have produced hypothermia in almost all cases. In smaller animals, with larger surface:mass ratios and higher rates of heat loss, somewhat larger relative doses of ethanol usually produce hypothermia even at normal T,, and hypothermia becomes very marked during severe cold exposure. In contrast, at high Ta ethanol produces hyperthermia in rats and mice, and increases the lethality of raised Ta; corresponding observations in humans are lacking. These findings suggest that ethanol tends to impair the efficacy of thermoregulation in both directions, and therefore to produce poikilothermia, rather than hypothermia (Myers, 1981a). To explore this idea further, it is necessary to see how ethanol affects the various processes that contribute to the maintenance of normal thermal balance. 3. T H E R M O R E G U L A T O R Y MECHANISMS AND THEIR MODIFICATION BY ETHANOL 3.1. BODY TEMPERATURE AND DYNAMIC THERMAL EQUILIBRIUM

In the past 25 years, numerous excellent reviews of the physiology of thermoregulation have appeared. To cite only a few, the comprehensive reviews by Benzinger (1961), Hardy (1961), von Euler (1961), Hemingway (1963), Carlson (1964), Bligh (1966), Borison and Clark (1967), Cremer and Bligh (1969), Freund (1979), and Lomax and Sch6nbaum (1979) provide a very complete view of the evolution of knowledge about thermoregulatory mechanisms, both central and peripheral. In addition, several symposia (Sch6nbaum and Lomax, 1973; Lomax and Sch6nbaum, 1975; Cooper et al., 1977; Cox et al., 1980) provide excellent detailed discussions of specific aspects of the topic. Therefore no attempt will be

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made here to review the basic physiology of thermoregulation in any depth. We shall simply recall the main elements, as a guide to systematic review of the effects of ethanol. As von Euler (1961) pointed out, it has been recognized for about 200 years that homeotherms achieve their constancy of temperature by maintaining a dynamic equilibrium between heat production and heat loss. He cited Bergman (1845) who proposed that the vasomotor reactions which play a major role in controlling cutaneous heat loss are governed by thermosensitive structures in the brain, responding to temperature information relayed to them from other parts of the body, and capable of altering their sensitivity under various physiological conditions. Since then, the picture which has evolved contains the following major elements: (1) Heat is produced by metabolic activity in all tissues, under the modulatory influence of insulin, thyroid hormone, epinephrine, adrenocorticoids, and other hormonal factors (nonshivering thermogenesis). (2) During sudden cold stress additional heat is generated by involuntary muscular contraction, i.e. shivering. (3) Heat loss through the expired air depends upon the temperature gradient between inspired air and the lung, and the respiratory depth and frequency. (4) Heat loss by radiation and conduction from the skin depends upon the temperature gradient between the skin and the surrounding medium (air or water), the cutaneous blood flow and the amount of insulation in the form of subcutaneous fat, fur and clothing. (5) Heat loss by evaporation depends upon the rate of sweat secretion and the relative humidity of the surrounding air. (6) Integrated responses of these various elements are brought about by changes in firing rates of certain neurons in the hypothalamus, in response to changes in temperature of the blood perfusing the brain and to information brought by sensory pathways from thermosensitive end-organs in the skin and possibly other organs. (7) Some property of the relevant hypothalamic neurons determines the 'set-point' with which the blood temperature is compared, and the magnitude and direction of the difference determine the appropriate compensatory responses. The sensitivity, i.e. the minimum difference required to initiate the compensatory response, is apparently modified by numerous neurotransmitters, peptide neuromodulators, prostaglandins, ions and other physiological factors. (8) These intrinsic physiological responses can be supplemented by behavioral responses which either modify the environment, or move the individual from a more taxing to a more favorable environment. Alcohol and other drugs can modify all of the major thermoregulatory elements listed above. The evidence will be reviewed in the following sections. One point that should be borne in mind, as the reader encounters one instance after another of contradictory data bearing on each possible mechanism, is the little-heeded diurnal variation in ethanol effect on body temperature (Section 2.3.1). This variation could conceivably account for a substantial part of the apparent contradiction, depending on the time of day at which tests are done. However, for practical reasons most studies are done in rodents and most experimental interventions are made during daylight hours. These may be unfavorable circumstances for studying the effects of alcohol on normal thermoregulatory mechanisms. 3.2. EFFECTS OF ETHANOL ON WHOLE-BODY THERMAL BALANCE 3.2.1. Heat Production Exposure of non-acclimated homeotherms to a cold environment leads to a sharp increase in the rate of 02 consumption, which reaches values up to five or more times the basal rate (Hemingway, 1963). This sudden and marked increase is due primarily to shivering, with the attendant increase in oxidative metabolism in skeletal muscle. When

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the brain temperature falls below 30°C 402 falls progressively as shivering ceases, and this contributes to further fall in body temperature (Grosse-Brockhoff and Schoedel, 1943). In species which are not provided with highly efficient insulation in the form of thick fur or subcutaneous fat, there is also an increase in non-shivering thermogenesis in liver and other viscera, especially in individuals that have become acclimated to a cold environment (Sellers, 1957; Hardy, 1961; Carlson, 1964; Irving, 1966; Cremer and Bligh, 1969). Epinephrine and norepinephrine facilitate these thermogenic responses in various ways, including the activation of adenylate cyclase. This results in increased levels of cAMP and a consequent increase in the mobilization of glucose from glycogen and of free fatty acids from adipose tissue (Cremer and Bligh, 1969). Since ethanol has been shown to affect all these processes individually, it would be expected to affect thermogenesis in the cold. However, the observed changes are by no means simple or consistent. In humans, ethanol has been reported to delay the onset of shivering and reduce its duration during exposure to cold air or immersion in cold water and during subsequent recovery in air at normal Ta (Hobson and Collis, 1977; Martin et al., 1977; Fox et al., 1979; Graham and Baulk, 1980; Risbo et al., 1981). This is consistent with the subjective feeling of being less cold under these conditions after ingestion of ethanol (Section 2.2.2.1). In rats ethanol (2.5 g/kg i.p.) inhibited post-halothane shivering and thus delayed recovery from hypothermia (Nikki et al., 1971). Reduction or suppression of shivering by ethanol appears analogous to the effects of other anesthetic and hypnotic agents and would be expected to contribute to a fall in body temperature during severe cold stress. On the other hand, ethanol itself has been reported to have a calorigenic effect under certain conditions. Rosenberg and Durnin (1978) reported that moderate doses of ethanol (230 ml of wine), taken either alone or with food, produced a significant (10-30~o) increase in e¢o2 in a group of healthy female volunteers at normal Ta. Similar but less clear results were produced by the consumption of two ounces of rum by healthy males exposed to winter cold (Gupta, 1960). In conscious rats in a thermoneutral environment, ethanol reduced the 02 consumption, apparently by sedating or anesthetizing the animals; however, when the rats were first lightly anesthetized with pentobarbital to bring the 02 consumption to a stable uniform rate, injection of ethanol produced a modest but significant increase in 02 consumption (Kalant et al., 1963). Such an effect would be consistent with the metabolism of ethanol itself (Kalant et al., 1963) or with an ethanol induced shift of oxidative metabolism in muscle from primarily glucose to primarily fatty acids (Juhlin-Dannfelt et al., 1977). However, an increase in 02 consumption does not necessarily mean an increase in heat production, since the caloric equivalent of a mole of O 2 depends upon which substrate is being oxidized. As noted elsewhere (Kalant et al., 1963), the production of the same amount of heat from ethanol as from glucose requires 12~o more oxygen for the ethanol. If fat is the principal substrate, the oxygen consumption increases even more for equal calorie production. Therefore a definitive answer to this question requires whole-body calorimetry studies under thermoneutral conditions. Variable contributions by reduction of shivering, increased non-shivering thermogenesis and sedation probably account for the fact that other investigators have reported conflicting results. Graham and Baulk (1980) found that ethanol reduced the rate of recovery of total body heat content following a period of immersion in cold water. Fox et al. (1979) also observed a consistently lower metabolic rate after ethanol, during cold water immersion, though the difference from non-alcohol subjects was not statistically significant. In contrast, when cold-exposed subjects were performing exercise at a fixed rate (40~o of "~o2max), ethanol did not alter the metabolic efficiency as assessed from the 402 and RQ (Graham, 1981a,b). This was also true during equivalent exercise or at rest in air at normal Ta (Garland et al., 1960; Risbo et al., 1981). There is similar variability in the results of experiments in rats. Ethanol (2 g/kg i.p.) in unanesthetized rats produced a small and non-significant fall in O2 consumption by rats at normal Ta (Lomax et al., 1981). A dose of 1.25 g/kg produced a sharp fall in intrahepatic temperature, which could be partially overcome by epinephrine or norepinephrine

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(Molenda and Obrzut, 1967). A dose of about 0.6 g/kg by continuous i.v. infusion failed to alter the recovery of body heat content by anesthetized rats at a Ta of 27°C during recovery from deep hypothermia. All one can reasonably conclude from these studies is that ethanol can affect thermogenesis both directly and indirectly, and the direction and magnitude of change depends on the type of subject, the dose and the experimental conditions. Under most circumstances involving cold stress, it is likely that suppression of shivering thermogenesis outweights any possible thermogenic effect of ethanol metabolism. 3.2.2. Hypoglycemia and Thermoregulation Since hypoglycemia produced by excess insulin or by impaired gluconeogenesis can give rise to hypothermia (Kedes and Field, 1964) and can suppress shivering in cold exposed animals, Haight and Keatinge (1973) suggested that hypothermia following ethanol ingestion might be an indirect consequence of ethanol induced hypoglycemia. Since ethanol is known to inhibit gluconeogenesis in the liver (Krebs et al., 1969; Juhlin-Dannfelt et al., 1977), this action in combination with fasting or with excessive utilization of glucose (e.g. during strenuous physical activity) is known to produce hypoglycemia (Hawkins and Kalant, 1972). These are also the conditions in which ethanol induced hypothermia is most readily demonstrable in humans (Kedes and Field, 1964; Carter, 1976). The suggestion by Haight and Keatinge has therefore been put to experimental test by a number of investigators. Their results generally fail to support the hypothesis that hypoglycemia is a necessary condition for the development of hypothermia. In humans immersed in cold water (Graham and Baulk, 1980) no hypoglycemia occurred, even though the alcohol group had a greater fall in temperature. In subjects exercising vigorously in cold air, ethanol produced a relative hypothermia during the first 2 hr, while relative hypoglycemia did not occur until the third hour (Graham, 1981a,b). Nevertheless, administration of glucose together with the ethanol prevented the body temperature difference between the alcohol and control groups. This did not happen in rats exposed to normal Ta but receiving a relatively much larger dose of ethanol (Myers, 1981a). In guinea-pigs exposed to severe cold stress ethanol caused more rapid fall in core temperature and earlier death but the blood glucose level at the time of death was higher than in the non-alcohol group (Hirvonen and Huttunen, 1977; Huttunen, 1982; Huttunen et al., 1980). Finally, Oliveira Souza and Masur (1982) reported that at a T,d of 28°C, at which ethanol did not produce hypothermia in the rat (Section 2.3.3), it also failed to cause hypoglycemia; indeed, it caused hyperglycemia, probably by norepinephrine induced glycogenolysis. The foregoing findings suggest that while a calorigenic effect of glucose may offset a very mild hypothermia after small doses of ethanol, hypothermia is not a direct consequence of hypoglycemia. Indeed, there is rather more persuasive evidence that severe and prolonged hypothermia may cause hypoglycemia, perhaps after a sustained adrenergic response has exhausted the hepatic glycogen reserve. 3.3. EFFECTSOF ETHANOLON BODY HEAT LOSS 3.3.1. By Circulatory Mechanisms It is obvious that the skin is a major site of heat transfer between the body and the external environment and that delivery of deep body heat to the skin via the cutaneous blood flow is a major determinant of the rate of heat transfer (Bowman and Rand, 1980). In view of the common experience of flushing and warmth after the ingestion of ethanol it is not surprising that many investigators have studied the effects of ethanol on cutaneous circulation, skin temperature, and heat loss. Many of the studies form part of a broader examination of the effect of alcohol on thermoregulation, but many have been concerned with the potential therapeutic value of ethanol in peripheral vascular disease, or in the prevention of frostbite.

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3.3.1.1. Effects on cutaneous blood flow. The direct effects of ethanol upon peripheral vascular muscle tone depend upon the concentration of ethanol used. In healthy human subjects, continuous infusion of ethanol into the brachial artery (Fewings et al., 1966; Gillespie, 1967), at rates of 150 mg/min or higher, produced arterial spasm and consequent reduction of blood flow through the hand and forearm. Since total blood flow through the arm was not stated it is impossible to estimate accurately the alcohol concentration in the blood perfusing the arm, but it was probably in the range of at least 100-300 mg/dl. In contrast, perfusion of the rabbit's ear with 2-3~o solutions of ethanol in Ringer's solution produced vasomotor paralysis and non-reactivity to epinephrine (Genuit, 1940). Such a concentration would, in the systemic circulation, be lethal. It is obvious, therefore, that the vasodilatation produced by ethanol at low concentrations in vivo must be indirect, as shown many years ago by the elegant cross-circulation experiments of Siems and Rottenstein (1950). The occurrence of such vasodilatation after oral or i.v. administration of ethanol to humans has been confirmed repeatedly by the use of capacitance or venous occlusion plethysmography, heat conduction techniques, and digital pulse wave amplitude and velocity measurements (Horwitz et al., 1949; Graf and Strrm, 1960; Conrad and Green, 1964; Fewings et al., 1966; Ghiringhelli et al., 1966; Gillespie, 1967; Downey and Frewin, 1970; Allison et al., 1971). Consistently, the increase in blood flow has been shown to be selective, involving chiefly the skin of the extremities and face, and being offset in resting subjects by a reduction in flow through the deeper vessels in the muscles. In subjects engaged in vigorous exercise, the muscle blood flow did not fall below the alcohol-free values, but it did not undergo the large increase shown by cutaneous blood flow (Graf and Strrm, 1960). The pattern is essentially that of a thermoregulatory vasodilatation response, and is consistent with the feeling of warmth and increased sweating that follow alcohol consumption at normal Ta. The effect of this vasodilatation on heat exchange has been measured directly by calorimetry of the hand at normal Ta (Keatinge and Evans, 1960) and during immersion in cold water (Keatinge and Evans, 1960; Goldman et al., 1973). Ethanol produced a 72~ increase in heat loss from the hand at normal Ta and a 22~ increase during the first 15 min of cold exposure. This was accompanied by a higher skin temperature during the same period. During continued immersion the skin temperature fell rapidly to that of the surrounding water but the spontaneous rhythmic vasodilatation, resulting in rhythmic episodes of skin warming, that occurs at such temperatures was greatly enhanced by ethanol. The same phenomenon occurred after alcohol during exposure of the hand to air at - 2 ° C to -4°C, but not at -16°C to -18°C (Schulze, 1947).

3.3.1.2. Effects on skin temperature. Most investigators have relied on measurements of skin temperature, rather than of actual heat exchange, and the results have been somewhat less consistent. In human subjects at normal Ta ethanol induced a substantial rise in skin temperature of the face and extremities (Miles 1924; Cook and Brown, 1932; Risbo et al., 1981) that paralleled in time and magnitude the measured change in blood flow (Ghiringhelli et al., 1966). When subjects were adequately dressed, and only the hand or foot was exposed to cold, the cold exposed extremity showed a rapid fall in skin temperature (Gupta 1960; Little, 1970) and in blood flow rate (Downey and Frewin, 1970), but the fall was less marked or even reversed after consumption of ethanol and the subsequent rise in temperature during the recovery period was more rapid (Little, 1970). Not surprisingly, subjects who had to remove their gloves to perform fine manual work at a Ta of --40°C required less time to complete the task after ingesting alcohol, because of taking less time to warm their hands during the work (McCleary and Johnson, 1954). During whole-body exposure to cold, effects of ethanol on skin temperature change are somewhat less predictable. In humans at an air temperature of 10--15°C the skin temperature of fingers and toes fell rapidly, and the fall was greater after a dose of 2 g/kg than after 0.8 g/kg (Risbo et al., 1981). This effect of ethanol was even greater when

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subjects were exercising vigorously in air at +5°C to -15°C (Graham, 1981a,b, 1983; Graham and Dalton, 1980). In contrast, during whole-body immersion at 15°C the mean skin temperature reached 16°C within 5 rain, both with and without previous ethanol ingestion (Martin et al., 1977). This is to be expected since heat conduction from skin to water is much more efficient than to air, and the resulting rapid cooling of the skin, and possibly a vasoconstriction response to this cooling, could well mask the vasodilatation response to ethanol. One group, using both skin temperature measurement by thermistors and heat radiation measurement by infra-red scanning, found no effect of ethanol (approximately 0.6 g/kg) in nearly nude subjects at air temperatures of 25°C or 30°C, or in clothed subjects with face exposed in air at -23°C (Livingstone et al., 1980). The same group also found no effect on total heat loss during whole-body immersion calorimetry in water at 25°C. No reason is immediately apparent for the difference between these results and those obtained by other investigators, except for the possibility that the ethanol dose was too small, or that it might not have been administered in the post-absorptive state. In summary, the great preponderance of evidence indicates that ethanol produces a cutaneous vasodilatation, leading to increased loss of heat to the environment. If the exposure is to extremely cold air ( - 15°C to -40°C), or to cold water, cooling is rapid and reflex vasoconstriction is so prompt that skin temperature measurements may not reveal the elevated heat loss induced by ethanol. The pattern of vascular response to ethanol is that of a heat-dissipating thermoregulatory response of central origin, and its mechanism must be sought centrally. 3.3.2. By Respiratory Exchange Depending upon the species, varying amounts of body heat may be lost by increased pulmonary ventilation or by panting. In humans, sweating is a major response to hyperthermia, while in species which do not sweat the equivalent response is panting (Hardy, 1961). In the rabbit, for example, exposure to air at 35°C induces vigorous panting with a four-fold increase in respiratory rate, that is apparently sufficient to keep the brain temperature within normal limits (Hellon, 1981). However, in all species heat can be lost through normal respiration, partly by direct heat exchange between lung and inspired air, and partly by evaporation. Various investigators have found a progressive stimulation of ventilation rate with increasing body and brain temperature (Hardy, 1961; Boulant, 1981). Mercer and Jessen (1979) found a linear increase in respiratory evaporative heat loss with increasing core temperature beyond a threshold value of about 39°C in the goat. The additional heat loss amounted to about 1 W/kg per °C rise of core temperature. Therefore, if the effect of ethanol on thermoregulation is comparable to a heat-dissipating thermoregulatory response, one might expect to find that ethanol, in low doses, also provokes increased respiration. This is indeed the case, as shown in earlier studies reviewed by Wallgren and Barry (1970). The stimulatory effect is apparently exerted upon the glomus and the carotid sinus. Human volunteers who ingested 1.2 g of ethanol/kg of body weight over a 5 hr evening drinking session maintained an elevated respiratory rate throughout the night (Marbach and Schwertz, 1964). In rats stabilized at Za 26°C, injection of a larger dose of ethanol (2 g/kg) i.p. produced an initial slight fall in respiratory rate and minute volume, but recovery was evident by 25 min and the respiratory volume was actually increased above baseline (Lomax et al., 1981). This probably represents an acute (within-session) tolerance to the initial respiratory depressant effect, perhaps unmasking the stimulant effect. A different effect on ventilation was seen, however, in healthy young volunteers immersed in water at 12°C, 22°C and 30°C. At 22°C, the initial gasp response and the subsequent sustained elevation of expiratory ventilation rate were attenuated by ethanol (0.7-1.0g/kg), with corresponding increase in the end-expiratory pCO2 (Martin and Cooper, 1978). It seems likely that this represents a sedative action of ethanol, rather than a direct inhibition of respiratory control centres. When subjects were tested at 30°C, a

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temperature which tends to have a relaxing effect of its own, the hyperventilation was no longer visible in the control trials, and ethanol now induced a slight depression of respiration and an increase in end tidal pCO2 above baseline. During trials at 12°C, on the other hand, the respiratory stimulus due to the cold was so strong that the ethanol effect was almost completely overridden (Martin et al., 1977). These rather limited findings do not permit any firm conclusion about the possible role of respiratory changes in the effects of ethanol on thermoregulation. They suggest, however, that if such a role exists, it is minor. Increase in respiration would be a homeostatic response to increased body heat load, and if respiration is increased by ethanol it might theoretically contribute to fall in body temperature. However, with large doses of ethanol, that are most likely to cause hypothermia, the predominant effect on respiration is a depressant one, which would tend to conserve heat. This appears to be particularly true when the ethanol is taken before exposure to an environmental cold stress.

3.4. EFFECTS OF ETHANOL ON CENTRAL THERMOREGULATORY MECHANISMS 3.4.1. Electrophysiological Effects on Hypothalamic Neurons Over the past 40-50 years there has been a progressive accumulation of evidence that the main locus of central thermoregulatory control is in the hypothalamus (for reviews, see Benzinger, 1961; Hardy, 1961; Hemingway, 1963; Bligh, 1966; Boulant, 1981; Hellon 1981). There is now little doubt that the major integration of thermoregulatory mechanisms is a function of neurons in the preoptic and anterior hypothalamic nuclei (POAH). Shivering, and possibly non-shivering thermogenesis, appear to be directly controlled by neurons in the posterior hypothalamus, but these are probably under the modulatory influence of POAH units (Hemingway, 1963; Bligh, 1966; Hellon, 1974, 1981) and come into play as a consequence of change in this modulation. The integrative role of the POAH implies the convergence upon this area of afferent temperature information, including the temperature of arterial blood perfusing the area, as well as afferent impulses from multiple sites in the periphery as well as in the central nervous system. It also implies the existence of efferents from the POAH to these sensors as well as to the thermoregulatory effector systems (Hardy, 1961; Cremer and Bligh, 1969). These interconnections constitute a complex system of reciprocal feedback controls, in which peripheral stimuli can over-ride central influences or vice versa, depending upon the relative in~ensities of peripheral and central thermal disturbances. For example, local cooling of the POAH by means of a water perfused implanted thermode initiated heat conserving (cutaneous vasoconstriction) and thermogenic (shivering) responses even though the rectal temperature was normal (Marques et al., 1981; Inomoto et al., 1982). However, when the animal was exercised and the body temperature rose, heat dissipating mechanisms (cutaneous vasodilatation, panting) were activated even though the local temperature of the POAH was kept subnormal by a thermode (Mercer and Jessen, 1979). Under normal conditions, the central control system appears to function in an 'on-off' fashion, alternately activating heat conserving and heat dissipating mechanisms so as to maintain the hypothalamic temperature within oscillation limits of 0.2-0.4°C (Young and Dawson, 1982). The thermoregulatory roles of the POAH have been shown to be subserved by different types of neurons, some (cold sensitive units) showing a rise in firing rate in response to local cooling, and others (warm sensitive units) responding similarly to local warming (Bligh, 1966; Jell and Gloor, 1972; Boulant, 1981; Lin and Simon, 1982). These differential responses do not appear to be due to changes in input from remote sensors, because they can be reproduced by local warming or cooling of isolated hypothalamic tissue, either in deafferented islands (Ford, 1974) or in slices in vitro (Kelso et al., 1982). However, this fact does not preclude the possibility of reciprocal inhibitory influences between adjacent cold and warm sensitive units within the same slice, and it also seems likely that afferent

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stimuli from remote sensors modulate the temperature thresholds and firing rates of POAH units (Boulant, 1981; Werner et al., 1981; Kelso et al., 1982). It is not yet clear exactly how these temperature-sensitive hypothalamic units actually detect the temperature change. However, the most credible hypothesis to date is that such units show distinctive temperature-dependent alterations in membrane potential (Hellon, 1981) and other membrane properties related to the initiation and propagation of action potentials and release of neurotransmitters (Joyner, 1981). All neurons show such temperature dependent changes in accordance with the predictions of the Hodgkin-Huxley equation (Joyner, 1981), but differential patterns of Q10 for the various membrane processes might explain different patterns of thermosensitivity. For example, Carpenter (1981) has proposed that a high Q~0 for sodium pump activity might enable a neuron to function as a cold receptor, while a higher Ql0 for Na t conductance than for K + conductance might explain the properties of a warm receptor. To date, this suggestion does not appear to have been tested adequately. Comparison of Q10 values for firing rates of cold and warm sensitive units (Lin and Simon, 1982) does not really address this hypothesis. The foregoing mechanisms and functional relations are potentially testable in the specific context of alcohol and thermoregulation. Existing techniques would make it quite feasible to explore, for example, the interaction of TpOAHand blood or rectal temperature on thermoregulatory responses, in the presence and absence of varying concentrations of ethanol; the effect of ethanol on the threshold and Q~0 of warm and cold sensitive neurons; the dose-response relations for ethanol modification of membrane potential and ionic conductances of such units; or the modification of ethanol actions on POAH neurons by ouabain or other inhibitors of cell membrane (Na+/K ÷ )-ATPase. Present-day techniques do not yet appear to be good enough to study, in membrane preparations from different types of thermosensitive neurons, the physical changes produced by ethanol and other 'membrane-fluidizing' agents (see Section 4.3.3), but it is not improbable that methods for such studies on single units in situ will be developed before long. Unfortunately, questions of this type appear to have been explored very little so far. Seventy years ago, Barbour and Wing (1913) reported that direct local injection of 0.2 ml of 5~o ethanol into the base of the rabbit brain produced a fall in body temperature, but no one appears to have used modern stereotaxic and microinjection techniques to examine the direct effects of ethanol on thermoregulatory mechanisms. None of the reports we have seen concerning the effects of ethanol on single-unit activity in the hypothalamus (e.g. Wayner et al., 1975) included thermosensitive POAH units. A few studies have been carried out, of the effects of ethanol on neuronal membrane properties that might be related to the thermoregulatory set-point. It has been proposed that the set-point of the effector systems controlled by posterior hypothalamic neurons depends upon the ratio o f N a ÷ to Ca 2+ ions (Myers and Ruwe, 1982). According to this hypothesis, an increase in the Na+:Ca 2+ ratio causes hyperthermia, and a decrease causes hypothermia, presumably by altering the excitability of the neurons with respect to afferent impulses from thermal sensory units. In support of the view that ethanol alters the Na t :Ca 2+ ratio, it has been found that i.c.v, injection of the calcium chelator, EGTA, blocked the hypothermic effect of ethanol in the rat at 22°C (Myers and Ruwe, 1982). Conversely, ethanol decreased the toxicity due to EGTA, i.e. raised the LDs0 of EGTA injected icv (Harris, 1979). Yet i.c.v, injection of Ca 2+, though causing hypothermia itself, had no effect on ethanol induced hypothermia, even though it prolonged ethanol narcosis (Erickson et al., 1978; Harris, 1979). It follows that different effects of ethanol may involve different cellular mechanisms, so that an intervention that modifies the effects of ethanol on motor or endocrine function will not necessarily affect thermoregulation in the same way. So far, there is no evidence concerning the subcellular localization of the Ca 2+ pool presumably affected by EGTA, nor can the failure of Ca 2~ to alter ethanol hypothermia be explained in terms of specific neuronal membrane processes. Another possible mechanism of action is inhibition of the neuronal membrane (Na+/K+)-ATPase, which is believed to affect the release of norepinephrine, acetylcholine

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and possibly other neurotransmitters (Vizi et al., 1982). Chai et al. (1971) reported that acetylstrophanthidin, an inhibitor of (Na+/K+)-ATPase, when injected directly into the preoptic area caused a marked fall in body temperature. Ethanol also inhibits (Na+/K+)-ATPase (Israel et al., 1965, 1966; Kalant et al., 1978b), and Chai et al. (1971) found that the ethanol vehicle used for the acetylstrophanthidin (10/~1 of 29~o ethanol, or approximately 3 mg) also caused some fall in temperature. In contrast, 20 #1 of pure ethanol (i.e. approximately 16 mg) injected into the lateral ventricle of the rabbit had no effect, probably because of excessive dilution in the CSF (Cranston et al., 1976). Chai et al. did not investigate systematically the dose-response relations nor the interaction between ethanol and acetylstrophanthidin. However, injection of cadmium which, like ethanol, inhibits synaptosomal (Na+/K + )-ATPase noncompetitively with respect to ATP, also increases ethanol induced hypothermia additively in the rat (Magour et al., 1981). There is clearly a pressing need for research into the effects of ethanol on all neurophysiological aspects of POAH thermoregulatory function. 3.4.2. Hypothalamic Blood Flow In an elegant study by Hayward and Baker (1969), it was shown in five different species of conscious, freely-moving normothermic animals that the cerebral arterial blood is cooler than the deep brain sites it supplies. In the monkey, rabbit, cat, dog and sheep, therefore, the arterial blood cools the tissue by carrying away heat generated by neuronal metabolic activity. It seems reasonable that alterations in this relationship constitute the central thermal stimuli which, together with neural impulses from peripheral thermal sensors, control the activity of central thermoregulatory neurons. In addition to cooling the arterial blood reaching the brain (by decreasing heat production in muscle, and increasing heat loss through cutaneous vasodilatation), it is conceivable that ethanol could impair heat exchange in the POAH by selectively reducing blood flow in the central or ganglionic branches of the anterior cerebral artery supplying the POAH region. In theory, this would increase the local temperature and thus activate heat loss mechanisms and produce a fall in body temperature. However, no evidence has so far been provided on this point. An alternative possibility would be that, since the anterior hypothalamus is said to have a particularly rich vascular supply, and correspondingly high rate of blood flow (Borison and Clark, 1967), a general reduction in total cerebral arterial flow would have a particularly large effect on the POAH. In humans, small doses of alcohol produce inconsistent changes in total cerebral blood flow (Nelson et al., 1980), while large intoxicating doses produce a significant fall in man (Battey et al., 1953; Fazekas et al., 1955; Sutherland et al., 1960) and rat (Hemmingsen and Barry, 1979). In addition, scintillation camera techniques have revealed regional flow disturbances in the brains of alcoholics in various stages of intoxication and withdrawal (Heiss, 1977). With sufficient refinement of technique, it might be useful to re-examine rostral hypothalamic blood flow during alcohol intoxication in large experimental animals, and correlate the change with alternations of thermoregulatory function. 3.4.3. Effects on Neurotransmitters Involved in Thermoregulation 3.4.3.1. Dopamine. There is abundant evidence to suggest that dopamine (DA) may be a neurotransmitter in a central pathway responsible for the production of hypothermia in various species (Cox, 1979; Cox and Lomax, 1977). For example, hypothermia has been produced in the rat by intracerebroventricular (i.c.v.) injection of DA (Lomax et al., 1978), and this effect was blocked by i.c.v, pretreatment with phentolamine and with haloperidol. A similar effect in the cat was produced by injection of DA directly into the POAH (Ruwe and Myers, 1982). Others ligands, known to act as agonists at dopamine receptors, also produce hypothermia. For example, i.p. injection of piribedil in the mouse (Hoffman and Tabakoff, 1977) and of apomorphine in the rat (Lee and Cox, 1980) produce dose-

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dependent falls in rectal temperature. Bromocriptin, lergotrile and compound 25-397 also produced hypothermia in the cold exposed rat, which could be blocked by haloperidol and (in the case of bromocriptin) by ct-methyl-p-tyrosine (e-MT) (Silbergeld et al., 1977). The effect of the dopaminergic agonist apomorphine, whether injected i.p. or directly into the hypothalamus, is to initiate a coordinated heat loss response, including vasodilatation of the tail, increased evaporative heat loss, and decreased metabolic thermogenesis (Lee and Cox, 1980; Cox et al., 1981). This response, like that produced by ethanol, occurred at a Ta of 17°C and to a lesser degree at 25°C, but not at 35°C. Also, as in the case of ethanol, administration of apomorphine at Ta of 35°C resulted in hyperthermia, with a consequent increase in evaporative heat loss, apparently as a compensatory response. In view of the similarities between the effects of ethanol and dopaminergic agonists on thermoregulation, it might be expected that interference with dopaminergic mechanisms would abolish or reduce the hypothermic effect of ethanol. The results of such studies are, however, contradictory. Pimozide, a DA receptor blocker (Seeman, 1980), was reported to diminish the hypothermic effect of ethanol in the rat (Mullin and Ferko, 1981), and a DOPA decarboxylase inhibitor did the same in mice (Str6mbom et al., 1977). In contrast, ethanol hypothermia in rats was not diminished by butaclamol (Myers and Ruwe, 1982), by e-MT or haloperidol (Pohorecky et al., 1976; Silbergeld et al., 1977), even though e-MT blocked the hypothermic effect of bromocriptin (Silbergeld et al., 1977). Injection of 6-hydroxydopamine (6-OHDA) directly into the posterior mesencephalon of rats reduced the hypothermic effect of ethanol, even when noradrenergic terminals were protected by protriptyline (Kiianmaa, 1980); yet i.c.v, injection of 6-OHDA in mice, similarly protected with desmethylimipramine, did not affect ethanol hypothermia (Tabakoff and Ritzmann, 1977; Melchior and Tabakoff, 1981). Apomorphine, in a dose presumed to act agonistically only at presynaptic DA autoreceptors, produced slight hypothermia in mice, but failed to modify the hypothermic effect of ethanol (Str6mbom et al., 1977). Similar contradictions are found in relation to the effects of ethanol on DA metabolism. If ethanol produces hypothermia via a dopaminergic pathway, one might expect ethanol to increase the synthesis, release or receptor actions of DA in some relevant region of the brain. Griffiths et al. (1974) reported that a single exposure to low concentrations of ethanol decreased mouse brain DA level during the same period of time in which it produced its behavioral effects; the reduction in dopamine level might thus imply an ethanol induced release of DA from axon terminals. However, a single (but higher) dose of ethanol in the rat (Nikki et al., 1971) or guinea-pig (Huttunen, 1982) did not lower brain DA concentration, or even raised it. This would appear to reflect decreased release, since a similar dose of ethanol failed to increase DOPA levels in mice pretreated with a DOPA-decarboxylase inhibitor, and actually enhanced the inhibition of synthesis produced by a low dose of apomorphine acting presynaptically (Str6mbom et al., 1977). K+-stimulated release of DA from isolated slices of caudate nucleus was increased in preparations obtained from animals with low blood alcohol levels, and decreased in those from animals with high alcohol levels (Darden and Hunt, 1977). Part of the apparent contradiction may be due to regional differences. Bacopoulos et al. (1978) found that ethanol (2 g/kg) reduced the turnover of DA in the rat caudate nucleus, increased it in the olfactory tubercle, and had no effect in the nucleus accumbens, amygdala and hypothalamus. Differences in the net balance of these effects with different doses of ethanol, different species, and different states of arousal and activity, might well account for the discrepancies among findings reported in the literature. Finally ethanol, added in vitro, was reported to enhance the DA stimulation of striatal adenylate cyclase activity in preparations from rat (Fish et al., 1979) and mouse (Rabin and Molinoff, 1981). However, this effect was not blocked by the DA receptor blocker, butaclamol, and Tabakoff and Hoffman (1979) were unable to reproduce this effect with either a single dose of ethanol in vivo, or addition of 50 mM in vitro. In view of these sharp disagreements, it is impossible to conclude that the effects of ethanol on thermoregulation are mediated chiefly or exclusively by dopaminergic systems. Kiianmaa (1980) found that the degree of ethanol induced hypothermia correlated better

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with the DA content of the striatum than with that of the limbic forebrain, and Reggiani et al. (1980) reported that a single dose of ethanol increased the production of dihydroxyphenylacetic acid (DOPAC) selectively in the striatum, and not in the accumbens or substantia nigra. These findings recall the early observation (Barbour and Wing, 1913) that injection of ethanol into the striatum could produce hypothermia. If the effects of DA and of ethanol are mediated via the POAH, however, it seems clear that striatal dopaminergic fibers must constitute only one of many inputs that are integrated by POAH neurons. It is therefore necessary to examine the evidence concerning ethanol effects on other neurotransmitters that may act in the POAH. 3.4.3.2. Norepinephrine. As in the case of DA, there is ample evidence to implicate norepinephrine (NE) in central thermoregulatory mechanisms (Bruinvels, 1979), but again the nature of its role is not wholly clear, and much of the evidence is contradictory. Injection of NE i.c.v, in the rat produces hypothermia, which can be blocked by phentolamine (Burks and Rosenfeld, 1979). Another noradrenergic agonist, clonidine, in a large dose (500/~g/kg) i.p. which may have acted on post-synaptic ~-receptors in the periphery, also produced hypothermia in the mouse (Hofl'man and Tabakoff, 1977). On the other hand, a dose of clonidine (50 ~tg/kg) s.c. which probably affected primarily presynaptic ~2-receptors, also produced hypothermia in the rat, and this effect was inexplicably enhanced by the DA blocker pimozide (Mullin and Ferko, 1981). Hypothermia also follows direct microinjection of NE into the POAH of rats at normal T, (Veale and Whishaw, 1976; Poole and Stephenson, 1977; Myers and Ruwe, 1978). However, injections at the same site produced hyperthermia at Ta of 5°C and 35°C (Veale and Whishaw, 1976), while injection of 6-OHDA at that site produced a failure of thermoregulation against both cold (8°C) and heat (35°C) (Myers and Ruwe, 1978). These seemingly discordant findings may be clarified to some extent by single unit electrophysiological studies. Most thermosensitive units in the cat POAH respond to local injection of NE (Jell and Sweatman, 1975). Cold sensitive units in the rabbit POAH increased their firing rate in response to i.c.v, or local injection of NE, and also changed their pattern of thermosensitivity to that of warm sensitive units (Gordon and Heath, 1981a). This would be consistent with a change in set-point, but the direction of change is opposite to that described by Satinoffand Hackett (1977). The latter investigators argued that NE produces a rise in set-point, since brain temperature and whole-body oxygen consumption rose, and tail vessels constricted, at all Ta tested (5°C, 25°C or 31°C). Higher doses of NE, however, produced hypothermia not by lowering the set-point, but by disorganizing the effector responses, e.g. by reducing thermogenesis while still maintaining vasoconstriction. Conceivably this distinction between physiological and pharmacological effects may explain the observation by Borison and Clark (1967) that exogenous administration of a neurotransmitter may not reproduce the effects of its endogenous release. For example, intracerebral injection of NE produces hypothermia, while release of endogenous NE by amphetamine or impairment of its reuptake by cocaine result in hyperthermia. Accurate knowledge of endogenous concentrations in the synaptic cleft and of effective local concentrations after microinjection may be necessary for resolution of this question. Such considerations might also explain the marked species differences in thermoregulatory effects not only of NE but also of DA and 5-HT (Feldberg, 1970; Bowman and Rand, 1980). In view of the uncertainty about the exact role of NE, it is not surprising that the same uncertainty attaches to its role in the effects of ethanol on thermoregulation. No change in plasma catecholamines was found in humans during mild cold exposure after ethanol ingestion (Risbo et al., 1981), and guinea-pigs exposed to a T~ of - 2 0 ° C had less epinephrine in the urine if they had received ethanol than if they had not (Huttunen et al., 1980). These findings are consistent with the observations (Section 2.2.2.1) that cold exposed subjects are less uncomfortable if they have consumed alcohol, and probably reflect a diminished peripheral sympathetic stress response.

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More direct evidence concerning a central role, unfortunately, is also contradictory. Systemic administration of ethanol has produced, in different studies, a rise, fall, or no change in brain concentrations of NE (for references, see Huttunen, 1982). Incorporation of [3H]tyrosine into [3H]NE and its rnetabolites in rat hypothalamus was increased after low doses of ethanol that raised body temperature, but reduced when ethanol produced hypothermia (Pohorecky and Jaffe, 1975). In addition, marked regional differences have been reported, including a decrease in NE turnover in hypothalamus, an increase in pons medulla, and no change elsewhere (Bacopoulos et al., 1978). In the guinea pig at a Ta of 20°C, ethanol induced hypothermia was accompanied by large increases in the hypothalamic concentrations (decreased release?) of both NE and epinephrine, but at - 2 0 ° C NE was increased while epinephrine was not (Huttunen, 1982). Ethanol-induced hypothermia in the rat was not affected by icy injection of phenoxybenzamine (Pohorecky et al., 1976) or phentolamine (Myers and Ruwe, 1982), but in the mouse was reduced by pretreatment with a-MT (Melchior and Tabakoff, 1981). Amphetamine and DH-524, administered systemically, have been reported to intensify the hypothermic effect of ethanol in the gerbil (J/~rbe and Ohlin, 1977) but to decrease it in the mouse (Abdallah and Roby, 1975). In one study, intracerebral injection of 6-OHDA in the mouse increased the hypothermic effect of ethanol at 23°C (Melchior and Tabakoff, 1981); in another, it decreased ethanol hypotherrnia in the rat, especially if NE terminals were not protected by protriptyline (Kiianmaa, 1980); and in a third it had no effect on ethanol hypothermia in the mouse, with or without desmethylimipramine protection (Tabakoff and Ritzmann, 1977). 3.4.3.3. Sermon&. In a review of the literature up to 1965, Borison and Clark (1967) concluded that 5-hydroxytryptophan (5-HTP), the biosynthetic precursor of serotonin (5-HT), increased heat production after either central or peripheral administration. In later reviews, Feldberg (1970), Hellon (1974) and Jacob and Girault (1979) concluded that i.c.v. or intrahypothalamic injection of 5-HT altered body temperature in almost all species, but that evidence concerning the direction of change was contradictory, and varied greatly from one species to another. For example, Gardey-Levassort et al. (1974) observed that 5-HTP in the rabbit produced hyperthermia that was intensified by probenecid and reduced by a decarboxylase inhibitor. This suggested that 5-HT centrally caused hyperthermia. However, the drugs were given i.p., and the effect of the decarboxylase inhibitor may have been to increase the supply of 5-HTP reaching the brain, as suggested by the finding that hypothalamic 5-HT levels were increased. From this, one might conclude that 5-HT centrally produces hypothermia. Indeed, in the rat (Burks and Rosenfeld, 1979) and rabbit (Tangri et al., 1980) i.c.v. injection of 5-HT caused hypothermia which was prevented by methysergide, but not by phentolamine or haloperidol. In addition, Lilly 110140, an inhibitor of 5-HT reuptake into presynaptic terminals, also produced hypothermia, and p-chloroamphetamine, an inhibitor of 5-HT synthesis, produced hyperthermia (Pohorecky et al., 1976). The mouse also responded to 5-HT by a fall in temperature which was increased by probenecid (Ritzmann and Tabakoff, 1976), yet pretreatment with 5,7-dihydroxytryptamine (5,7-DHT) plus desmethylimipramine (to selectively deplete brain serotonin) had no effect on body temperature (Melchior and Tabakoff, 1981). In the cat, Ruwe and Myers (1982) found that injection of 5-HT directly into the POAH produced either hypo- or hyperthermia, depending on the site. This finding is consistent with the suggestion that there are two distinct sets of hypothalamic neurons responding to 5-HT, a more rostral set triggering heat conservation mechanisms and a more caudal set mediating heat loss (Hellon, 1974). However, where 5-HT produced hypothermia, this effect could be blocked by methysergide, butaclamol or phentolamine, but where it caused hyperthermia it could be blocked only by methysergide. These observations have been interpreted as evidence that hyperthermia is a specific effect of 5-HT, but that hypothermia following injection of 5-HT may be at least partly due to uptake of excess 5-HT into catecholaminergic neurons (Ruwe and Myers, 1982).

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An alternative hypothesis is that hypothermia is due not to 5-HT itself, but to its deaminated and reduced metabolites, the tryptophols (Barofsky and Feldstein, 1970), since pretreatment with the MAO inhibitor phenelzine markedly reduced the hypothermia produced in mice by 5-HTP, the precursor of 5-HT. However, Ritzmann and Tabakoff (1976c) found no hypothermia after i.c.v, injection of 5-hydroxytryptophol in a different mouse strain. In a recent review, Myers (1981b) concludes that almost all species show a h y p e r t h e r m i c response to i.c.v, or intrahypothalamic injection of small doses of 5-HT, and that when hypothermia occurs, it is due to peripheral 'side effects' of large doses. This is consistent with the observation that when 5-HT-sensitive neurons are functionally deafferented by i.c.v, injection of 5,7-DHT or by chronic administration ofpCPA or metergoline, the basal body temperature is reduced (Frankel et al., 1978), and the consequent receptor hypersensitivity results in a greatly enhanced hyperthermic response to the serotoninergic agonist quipazine (Carruba et al., 1981). All of the foregoing findings are difficult to reconcile with the very limited data on the effects of 5-HT on activity of thermosensitive POAH neurons. The majority of both thermosensitive and thermoinsensitive POAH units respond to direct local injection of 5-HT, but the response patterns differ (Jell and Sweatman, 1975; Gordon and Heath, 1981a). Intraventricular injection of 5-HT tended to excite warm sensitive POAH units and inhibit cold sensitive ones, which would apparently fit with a hypothermic effect. However, direct injection into the POAH tended to inhibit both warm and cold sensitive units and to abolish their thermosensitivity (Gordon and Heath, 1981a). This would suggest a poikilothermic rather than a hypo- or hyperthermic action. If ethanol induced disturbances of thermoregulation are mediated chiefly by serotoninergic mechanisms, as has been claimed (Pohorecky et al., 1976), one might expect to find that ethanol alters 5-HT metabolism or 5-HT receptor properties, and that ethanolinduced temperature changes can be modified by serotoninergic agonists or blockers. Once again, however, the results of such studies are as variable as those of 5-HT itself. Ritzmann and Tabakoff (1976c) found that ethanol induced hypothermia in the mouse was enhanced by i.c.v, injection of 5-HT (though this effect may have been merely additive) and Frankel et al. (1978) found that depletion of rat brain 5-HT by p-chlorophenylalanine (pCPA) decreased the hypothermic effect of ethanol. In contrast, depletion of rat brain 5-HT by p-chloroamphetamine increased the magnitude of ethanol induced hypothermia, and inhibition of 5-HT reuptake decreased the hypothermia (Pohorecky et al., 1976). Inexplicably, the same investigators found that probenecid enhanced hypothermia produced by ethanol (Pohorecky et al., 1976), just as it did with that caused by 5-HT (Ritzmann and Tabakoff, 1976c). Still others have found ethanol hypothermia to be unaffected by i.c.v. injection of 5,7-DHT plus desmethylimipramine in the mouse (Melchior and Tabakoff, 1981) and rat (L6 et al., 1980), by a large dose of tryptophan which elevates brain serotonin level (L~ et al., 1979a) or by i.c.v, injection of methysergide in the rat (Myers and Ruwe, 1982). Equally contradictory are the reported effects of ethanol on brain 5-HT metabolism. Conflicting results in the earlier literature have been summarized by Frankel et al. (1974) and Pohorecky et al. (1976, 1978). In brief, single doses of ethanol in rats and mice have been reported to increase brain 5-HT (Pohorecky et al., 1974), decrease it (Griffiths et al., 1974) or leave it unchanged (Nikki et al., 1971; Frankel et al., 1974; Tabakoff et al., 1975; Pohorecky et al., 1978; Fukumori et al., 1980), though there is agreement that ethanol inhibits the active transport of 5-HIAA out of the brain, thus raising its concentration there (Tabakoff et al., 1975; Pohorecky et al., 1978; Fukumori et al., 1980). Once more, therefore, it is necessary to conclude that no clear case has been made for a predominant role of 5-HT in the central thermoregulatory effects of ethanol. 3.4.3.4. Histamine. Although a neurotransmitter role for histamine in the CNS has not yet been clearly proven (Blatteis, 1981), there is a reasonable amount of evidence that exogenous histamine injected icv, or endogenous brain histamine formed from systemically injected histidine, causes a dose-dependent hypothermia in various species (Lomax and L P T . 23/3

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Green, 1979, 1981; Tangri et al., 1980; Dhawan et al., 1982). This action is exerted through both H1 and H2 receptors, but it is also blocked by methysergide (Tangri et al., 1980) and by p CPA (Pilc and Nowak, 1980), though not by a fl-adrenoreceptor blocker (Dhawan et al., 1982), so that histamine may be acting indirectly through serotoninergic neurons. This idea is supported by the observation that histamine induced hypothermia is accompanied by a fall in brain 5-HT and an increase in 5-HIAA, indicative of increased 5-HT turnover (Pilc and Nowak, 1980). However, there is as yet very little evidence to link histamine to ethanol induced temperature change. Papanicolaou and Fennessy (1980) found a dose-dependent biphasic effect of ethanol on both body temperature and whole-brain histamine levels in mice. A low dose of ethanol (0.175 g/kg) i.p. raised the body temperature and brain histamine level, while higher doses reduced both. In the rat, doses of 0.8-2 g/kg of ethanol by gavage, which are probably comparable to the low i.p. doses in the mouse in terms of rate of rise of brain alcohol level, also raised the histamine concentration of whole brain (Rawat, 1980) and specifically of the hypothalamus (Subramanian et al., 1978). The increase appeared to be due primarily to inhibition of histamine release, at least as tested in vitro (Subramanian et al., 1978). There is insufficient experimental evidence, especially with larger doses and other routes of administration, to indicate whether alcohol induced changes in histamine turnover correlate well, in time and magnitude, with changes in body temperature. 3.4.3.5. Acetylcholine. In reviewing a rather large body of experimental literature on the effects of acetylcholine (ACh), pilocarpine, carbachol and other cholinergic drugs on body temperature, Crawshaw (1979) has pointed out lucidly that ACh acts in so many neural systems in the brain that it is extremely difficult to separate direct from indirect actions on thermoregulatory neurons. Jell and Sweatman (1975), injecting ACh directly into the POAH of the cat, found many units which responded, but these included both thermosensitive and insensitive units, and there was extensive overlap of unit responsiveness to ACh, 5-HT, NE and prostaglandins. For this very reason the specific role of ACh in thermoregulatiov remains unclear. Feldberg (1970) accepted the hypothesis that ACh provides the first link in the neuronal chain constituting the effector pathway for thermogenesis (Myers and Yaksh, 1969). However, like the other neurotransmitters discussed above, ACh has produced both hyper- and hypothermia, depending upon species, dose, route and ambient temperature (Crawshaw, 1979). Ethanol produces different effects on ACh release, depending not only on dose but also on type of synapse involved. At the peripheral neuromuscular junction it increases spontaneous release of ACh (Gage, 1965; Curran and Seeman, 1979). However, in the isolated longitudinal muscle myenteric plexus preparation from guinea-pig ileum, ethanol decreases electrically stimulated contractions, which are known to be mediated by ACh (Mayer et al., 1980). The same is true for electrically stimulated release from isolated cerebral cortical slices (Clark et al., 1977), and for spontaneous release from the cerebral cortex as studied by cortical superfusion in the conscious moving animal (Phillis and Jhamandas, 1971) and from the midbrain reticular formation as studied by perfusion through the push-pull cannula (Erickson and Graham, 1973). Since the doses of ethanol used in these studies characteristically produce hypothermia in the rat at normal room temperature, one must conclude either that ACh does act primarily to maintain heat production in this species, or that the effect of ethanol on ACh release in the POAH differs from that in the cortex and other sites studied. To date, there is no direct evidence to permit a confident answer. 3.4.3.6. A m i n o acids. In the last few years, a number of amino acids have been studied intensively with respect to their possible roles as central neurotransmitters. There is a considerable amount of evidence to support the view that aspartate and glutamate function as excitatory transmitters and glycine, taurine and 7-aminobutyrate (GABA) as inhibitory transmitters. This appears to be true in many parts of the central nervous system, including

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the hypothalamic thermoregulatory pathways (Blatteis, 1981; Bligh, 1981) where they appear to modulate not only the afferent paths from peripheral cold sensors but also efferent impulses to the effector systems (Bligh, 1981). Ethanol could conceivably modify these amino acid transmitter functions in various ways: (1) by inhibiting active transport of amino acids across the blood-brain barrier; (2) by modifying local synthesis of amino acids, such as GABA, that are formed in situ; (3) by modifying the release of transmitter amino acids from axon terminals; (4) by altering binding to their receptor sites. There is, regrettably, very little information available about these possible actions. Effects of ethanol on cerebral uptake of amino acids have not been explored in depth (Chin, 1979; Pratt, 1980). In confirmation of earlier observations by Pohorecky et al. (1978), Eriksson and Carlsson (1980) found that ethanol increased the brain:blood ratios of large neutral amino acids in rats injected with amino acids i.p., but the results do not permit any conclusion about whether this reflects increased absorption from the injection site, increased influx to the brain or decreased active transport out of the brain. Studies of ethanol effects on synthesis of amino acids in situ are largely confined to GABA, for which the results are variable and inconclusive (Hunt and Majchrowicz, 1979). Effects of ethanol on GABA release have been studied in electrically stimulated slices of cerebral cortex (Carmichael and Israel, 1975), but not of hypothalamus, nor POAH specifically. The same is true with respect to receptor binding. Virtually the only evidence directly relevant to neurotransmitter amino acids and ethanol induced temperature change is the observation that hypothermia produced by ethanol (3 g/kg) i.p. in mice was not significantly altered by i.p. injection of taurine (Boggan et al., 1978). Since this was not even an i.c.v, injection, and the fate of the taurine in the brain is not known, we must conclude that this topic remains practically unexplored. 3.4.3.7. Discussion. From all the foregoing evidence, it is clear that every proven or putative neurotransmitter examined to date has been able to produce hypothermia in some circumstances and hyperthermia in others, and that their effects are inter-related. A schematic model that attempts to account for all the observed effects, first proposed by Bligh and his collaborators and modified by Tabakoff et al. (1978a), is shown in Fig. 1. It proposes that the set-point for thermoregulatory responses is determined by the effect of afferent stimuli from heat and cold receptors upon the tonic activity of dopaminergic neurones, which in turn modifies the activity of effector systems for heat loss and heat generation and conservation. Unfortunately the difficulties for such a scheme are legion. It does not explain, for example, how 5-HT injection can cause both hypo- and hyperthermia, nor why ethanol effects on the DA neuron would impair both heat conservation at low Ta and heat loss at high Ta. It makes no provision for the observed effects of histamine, amino acids, peptides or calcium. The most reasonable interpretation of the experimental observations is that no single transmitter acts exclusively upon POAH neurons to regulate the set-point. All of them may also influence afferent and efferent thermoregulatory pathways. The balance of all these influences presumably determines the net change in body temperature. Since ethanol can ,EAT

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FIe. 1. Schematic model of neural connections in the central thermoregulatory mechanisms of the preoptic area and anterior hypothalamus, as proposed by Tabakoff et al. (1978). 'Heat' and 'cold' designate inputs from peripheral thermal sensors. The DA neurone is viewed as the set-point controller, which would act tonically to set the balance of activity in the effector systems for production, loss and conservation of heat. Reproduced with the permission of the authors, and of Grune & Stratton, publishers.

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affect the synthesis, release, degradation and re-uptake of most or all transmitters, depending upon the dose, concentration, route of administration, specific CNS locus and behavioral state of the subject, the effect of ethanol on body temperature in any individual case probably also depends on the balance of all these alterations. A finer analysis of the mechanisms of ethanol action must await a major improvement in the techniques for studying direct local effects of ethanol on individual components of the thermoregulatory system. 3.4.4. Neuropeptides Over the past decade, numerous peptides have been found to be present in the central nervous system and to exert potent effects there, probably as modulators of neuronal activity. A number of these have been shown to act on thermoregulatory mechanisms. Among these are bombesin, somatostatin, melanotropin release inhibiting factor I (prolyl-leucyl-glycinamide),.~-melanotropin and neurotensin (Yehuda and Kastin, 1980; Brown, 1981; Lipton et al., 1981; Morley et al., 1982) which have so far scarcely been studied with respect to their possible role in the effects of ethanol. Only three neuropeptides have been studied more than once in this connection: fl-endorphin, vasopressin and thyrotropin releasing hormone (TRH). 3.4.4.1. fl-Endorphin. It has been known for many years that small doses of exogenous morphine-like opiates produce a dose-dependent hyperthermia in many species (French et al., 1978a), and that larger doses cause an initial hypothermia followed later by hyperthermia (Lotti et al., 1965a; Rosow et al., 1980). This is true even when the drugs are injected icv (Clark 1979, 1981) or directly into the POAH (Lotti et al., 1965a). When specific endogenous opioid peptides were discovered, their effects on central thermoregulatory mechanisms were soon tested and fl-endorphin (/3-E) also was found to produce a dose-dependent hyperthermia when injected i.c.v, in both rat and mouse (Holaday et al., 1978a; Clark, 1981; Clark and Bernardini, 1981). As with morphine, larger doses of fl-E produced an initial hypothermia. The hyperthermia occurred at low and high ambient temperatures as well as at normal room temperature (i.e. from 0°C to 34°C), and was therefore interpreted as an indication of an upward adjustment of the set-point of the thermoregulatory system. Further support for this concept is provided by the observation that icv injection of fl-E in rats almost immediately produced a huddled posture (heat conservation) and wet-dog shakes (heat generation), followed shortly afterwards by a rise in colonic temperature, which was more marked at 34.5°C than at 26.5°C (Holaday et al., 1978a). Hyperthermia at both low and high ambient temperatures also occurred when fl-E was injected directly into the POAH of the rat (Thornhill and Wilfong, 1982) and rabbit (Rezvani et al., 1982). In the POAH of the rabbit it also affected the firing rates of single units responding to cutaneous heat stimuli (Gordon and Heath, 1981b). Similar effects were seen in the conscious unrestrained cat after i.c.v, injection of an enkephalin analog (o-ala2)-methionine-enkephalinamide or 'DAME' (Clark and Ponder, 1980). In contrast, the x-agonist ketocyclazocine and the a-agonist SKF 10,047 caused hypothermia at an ambient temperature of 0°C and 22°C, but hyperthermia at 34°C (Clark et al., 1981); this was suggestive of impairment of thermoregulatory mechanisms, rather than change in set-point. Since the effects of morphine, DAME and fl-E on thermoregulation were blocked by naloxone in most (Clark and Ponder, 1980; Clark et al., 1981; Clark and Bernardini, 1981; French et al., 1978a; Rezvani et al., 1982), although not all cases (Thornhill and Wilfong, 1982), it was reasonable to examine the effects of naloxone and other blockers alone, as a means of exploring the possible physiological role of endogenous opioids in normal thermoregulation. Lotti et al. (1965b) has observed that nalorphine, injected into the anterior hypothalamus of the rat, blocked morphine-induced hypothermia, but caused mild hyperthermia by itself. Nalorphine is a mixed agonist-antagonist, and it now seems

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likely that the hyperthermia was a #-agonist effect. Similar results have been obtained with a variety of other mixed agonist-antagonists (Rosow et al., 1982b). In contrast, in the rat not previously treated with opiate, naloxone or naltrexone caused no effect in some studies (Lin et al., 1980); hyperthermia in others, especially after acute or chronic heat stress (Holaday, 1978b); and a mild hypothermia in others (Thornhill et al., 1980; Rosow et al., 1982a; Morley et al., 1982). These diverse findings are considered by some investigators to support the concept of a physiological role of endogenous opioids in maintaining normal body temperature. A possible explanation of the discrepancies is suggested by the finding that handling stress causes a hyperthermia which can mask the hypothermic effect of naloxone, unless the latter is given before the stress is applied (Stewart and Eikelboom, 1979). In hypophysectomized rats, stress failed to block naloxone hypothermia. Therefore it seems likely that an endogenous opioid in the brain itself does exert a tonic influence on temperature regulation in the rat. In the cat, i.c.v, injection of naloxone (100 or 250 #G) failed to block stress induced hyperthermia (Clark et al., 1983). Since the same doses, however, also failed to produce hypothermia in morphine tolerant cats, it is possible that these doses of naloxone were too small to permit diffusion to the appropriate sites of action in the cat brain. In the mouse, ethanol induced hypothermia was potentiated by icv injection of fl-E (1.8 nmol); neurotensin was even more potent (Luttinger et al., 1981). If the ratios of brain weight and ventricular volume to body weight are roughly equal in mouse and cat, this dose of fl-E would be equivalent to about 200 nmol in the cat, a dose large enough to produce hypothermia by itself. In the rat a single injection of ethanol (2.5 g/kg) i.p. was reported to increase the concentration of fl-E in the hypothalamus at 20 and 60min post-injection, and of methionine-enkephalin in the striatum at 60 min (Schulz et al., 1980). In unpublished studies in this laboratory (N. Woo and H. Kalant) we have been unable to confirm this finding. However, if it is valid, and if it reflects decreased release of opioids at axon terminals on POAH units, it might suggest that ethanol induced hypothermia is due in part to suppression of a tonic hyperthermic influence of the peptides. In addition, it has been reported that ethanol, like naloxone, can inhibit both unconditioned and conditioned stress induced hyperthermia (York and Regan, 1982); presumably this is consistent with ethanol suppression of an endogenous opioid mechanism. However, this is not consistent with the finding that ethanol induced hypothermia in mice was, if anything, slightly decreased by naloxone (3 mg/kg) i.p, (Boggan et al., 1979). Moreover, in experiments with SS and LS mice (which were genetically selected for low and high sensitivity respectively to ethanol) the SS strain was reported to show the expected small hypothermic response to ethanol, compared to the LS strain, but their order of sensitivity to morphine hypothermia was reversed. Naloxone attenuated ethanol hypothermia only in the SS strain (Brick and Horowitz, 1982). In our own laboratory (J. M. Khanna et al., unpublished results) the LS strain showed a greater hypothermic response to both morphine and ethanol than did the SS strain. At this stage, one must conclude that a role for endogenous opioids in the hypothermic effect of ethanol is possible but not conclusively proven. 3.4.4.2. Thyrotropin releasing hormone. In contrast to the literature on endogenous opioids, that on TRH is remarkably consistent, although (or perhaps because) it is less in amount. Injection of TRH (50 #g) i.c.v, in the rat causes a clear sustained hyperthermia (Cohn et al., 1976), though a dose of 10 #g fails to do so (Morley et al., 1982). However, even low doses have shown a striking ability to reverse the narcosis and hypothermia produced in rats and mice by ethanol, pentobarbital, amobarbital and naloxone (Breese et al., 1974; Cohn et al., 1976; Cott et al., 1976; Morley et al., 1982). The same effect can be shown after i.p. or even oral administration, if the dosage is increased appropriately (Breese et al., 1974; Cott et al., 1976; Porter et al., 1977). The reversal of ethanol induced narcosis and hypothermia did not require the presence of the hypophysis or thyroid, and was not prevented by ~ or fl adrenergic blockers. However, it was prevented by i.c.v, injection of atropine and enhanced by D-tubocurarine

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and hexamethonium (Cott et al., 1976). These findings suggest that TRH either increases ACh output in central muscarinic synapses, or sensitizes the muscarinic receptors. Conceivably these are receptors in the cholinergic thermogenic effector pathway proposed by Myers and Yaksh (1969). This effect of TRH would appear to be independent of any general analeptic action since the doses required to reverse alcohol induced hypothermia differed substantially from those required to reverse alcohol narcosis or impairment of various psychomotor performance tasks (Porter et al., 1977). It is still not clear, however, whether these actions of TRH should be considered physiological or pharmacological. In the conscious goat local cooling of the POAH by an implanted thermode led to sharp increases in serum TSH and triiodothyronine during the period of rise in body temperature (Marques et al., 1981). These changes, which imply increased release of TRH, suggest that TRH plays a role in the maintenance of normal body temperature during cold exposure. However, the reversal of ethanol hypothermia by TRH in the mouse did not require the presence of the hypophysis or pituitary (Cott et al., 1976). This might represent a species difference in thermoregulatory mechanisms, or it might mean that TRH can stimulate thermogenesis by both humoral (nonshivering?) and neural (shivering?) mechanisms. This remains to be explored. 3.4.4.3. Vasopressin and related peptides. Veale et al. (1981) have reviewed evidence suggestive of a thermoregulatory role for arginine vasopressin. In sheep it appears to act centrally, in the septal area, to limit the extent of pyrogen induced fever, but to have no effect when injected into the POAH or caudal hypothalamus. When injected i.c.v, in the rat, it is reported to lower body temperature. However, there is so little firm evidence concerning its possible role in normal thermoregulation that the subject is still largely conjectural. Not surprisingly, therefore, very little attention has been paid to its acute interaction with ethanol on body temperature. We have found only two reports from the same group, one dealing with oxytocin (Rigter et al., 1980c) and one with desglycinamideg-arginine8-vasopressin or DGAVP (Crabbe et al., 1980). Oxytocin (s.c.) did not affect baseline temperature in mice, but produced a non-significant enhancement of ethanol hypothermia. In this study, DGAVP did not affect either baseline temperature or ethanol hypothermia, but in a comparison of several inbred strains of mouse (Crabbe et al., 1980) DGAVP tended to decrease the inter-strain differences in hypothermic response to a single dose of ethanol. Obviously, this field is still in the very earliest stage of exploration. 3.4.5. Prostaglandins Evidence concerning the role of prostaglandins of the E series (PGEs), in the intrahypothalamic mediation of fever production by pyrogens, has been reviewed by Cooper et al. (1979). Despite the contrary view of Cranston et al. (1976), most investigators accept the likelihood of this role. According to Bowman and Rand (1980), i.c.v, injection of PGEs produced a rise in body temperature of all species tested up to that point. Injection of PGE~ directly into the POAH of the conscious rat produces a dose-dependent hyperthermia, at ambient temperatures ranging from 5°C to 35°C (Veale and Whishaw, 1976). This is suggestive of an upward shift of the set-point. Thermosensitive single units in the POAH of the cat generally respond to local microinjection of PGEI or PGE2 (Jell and Sweatman, 1975); cold-sensitive units (presumably those initiating heat conservation or thermogenic responses) tend to be facilitated, while warm-sensitive units are inhibited (Gordon and Heath, 1980). In view of the evidence that ethanol tends to produce hypothermia in most species at normal or low ambient temperatures (Sections 2.3.1 and 2.3.2), one might expect that ethanol would, if anything, inhibit the production and release of PGEs in the POAH. Over 40 years ago, Genuit (1940) reported that systemically administered short-chain aliphatic alcohols reduced colitoxin fever in the rabbit, in sub-narcotic doses. This would be consistent with inhibition of PGE synthesis. Unfortunately there is no direct in vivo

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evidence bearing on this point, and such evidence as is provided by in vitro studies suggests that the direct short-term effect of rather high concentrations of ethanol is to increase prostaglandin synthesis (Collier et al., 1975; Horrobin, 1980). In addition, there is disagreement concerning the effect of prostaglandin synthetase inhibitors on the hypothermic effect of ethanol. In the LS and SS mouse strains, bred selectively for differences in CNS sensitivity to ethanol, pretreatment with indomethacin or aspirin decreased ethanol hypothermia (George et al., 1981), while in Sprague-Dawley rats receiving a smaller dose of ethanol, indomethacin had no effect (Pohorecky et al., 1976). George et al. (1981) suggest that the ethanol-indomethacin interaction may be on peripheral rather than central prostaglandin metabolism since peripheral administration of PGE 1 was reported to cause hypothermia by inducing vasodilatation and decreasing metabolic rate (Lin, 1979). Clearly, no conclusion is possible until the interaction of ethanol and PGEs is studied in the POAH itself. 3.5. BEHAVIORALTHERMOREGULATION In addition to the physiological mechanisms governing heat production and heat loss described in preceding sections, animals can also affect their own body temperature by voluntary and involuntary modifications of the physical relationship to their environment. An example of an involuntary modification is the growth of longer fur or thicker subcutaneous fat, with consequent decrease in transcutaneous heat loss, in animals exposed chronically to ambient cold (Irving, 1966; Hensel, 1981). Obviously these cannot change rapidly and therefore cannot be related to the acute effects of ethanol or other drugs on body temperature. In contrast, voluntary measures such as putting on or taking off clothing, or seeking a warmer or cooler environment, can readily be affected by psychoactive drugs. Indeed, some of the cases of alcohol induced accidental hypothermia in humans (Section 2.1) are directly attributable to a failure to employ such measures. In experimental animals, behavioral mechanisms of this type can be studied in various ways (Cox et al., 1975; Satinoff, 1979; Rigter et al., 1980b; Clark and Bernardini, 1982). Essentially, the animal learns either to escape from an excessively hot or cold environment, or to perform work to obtain additional heat (e.g. from heat lamps) or cooling (e.g. by turning on a fan or a shower). The great advantage in these methods is their ability to distinguish between a change in set-point and a disruption of thermoregulatory effector systems. If the animal fails to make an appropriate behavioral response until the body temperature has shifted to a new value which is then maintained by integrated physiological and behavioral means, one may infer a shift in set-point. If the animal's body temperature changes, but the behavioral responses are appropriate to the imposed thermal load, one may infer an unchanged set-point but impaired physiological response mechanisms. Such methods have been used profitably to study the thermoregulatory effects of opioids (Clark, 1979; Clark and Bernardini, 1982), hydralazine (Rigter et al., 1980b), diphenhydramine and oxotremorine (Cox et al., 1975), and a variety of other drugs. In the rat, a dose of ethanol (1.5 g/kg i.p.) significantly reduced the latency to escape from radiant heat, even though the rectal temperature was falling below normal under the influence of the ethanol (Lomax et al., 1981). This implies a reduction in set-point, yet the effects observed after ethanol at different ambient temperatures (Section 2.3.3) suggested either impairment of thermoregulatory response mechanisms or reduced accuracy or sensitivity of POAH neurons ('broadening of the set-point'). Since elevated Ta has been shown to increase the narcotic and lethal effects of ethanol (Section 2.3.3; also Li et al., 1977), it is possible that two different mechanisms are operating in the different environmental circumstances. 4. CHRONIC ETHANOL EFFECTS: TOLERANCE AND PHYSICAL DEPENDENCE The effects of chronic ethanol ingestion on thermoregulation may arise from two distinct processes, viz (a) organic disease caused by ethanol or by malnutrition or other

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consequences of its use, and (b) functional adaptation to the repeated or prolonged action of ethanol. It is known, for example, that hypothermia (or, more broadly, defective thermoreguation) is one of the signs of Wernicke's encephaiopathy (Lipton et al., 1978; Editorial, Lancet, 1979; Macaron et al., 1979) which may improve dramatically on administration of thiamine. An observation that is possibly related to these is that the offspring of rats that consumed ethanol during pregnancy showed a greater hypothermic reaction to ethanol injections when tested at 52-120 days of age (Taylor et al., 1981).This effect of in utero exposure appeared to be less if the pregnant rats received a casein supplement in their ethanol containing diet, and may therefore be a consequence of fetal malnutrition. However, a detailed examination of thermoregulatory disturbances in all the disease states associated with alcoholism is beyond the scope of this review. The remainder of this section therefore deals only with the functional effects of adaptation to ethanol, i.e. tolerance and physical dependence. 4.1. TOLERANCE TO ETHANOL INDUCED HYPOTHERMIA

Although there is some disagreement in the literature concerning the extent and circumstances of production of hypothermia after a single dose of ethanol, especially in humans (Sections 2.2 and 2.3.1), there is almost unanimity that prolonged or repeated exposure to ethanol produces tolerance to this effect in all species. Human heavy drinkers showed the same slight hypothermia after i.v. infusion of 2 g/kg that light drinkers did after 0.8 g/kg (Risbo et al., 1981). Rats developed tolerance to ethanol hypothermia after prolonged exposure by repeated gastric intubation, liquid diets, or inhalation of ethanol vapor (Nikki et al., 1971; Ferko and Bobyock, 1978, 1979; Frankel et al., 1978; L~ et al., 1979a; Rogers et al., 1979; Mullin and Ferko, 1981). Mice have shown similar tolerance after ethanol given as repeated i.p. injection, liquid diets or vapor (Ritzman and Tabakoff, 1976a,b; Crabbe et al., 1979, 1981; Rigter et al., 1980a,b). The tolerance takes the form of a parallel displacement of the LDR curve to the right (Ritzmann and Tabakoff, 1976a; Crabbe et al., 1979; Rigter et al., 1980a), in keeping with the pattern of tolerance to other effects of ethanol (Kalant et al., 1971). In general, the degree of tolerance is proportional to the daily dose or intake of ethanol, and to the duration of the treatment (Ritzmann and Tabakoff, 1976a; Frankel et al., 1978; Ferko and Bobyock, 1978; L6 et al., 1979a). An apparent exception is the report that rats on an ethanol containing liquid diet for 16 days continued to show almost as much hypothermia as those receiving a single injection of 3.5 g/kg i.p. (Pohorecky et al., 1974), especially if exposed to a Ta of 4°C. However, these animals were probably consuming 10 g/kg or more daily, so that there may in fact have been some tolerance despite the demonstrated hypothermia. Although there is abundant evidence that the rate of ethanol metabolism is increased as a result of chronic ingestion of ethanol, this was insufficient to account for the degree of tolerance observed (Ferko and Bobyock, 1979). Moreover, the tolerance could be seen when the test dose of ethanol consisted of only 2/~1 injected i.c.v., which yielded no measurable ethanol level in the peripheral blood (Ritzmann and Tabakoff, 1976a). Therefore it must reflect a decrease in sensitivity of the central nervous system to ethanol. However, it is not yet clear whether there is more than one mechanism by which this comes about. This question relates directly to the issues of 'acute' vs 'chronic' tolerance, and 'physiological' vs 'behavioral' mechanisms of tolerance. 4.1.1. Acute vs Chronic Tolerance Tolerance to the effects of ethanol, including the hypothermic effect, has been produced within two very different time frames (Kalant et al., 1971; Cicero, 1978). In the past, the term acute tolerance has been used to designate tolerance occurring during the course of a single exposure to a drug. For example, tolerance to the effect of an i.p. dose of ethanol on a motor performance test in the rat was seen at 30 min and 60 min after the injection, in the form of a parallel displacement of the regression line of effect on brain ethanol concentration (LeBianc et al., 1975). This is generally interpreted as an inherent homeo-

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static response. A similar phenomenon was seen in the mouse exposed to ethanol vapor: during the course of a single 6 hr exposure to ethanol vapor mice of some strains developed progressively greater resistance to both hypothermia and loss of righting reflex (Grieve and Littleton, 1979). More recently there has been a number of reports of tolerance developing at an unknown rate, but demonstrable as a diminished hypothermic response to a second ethanol exposure soon after the first. Mice, for example, showed a diminished response to an injection of 3 g/kg (i.p.) 24 hr after a similar injection (Crabbe et al., 1979, 1981; Rigter et al., 1980a,b). Rats did not show such tolerance to a second injection 24 hr after a first; indeed, they showed an increased response (Lomax et al., 1980). However, rats exposed to ethanol vapor for 24 hr, and then removed, showed tolerance to an i.p. injection 48 hr later (Ferko and Bobyock, 1979; Mullin and Ferko, 1981). Most studies of tolerance, however, have employed repeated or continuous exposure to ethanol for periods ranging from one to five weeks or longer (Nikki et al., 1971; Ritzmann and Tabakoff, 1976b; Ferko and Bobyock, 1978; Frankel et al., 1978; L~ et al., 1979a; Rogers et al., 1979). Tolerance produced in this way is often referred to as chronic tolerance, and typically shows a progressive development during the course of the prolonged or repeated ethanol administration (e.g. Frankel et al., 1978). The question naturally arises as to whether these various time patterns of tolerance reflect the same or different processes. In relation to tolerance to sensorimotor impairment by ethanol in rats, there is some evidence to suggest that acute and chronic tolerance may be aspects of the same process (Kalant et al., 1971; Cicero, 1978; Littleton, 1980a). During the course of daily intubation with ethanol for 4 weeks the first sign of tolerance was a progressively faster development of acute (within session) tolerance on successive test days. This resulted in the appearance of a gradual progressive increase in tolerance when test performance at a fixed time after each dose was compared on successive days (Kalant et al., 1978a). However, a similar comparison does not appear to have been made for tolerance to hypothermia. Therefore indirect evidence must be used. For example, the tolerance observed in mice at 24 hr after a single i.p. injection is absent after 2-3 days (Crabbe et al., 1979). In mice withdrawn from ethanol, after ingesting it in a liquid diet for 7 days, the tolerance was also markedly diminished after 2 days and had completely disappeared before 6 days (Ritzmann and Tabakoff, 1976a). Similarly, in rats the tolerance lasted for 7-9 days, regardless of whether it was produced by 10 days of ethanol vapor inhalation (Ferko and Bobyock, 1978) or 5 weeks of daily intubation (L6 et al., 1982). This suggests that the tolerance, regardless of how it is produced, has a turnover time of its own that is characteristic of each species and analogous to the turnover times of structural proteins of the brain and other organs.

4.1.2. Physiological vs Behavioral Tolerance

Tolerance has been viewed traditionally as a physiological compensatory response, offsetting the effect of the ethanol when the latter is present, and giving rise to an overcompensation or withdrawal reaction when the alcohol disappears (Kalant et al., 1971). It has also been traditional to regard the compensatory response as an adaptation to the presence of the drug, and therefore to be fairly specific for a given drug or at least for a given pharmacological category. However, these views have been challenged in recent years, and a number of alternative hypotheses have been advanced: (1) The compensatory response is not to the presence of the drug itself, but to the functional disturbance that the drug produces, which in turn depends not only on drug and dose but also on the physiological, behavioral and environmental circumstances in which the drug is given and the particular test used (Kalant, 1977; LeBlanc et al., 1978). (2) Tolerance is a learned response which the subject acquires to offset the functional disturbance produced by the drug and therefore occurs when there is opportunity

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H. KALANTand A. D. L~ for repeated practice under the influence of the drug (Chen, 1968, 1979; Wenger et al., 1981).

(3) Tolerance is a conditioned response to cues habitually associated with the administration of the drug; the response is seen as being originally an unconditioned delayed response to the drug, which is initiated much earlier as a result of conditioning and thus offsets the drug action (Siegel, 1978). There is some experimental support for, and some against, the first and third of these hypotheses in relation to alcohol induced hypothermia. It seems most likely that these are not mutually exclusive but involve complementary processes which together determine the rate and degree of tolerance development under any given set of circumstances. In relation to ethanol induced hypothermia, support for the first hypothesis is of several kinds. In the study by Crabbe et al. (1981) the magnitude of the hypothermic response to the first dose of ethanol was positively correlated with the degree of tolerance to the second dose 24 hr later. Thus, although all the mice had the same previous 'practice' under ethanol those with the greatest functional disturbance developed the greatest tolerance. In the model used by Ferko and Bobyock (1978) and Mullin and Ferko (1981) there was almost no opportunity for operant learning or classical conditioning, since the development of tolerance occurred during a single continuous exposure to ethanol vapor, while the tolerance test involved an i.p. injection of ethanol in a different environment. Moreover, insertion of the rectal thermometer could not have served as a conditioned stimulus since it was done both at times during the vapor exposure, when the animals were hypothermic, and at times after removal from the vapor chamber when they were normothermic. An argument against this hypothesis, however, is that mice receiving ethanol i.p. twice at a Ta of 31 °C, at which they did not develop hypothermia, nevertheless showed tolerance when tested a third time at 22°C (Rigter et al., 1980a). No explanation can yet be offered for this finding on the basis of the first hypothesis. Evidence for the third hypothesis is provided by a number of similar experiments (L6 et al., 1979b; Mansfield and Cunningham, 1980; Crowell et al., 1981). In each of these, rats were injected repeatedly with ethanol in one environment, and with saline in a recognizably different environment. When tested with ethanol in both, they showed greater tolerance to the hypothermic effect in the habitually alcohol linked environment than in the saline linked one. When injected with saline in the alcohol linked environment they responded with hyperthermia which was interpreted as the conditioned compensatory response. Finally, the ethanol tolerance could be extinguished by repeated injections of saline in the previously alcohol linked environment. Similar, but much less complete, results have been obtained in the mouse (Melchior and Tabakoff, 1981). Despite this evidence, however, it must be noted that L6 et al. (1979b) found s o m e tolerance even when the test dose of ethanol was given in the saline linked environment. In other words, tolerance was enhanced by the appropriate environmental cues but could still occur in their absence. Taken together this whole body of evidence suggests that there is an innate adaptive capacity which produces acute tolerance to ethanol hypothermia as an unconditioned response but this tolerance can be accelerated and enhanced when it is transformed into a conditioned response to cues signalling impending drug administration. This might constitute the link between acute and chronic tolerance discussed above (Section 4.1.1).

4.1.3. Genetic Influences on Tolerance

The development of tolerance to the motor impairment effects of ethanol in the rat can be completely blocked by administration of cycloheximide in doses too small to induce non-specific toxicity (LeBlanc et al., 1976). Tolerance is accompanied by increased incorporation in vivo of [3H]leucine into protein of the heavy and light microsomal fractions of rat brain (Walczak, 1983). If these findings imply that the development of ethanol tolerance requires gene expression with respect to the formation of new protein

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in the brain, then it is not surprising that genetic influences on tolerance development have been reported. Grieve and Littleton (1979a) reported that C57B1 mice developed tolerance to the hypothermic effect during a single 5 hr exposure to ethanol vapor ('acute' tolerance), while DBA/2 mice did not. Moore and Kakihana (1978) observed the same difference between these two strains during eight successive daily i.p. injections of ethanol, 3 g/kg ('chronic' tolerance). Crabbe et al. (1980), studying crosses and recombinant inbred strains derived from C57B1 and BALb mice, reported that all showed tolerance to a second injection of ethanol given 24 hr after the first. However, their Figure 1 indicates that five of the recombinant strains did not show less hypothermia on the second test when the first test injection consisted of ethanol instead of saline. This suggests that there were indeed strain differences in the rate of tolerance development. The demonstration of a genetic influence on the development of tolerance sheds no light on the mechanism(s) of tolerance because these studies do not yet give any indication of the inherited factor. Nevertheless, it would be helpful, in studies of possible mechanisms, to use whenever possible strains with high and low capacities for developing tolerance. 4.1.4. Loss o f Tolerance During Continued Exposure Tolerance is a reversible phenomenon as shown by its disappearance after suspension of ethanol administration (Section 4.1.1). Much more surprising, however, is the observation that in rats undergoing prolonged administration of ethanol in a liquid diet tolerance to both the hypothermic effect and the loss of righting reflex produced by i.p. test doses began to diminish after 4-6 weeks, and disappeared completely by 10-14 weeks (Khanna et al., 1980a,b). This observation has been confirmed in a separate experiment in which the ethanol was given by daily gavage (Khanna and L~, unpublished). The time required for loss of tolerance corresponds reasonably well with the minimum time required to produce significant loss of hippocampal neurons in rats subjected to chronic ethanol treatment (Riley and Walker, 1978; Walker et al., 1980). It is therefore possible that loss of tolerance may be an early functional consquence of brain cell loss. If so, identification of the specific neurons involved could help to elucidate the mechanisms of tolerance and to identify what is specific to individual tests (e.g. hypothermia) and what is common to all manifestations of adaptation. 4.2. PHYSICAL DEPENDENCE

If physical dependence on ethanol, as manifested by a withdrawal reaction, is indeed another aspect of the same neuronal changes that give rise to tolerance (Section 4.1.2) then it would be reasonable to expect that it would be marked by hyperthermia under at least some conditions in which ethanol acutely causes hypothermia. Indeed, hyperthermia is a characteristic finding in alcoholic patients undergoing withdrawal reactions, especially in severe cases of delirium tremens (Isbell et al., 1955; Godfrey et al., 1958; Tavel et al., 1961; Rose et al., 1970; Thompson et al., 1975; Kramp et al., 1979). In general, the degree and duration of temperature elevation are directly related to the severity of the withdrawal reaction and to the duration of the preceding alcohol consumption. Many of the febrile patients had pneumonia, dehydration or other identifiable causes of fever (Tavel et al., 1961; Rose et al., 1970) but a substantial proportion had no recognizable cause other than the withdrawal reaction per se. This was particularly true in the experimental studies (Isbell et al., 1955; Gross et al., 1975) involving administration of ethanol in multiple doses daily, for 7-87 days; alimentation was well maintained, and no physical illness occurred other than the withdrawal reaction, yet temperatures as high as 41.4°C were recorded. In the clinical series the height of temperature elevation was an important prognostic sign and most of the adverse reactions and fatalities occurred in those with the highest temperatures on admission (Tavel et al., 1961; Thompson et al., 1975). Experimental studies in laboratory animals have not shown such consistency. Several observers have reported a brief rebound hyperthermia in mice at the time that blood

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ethanol concentrations fell to zero, after either a single injection (Kakihana, 1977; Sanders, 1980) or consumption of an ethanol solution for eight days (Kakihana and Moore, 1977). However, others have found either no temperature change during withdrawal (Brick and Pohorecky, 1977) or a definite withdrawal hypothermia in mice (Ritzmann and Tabakoff, 1976a; Tabakoff and Ritzmann, 1977; Harris, 1979) and rats (Pohorecky et al., 1974). Even though Brick and Pohorecky (1977) found no hypothermia, the rats showed a clear preference for a warm environment (30°C) over a cold (2°C) or intermediate (19°C) one, while controls and ethanol tolerant but non-withdrawn animals preferred the intermediate temperature. This combination of warm preference with normal body temperature suggests an elevation of hypothalamic thermoregulatory set-point during withdrawal; this seems appropriate as a rebound change, if ethanol acutely lowers the set-point (Lomax et al., 1980). Against this interpretation, however, is the observation that withdrawal hypothermia, like acute ethanol hypothermia, was exacerbated in a 4°C environment, and converted to hyperthermia at 34°C (Ritzmann and Tabakoff, 1976a). This argues for impairment of thermoregulatory mechanisms. In human subjects, the elevated temperature was accompanied by cutaneous vasodilatation (Godfrey et al., 1958) and profuse sweating (Isbell et al., 1955). As Freund (1979) has pointed out, this suggests that the temperature elevation is the result of intense muscular tremor and thermogenesis, rather than a raised set-point. However, these patients were not observed early and continuously enough to plot the relative time courses of core temperature, skin temperature and tremor activity, so that it is impossible to know the exact sequence of events. Therefore the question of set-point change remains open. In summary, the temperature alterations in human undergoing ethanol withdrawal are compatible with the idea that physical dependence and tolerance reflect the same adaptive change. In experimental animals the changes during early withdrawal are also consistent with this idea. Those instances in which tolerance to ethanol hypothermia followed a time course of disppearance different from that of physical dependence (e.g. Ritzmann and Tabakoff, 1976a,b) are probably best explained by the fact that tolerance and dependence were measured by different tests with different detection thresholds. If hypothermia supervenes later in withdrawal (Sanders, 1980) this might reflect secondary or peripheral mechanisms invoked by the withdrawal reaction, rather than intrinsic to it. This must be tested by much more complete time-course studies after different chronic alcohol regimens. 4.3. POSSIBLE MECHANISMS OF TOLERANCE TO ETHANOL HYPOTHERMIA 4.3.1. Changes in Neurotransmitter Function A number of recent reviews have dealt with changes in neurotransmitter turnover and receptor sensitivity in animals undergoing chronic ethanol treatment and withdrawal (Tabakoff et al., 1978; Hunt and Majchrowicz, 1979; Hoffman and Tabakoff, 1980; Tabakoff and Hoffman, 1980). These provide detailed coverage of the current state of knowledge and no attempt will be made to cover the same ground here. Only those papers will be cited which have a more or less direct bearing on the question of thermoregulatory aspects of ethanol tolerance and physical dependence. 4.3.1.1. Catecholamines. In Sections 3.4.3.1 and 3.4.3.2, literature concerning the possible roles of dopamine and norepinephrine in the acute thermoregulatory effects of ethanol was reviewed. A similar body of literature attests to alterations in the activity and metabolism of the brain catecholamines in ethanol tolerant animals. Unfortunately, however, the reported changes in tolerant and dependent animals are as contradictory as those reported to accompany acute alcohol hypothermia (Tabakoff and Hoffman, 1980). In animals continuing to receive ethanol chronically, both DA and NE levels in brain were normal or elevated in mouse (Griffiths et al., 1974), and rat (Nikki et al., 1971; Pohorecky et al., 1978). This could be taken as evidence of decreased catecholamine release because ~-MT did not affect the elevated levels (Griffiths et al., 1974). Moreover, the

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accumulation of DOPAC in the rat striatum that occurred with acute ethanol was no longer found during chronic ingestion (Barbaccia et al., 1980). These findings appear to indicate that the increase in catecholamine release that is at first produced by acute administration of ethanol in relatively low doses is offset during chronic ethanol exposure by the development of tolerance and that the decrease in release caused originally by high doses may still be present, though presumably also less marked, depending on the dose level. This would be consistent with the reports of decreased brain DA turnover in chronically exposed rats (Hunt et al., 1979) and of increased [3H]spiroperidol binding to DA receptors (Barbaccia et al., 1980; Reggiani et al., 1980) which occurs with functional denervation (Seeman, 1980). If these changes are part of the mechanism of tolerance, rather than consequences of it, they should be even more evident during withdrawal when the continuing influence of ethanol is removed. In keeping with this expectation striatal DA turnover in vivo was reported to be decreased further during the withdrawal reaction and K+-stimulated release of DA from striatal slices prepared from animals in withdrawal was reduced (Hunt et al., 1979). DA levels were increased even more than during chronic intoxication (Nikki et al., 1971; Griffiths et al., 1974). NE levels in rat brain, which had been normal during chronic ingestion of ethanol, were elevated at 20 and 30 hr after withdrawal (Pohorecky et al., 1978) which might signify a lower rate of release. The stimulation of cortical adenylate cyclase by NE was increased during withdrawal (French et al., 1975a,b), as expected if NE release is markedly diminished. All of this makes a coherent picture based on the concept that tolerance and physical dependence involve changes which reduce presynaptic release of DA and NE. Unfortunately there is almost as much evidence in the opposite direction. During chronic ethanol administration, K+-stimulated release of DA from striatal slices was even higher during chronic than during acute intoxication (Hunt et al., 1979). NE stimulation of cortical adenylate cyclase was decreased during chronic ethanol administration, suggesting a receptor response to high synaptic concentration of NE (French et al., 1975a,b). During withdrawal striatal adenylate cyclase showed a reduced stimulation by DA (Seeber and Kuschinsky, 1976; Hoffman and Tabakoff, 1977) and the hypothermic response to the dopaminergic agonists piribedil and apomorphine was reduced (Hoffman and Tabakoff, 1977; Rabin et al., 1980). The hypothermic effect of clonidine was reported to be reduced in ethanol tolerant rats (Mullin and Ferko, 1981) but not mice (Hoffman and Tabakoff, 1977). However, cortical flz-adrenergic receptors were reported to be decreased in number in mice undergoing ethanol withdrawal (Rabin et al., 1980). These findings tend to suggest increased release of DA and NE in tolerant and dependent animals. Finally, the behaviorally demonstrated preference of alcohol withdrawn rats for high Ta was unaffected by amphetamine, phenoxybenzamine, or pimozide (Brick and Pohorecky, 1977). This appears to suggest that neither NE nor DA plays a role in the disturbed behavioral thermoregulation following alcohol withdrawal. Yet NE (i.c.v.) and amphetamine (s.c.) were shown to alleviate some motor signs of the ethanol withdrawal reaction in the mouse (Collier et al., 1974). At present there is no way of explaining all these conflicting results. One can suggest that the changes observed in striatum, cortex, hypothalamus and other regions of the brain are not necessarily homogeneous, and indeed that they are probably quite different from region to region, or from one type of neuron to another. Moreover, the changes observed in any one region at any given stage of ethanol treatment or withdrawal may well be secondary to altered impulse flow from other loci, rather than primary adaptations to the local action of ethanol. For this reason, various investigators have opted for the strategy of producing a specific biochemical lesion affecting one neurotransmitter system, and then seeing how this lesion affects the ability to develop ethanol tolerance. This strategy, it is hoped, may provide greater insight into the role of the neurotransmitter in the development of tolerance. In relation to the catecholamines, two groups have used this approach. In mice, i.c.v. injection of 6-hydroxydopamine (6-OHDA) prevented the development of tolerance to

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ethanol hypothermia by 7 day feeding of ethanol in a liquid diet (Ritzmann and Tabakoff, 1976b; Tabakoff and Ritzmann, 1977; Melchior and Tabakoff, 1981). Tolerance development was also retarded by i.p. administration of ~-MT (Melchior and Tabakoff, 1981). These effects on tolerance were attributed to alterations in NE rather than DA function, because when the NE neurons were protected by injection of desmethylimipramine together with 6-OHDA, thus reducing only the DA content of the brain, hypothermia tolerance developed as in animals receiving only ethanol (Tabakoff and Ritzmann, 1977; Melchior and Tabakoff, 1981). In contrast, i.c.v, injection of 6-OHDA had no effect on an already established ethanol tolerance (Tabakoff and Ritzmann, 1977), so that the NE system appeared to be essential for the development, rather than the expression, of tolerance. In rats, on the other hand, 6-OHDA with or without desmethylimipramine had no effect at all on the acquisition of hypothermia tolerance during 20-25 days of ethanol administration (L~ et al., 1981a). This does not mean that noradrenergic systems play no role in tolerance in the rat. As will be described in Section 4.3.1.2, certain serotoninergic pathways play a very important role, but if both 5-HT and NE are depleted by injection of 5,7-DHT without desmethylimipramine hypothermia tolerance is completely blocked, while 5,7-DHT together with desmethylimipramine (depleting only 5-HT) merely retards it (L6 et al., 1981a). This suggests that in the rat, NE systems may play a contributory role, while in the mouse it may be primary.

4.3.1.2. Serotonin. There is rather less literature available on serotonin than on catecholamines with respect to changes in transmitter function associated with tolerance to ethanol hypothermia. Two week exposure of rats to an ethanol containing liquid diet led to modest increases in brain levels of tryptophan and 5-HT and of tryptophan hydroxylase activity which persisted for 12-24 hr after withdrawal (Pohorecky et al., 1974). Similar changes were observed in the brains of mice exposed to ethanol vapor for 3-10 days; the 5-HT level was moderately increased during chronic intoxication, rose still more early in withdrawal and then fell to normal in 5-8 hr (Griffiths et al., 1974) although the 5-HIAA level remained elevated for up to 20 hr after withdrawal (Tabakoff and Boggan, 1974). These findings suggest that chronic ethanol administration increases the turnover of 5-HT. However, there are some conflicting reports. Six weeks of ingestion of ethanol in a liquid diet did not affect 5-HT levels in the rat brain (Pohorecky et al., 1978). Three weeks of daily intubation with a small dose of ethanol (2 g/kg), followed by an injection of 2.5 g/kg (i.p.) decreased brain 5-HT levels in the rat, whereas the i.p. injection alone failed to do so (Nikki et al., 1971). Since the rats were sacrificed 85 min after the i.p. injection, the greater decrease of 5-HT in the chronic alcohol group may simply have reflected summation of the acute actions of the intubated and injected doses. French et al. (1975a) found that cortical slices prepared from chronically ethanol treated rats 3 days after withdrawal, showed significant stimulation of adenylate cyclase activity by 5-HT, blockable by methysergide, which was not encountered in slices from control animals. This apparent receptor supersensitivity suggests a decreased release of 5-HT during the later stages of withdrawal, at a time when the hypothermic response is over. During the early stages, however, there was presumably increased release which might have contributed to the preference for high Ta (Brick and Pohorecky, 1977) by an upward shift of the set-point. Yet previous administration of methysergide or ofp-chloroamphetamine (pCPA) did not alter the heat preference. These inconclusive findings do not provide very strong grounds for attributing to 5-HT an important role in tolerance to alcohol hypothermia. However, the alternative strategy mentioned in Section 4.3.1.1 has provided a much stronger affirmation of such a role. Frankel et al. (1978) reported that rats treated with pCPA, in a dose which reduced whole-brain 5-HT level by 95~o, showed a clear retardation of development of tolerance to the hypothermic effect of ethanol at normal T,, during 20 days of daily gavage or ingestion of an ethanol containing liquid diet. The same effect was produced by i.c.v.

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injection of 5,7-DHT together with desmethylimipramine, so that only 5-HT was depleted, and not NE or DA (L~ et al., 1980a). Elevation of brain 5-HT, by administration of large doses of tryptophan twice daily, had the opposite effect, i.e. it increased the rate of development of tolerance to ethanol hypothermia (L6 et al., 1979a). Lesions of the nucleus raph6 magnus or of the dorsal raph6 nucleus had no effect on tolerance development, so that serotoninergic fibers to the spinal cord, striatum and thalamus do not appear to be involved (L6 et al., 1981b). Only lesions of the median raphb nucleus, which projects to the hippocampus and septum, caused the same retardation of tolerance as seen previously with pCPA or 5,7-DHT plus desmethylimipramine. It appears, therefore, that the 5-HT influence involved is not directly upon the POAH thermoregulatory neurons, but upon a common adaptive mechanism which affects these neurons together with those mediating motor and other effects of ethanol. Unlike 5,7-DHT, 5,6-DHT did not retard, but actually accelerated the development of tolerance to ethanol hypothermia in the rat (Khanna et al., 1979b). It reduced brain 5-HT level by less than 20~o and in some way sensitized 5-HT receptors, as shown by a modest but significant increase in the hypothermic response to a test dose of 5-hydroxytryptophan. In the mouse, in contrast to the rat, icv injection of 5,7-DHT together with desmethylimipramine i.p., resulting in a large selective decrease in 5-HT level, failed to alter the development of hypothermia tolerance during four days of twice daily injection of ethanol (Melchior and Tabakoff, 1981). On the other hand, as noted in Section 4.3.1.1, 6-OHDA, which retarded tolerance development in the mouse, failed to so in the rat but 5,7-DHT without desmethylimipramine (reducing both 5-HT and NE in the rat brain) prevented tolerance completely. These findings suggest that both NE and 5-HT, in proportions varying from one species to another, are involved in some type of system for facilitating neuronal adaptation, which involves the thermoregulatory mechanisms as well as others. However, these effects have been studied only in rodents to date. It would be highly useful to test them in other species, in order to determine whether they represent a fundamental adaptational feature of mammalian brain. It is not unlikely that other classical neurotransmitters, such as acetylcholine, may also be involved in this function. There is a substantial literature on changes in acetylcholine turnover, and in muscarinic and nicotinic receptor sensitivity, during chronic ethanol treatment and withdrawal (Hunt and Majchrowicz, 1979; Hunt et al., 1979; Tabakoff and Hoffman, 1980; Pelham et al., 1980). However, the studies reported to date do not appear to be related specifically to thermoregulation, and will not be reviewed here. The same is true of histamine. Both acute and chronic ethanol administration have been reported to alter histamine metabolism in the fetal rat brain (Rawat, 1980) and in the hypothalamus of the adult rat (Subramanian et al., 1978). Both papers make reference to a possible significance with respect to thermoregulation, but contain no experimental results dealing with body temperature. 4.3.2. Neuropeptides In Section 3.4.4.1 the conclusion was drawn that, although endogenous opioid peptides might play a role in the effects of ethanol on thermoregulation, this was far from proven. The same can be said, even more emphatically, with respect to chronic ethanol administration. Schulz et al. (1980) reported that chronic ingestion of ethanol in the drinking fluid, by rats and guinea pigs, led to marked decreases in the levels of/%endorphin (fl-E) and methionine-enkephaline in most of the brain regions examined, including the hypothalamus and the neurointermediate lobe of the pituitary gland. However, the stronger ethanol solutions were aversive to the animals and caused a sharp decrease in fluid intake, so that the effects of ethanol are difficult to separate from those of dehydration. In addition, the reduced levels might indicate either decreased synthesis or increased release. When rats were made tolerant to both hypothermia and motor impairment by daily intubation with ethanol for three weeks, the isolated neurointermediate lobe showed

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significantly increased incorporation of [3H]phenylalanine into fl-E and its immediate precursors (Gianoulakis et al., 1981), as well as increased release of the labelled fl-E into the incubation medium. Whether this constitutes a chronic ethanol effect, or a result of ethanol withdrawal by transfer of the tissue to an alcohol-free medium in vitro, remains to be established. It also remains to be seen whether these findings can be replicated in the hypothalamus. In mice, ethanol ingestion for 21 days led to a selective increase in methionineenkephalin in the striatum, where it is postulated to act presynaptically to inhibit DA release (Reggiani et al., 1980). The DBA/2J strain, which is said to lack enkephalinergic modulation of striatal DA release, did not show any change in DA turnover after ethanol (Barbaccia et al., 1980). Again, such findings do not necessarily apply to the POAH. However, these preliminary observations at least leave open the possibility of endogenous opioid mediation of some ethanol effects on thermoregulation. In contrast, certain central effects of arginine vasopressin (AVP) and DGAVP have been linked more specifically to ethanol tolerance in relation to hypothermia. Both of these peptides have been shown to maintain a learned avoidance behavior for extended periods under extinction conditions in which the behavior would normally have disappeared rapidly (de Wied, 1976). Because of numerous resemblances between tolerance and learning, the peptides were tested in ethanol tolerant mice and were found to maintain tolerance to the hypothermic effect long after daily ethanol administration had ceased (Hoffman et al., 1978). The same effect was obtained with DGAVP in mice (Rigter and Crabbe, 1980; Rigter et al., 1980c) and rats (L6 et al., 1982). In contrast, an equimolar dose of oxytocin either was without effect (Hoffman and Tabakoff, 1979) or actually diminished the degree of tolerance expressed (Rigter and Crabbe, 1980; Rigter et al., 1980c). The effects of the peptides were not specific to hypothermia tolerance but applied also to tolerance to the hypnotic and motor effects of ethanol. Moreover, mice which received DGAVP during the development of tolerance showed increased severity of ethanol withdrawal seizures (Rigter et al., 1980d). This provides interesting support for the concept that tolerance and physical dependence are different manifestations of the same adaptive process (Section 4.1.2). These effects of the peptides, applicable to the maintenance of hypothermia tolerance but also of other manifestations of tolerance and dependence, are reminiscent of the effects of NE and 5-HT on the development of tolerance (Sections 4.3.1.1 and 4.3.1.2). Perhaps not surprisingly, these effects are related. In the mouse, in which NE appears to play a more important role than 5-HT, i.c.v, injection of 6-OHDA prevents the maintenance of ethanol tolerance by AVP (Hoffman and Tabakoff, 1979). In the rat, lesions of the median raph6 nuclei, which deplete hippocampal and septal 5-HT, prevent the corresponding effect of DGAVP on hypothermia tolerance (L6 et al., 1982). The nature of the interaction is currently under investigation, and should yield interesting insights into the nature of CNS adaptation, but the more general and fundamental the phenomenon proves to be, the less relevant it becomes to the specific control of thermoregulation.

4.3.3. N e u r o n a l M e m b r a n e M e c h a n i s m s One widely accepted explanation of the cellular actions of ethanol and other CNS depressants is that these agents 'fluidize' the lipids of the cell membrane, disturbing the interactions between the lipids and the protein inclusions in the membrane, such as receptors, ion conductance channels and membrane bound enzymes (Seeman, 1972; Chin and Goldstein, 1977a; Kalant and Woo, 1981). The higher the cholesterol content of the membrane, the less the effect of the drugs on membrane stability (Pang and Miller, 1978). Tolerance, in this hypothesis, is attributed to a change in the chemical composition of the membrane lipids, including the proportions of cholesterol and various fatty acids, and a corresponding change of their physical properties, rendering the membranes less susceptible to the fluidizing effect of ethanol (Chin and Goldstein, 1977b; Kalant and Woo,

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1981). This, in turn, would make the membrane proteins less susceptible to alcohol effects produced by allosteric interaction with the lipids (Kalant et al., 1978b; Rangaraj and Kalant, 1982). If the effect of ethanol on the set-point of POAH thermoregulatory neurons depends on such alterations of membrane properties (Section 3.4.1), one might expect to find countervailing alterations in membranes from tolerant animals. Unfortunately this has not yet been examined directly. Various groups have studied changes in lipid composition and physical properties of neuronal membranes from alcohol tolerant animals (for references, see reviews by Goldstein et al., 1980; Littleton, 1980a,b; Kalant and Woo, 1981). Indeed, Grieve et al. (1979) and John et al. (1980) have made an even more direct test by altering the membrane lipids by dietary and biosynthetic changes and demonstrating that these manipulations produced alcohol tolerance in mice not previously exposed to ethanol. Similar studies carried out on rats in our laboratory (F. Beaug~, T. Clandinin, N. Rangaraj and H. Kalant, unpublished observations) have yielded inconsistent results. In any case, the test of tolerance used by Grieve et al. (1979) and John et al. (1980) was loss of the righting reflex and the membranes under study were prepared from whole brain, predominantly the cerebral hemispheres. This approach does not allow for subtle differences between different types of neurons or different brain regions. Since cultured mammalian cells and E. coli show different patterns of changes in membrane lipids when exposed chronically to ethanol (Ingram et al., 1978), regional and neuron type differences are not at all inconceivable. Therefore the findings, though encouraging, cannot yet be linked directly to tolerance in thermoregulatory neurons. One expected consequence of altered membrane composition would be a change in binding sites for ligands of various types, including neurotransmitters, hormones, and ions. Ca 2+ has been studied extensively in this connection, and has been shown to have a striking effect on various manifestations of ethanol tolerance and dependence. In mice withdrawn after seven days of exposure to ethanol in a liquid diet, the withdrawal convulsions and hypothermia were markedly diminished by i.c.v, injection of Ca 2+ (Harris, 1979). Conversely, the toxicity produced by i.c.v, injection of EGTA was tripled in mice undergoing alcohol withdrawal; unfortunately this toxicity was measured only in terms of LDs0 and not of temperature changes. However, the fact that Ca 2+ enhanced the acute effects of ethanol (Section 3.4.1), and suppressed the effects of alcohol withdrawal, suggests that (a) the acute/~ction of ethanol is synergistic with a Ca2+-dependent process in the membrane, and (b) the development of tolerance and dependence involves either a loss of membrane Ca 2+ or a decrease in the Ca 2+ sensitivity of the process(es) involved. This is directly opposite to the changes reported by Ross (1980), but is consistent with the findings of several other groups (see Kalant and Woo, 1981, for references). It should be possible to study this question directly in the POAH by cytochemical methods, or by direct chemical analysis of tissue samples from acutely and chronically treated animals. A different, more indirect, approach to the functional changes in ethanol tolerance is a comparison of these changes with those produced by exposure to high or low temperature. Mammalian cells in tissue culture are readily killed by moderate elevation of the incubation temperature, and aliphatic alcohols enhance this effect by reducing the threshold temperature for thermal cytotoxicity (Li and Hahn, 1978). However, brief exposure to 43°C incubation or to 6~o ethanol, followed by a rest period of several hours, resulted in greatly increased resistance to the lethal effect of a second heat exposure, or of the cytotoxic agent adriamycin (Li and Hahn, 1978). It was therefore suggested that both heat tolerance and ethanol tolerance involve chemical changes in the membrane, leading to similar increases in resistance to membrane fluidization by either heat or ethanol. Unfortunately the examination of cross-tolerance in this study was incomplete in that the effect of preceding heat exposure on ethanol tolerance was not studied. In any case, such experiments are difficult to interpret because one does not know whether the first heat or alcohol exposure actually produced adaptation and increased tolerance in the cells or simply killed off the less resistant cells and left only the more resistant ones as survivors to undergo the second exposure. The same question may be raised about J.P.T. 23/3 C

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alterations in membrane composition in Tetrahymena, E. coli and other organisms exposed to heat and to ethanol (reviewed by Littleton, 1980). Cold adaptation, in contrast, has been studied in identifiable individual organisms, so that change in cold tolerance can be distinguished clearly from survival of innately tolerant individuals. In the abdominal ganglion of the sea snail (Aplysia californica) post-tetanic potentiation (PTP) disappears at a characteristic rate at the usual Ta of 15°C. Acute exposure to ethanol causes a much more rapid decay of PTP, presumably because of increased membrane fluidity, and tolerance that develops during prolonged ethanol exposure is characterized by a return to the normal duration ofPTP (Barondes et al., 1979; Traynor et al., 1979). The evidence cited above suggests that this tolerance involves an increase in the proportions of cholesterol and of saturated fatty acids, and a decrease of polyunsaturated fatty acids, in the neuronal membrane. On the other hand, exposure to lower Ta (as low as 6°C) prolongs the duration of PTP, presumably by causing greater rigidity of the membrane, and tolerance to the cold is characterized by a return to shorter duration of PTP. In Tetrahymena pyriformis adaptation to cold is accompanied by an increase in palmitoyl-CoA desaturase activity and in the proportion of palmitoleate relative to palmitate in the membrane (Nozawa and Kasai, 1978). This should render the membrane more fluid, and therefore more resistant to gel transition at low Ta. Yet cross-tolerance between ethanol and cold was seen in the Aplysia preparation (Barondes et al., 1979; Traynor et al., 1979). These findings are indeed difficult to explain. Cross-tolerance between ethanol and high Ta is understandable, since both tend to fluidize the membrane lipids, and the expected adaptive reaction to both might involve comparable increases in membrane rigidity. In contrast, cross-tolerance between ethanol and cold, which produce opposite acute effects on membrane fluidity and opposite adaptive changes in membrane lipid composition, is not at present explicable. Barondes et al. (1979) suggest that overall membrane fluidity is not the relevant property. Rather, they propose that changes in the boundary lipids, immediately adjacent to specific protein inclusions and interacting with them, are the functionally important changes and that some of these may be common to ethanol tolerance and cold acclimation of Aplysia. This suggestion is certainly compatible with changes in temperature-sensitivity of synaptosomal (Na +/K ÷ )-ATPase activity in preparations from ethanol-tolerant rats (Rangaraj and Kalant, 1982). Molenda (1967) observed that rats acclimated to a T~ of 4°C showed tolerance to the effect of ethanol on conditioned avoidance responses in rats, when these were tested at 20°C. Of even greater interest, Lomax and Lee (1982) found that rats acclimated to a Ta of 4°C for 7-21 days showed a progressive increase in tolerance to the hypothermic effect of ethanol (1 g/kg) i.p. at 4°C, that was not due to alteration in ethanol metabolism. We have also found that rats exposed to - 10°C for 2 hr daily for 10 days were cross-tolerant to the hypothermic effects of ethanol when tested at 22°C (A. D. L6 et al., unpublished). It seems highly unlikely that this cross-tolerance is related to that observed in Aplysia. The increased acyl-CoA desaturase activity in Tetrahymena during cold acclimation was produced by a reduction of Ta from 39.5 ° to 15°C (Nozawa and Kasai, 1978) that would affect these unicellular organisms immediately. In contrast, exposure of a normal adult rat to a T~ of 4°C produces scarcely any fall in core temperature because cutaneous vasoconstriction and shivering and nonshivering thermogenesis are able to maintain net thermal balance (Sellers, 1957). Therefore it is difficult to conceive of a change in set-point or other membrane properties of POAH neurons, resulting from whole-body exposure to a Ta of 4°C, that would be sufficient to explain the cross-tolerance to ethanol. It seems much more likely that the adaptive changes are initiated by the central consequences of afferent impulses from peripheral thermal sensors, rather than by direct temperature effects on the POAH neuronal membranes. 5. COMPARISON OF ETHANOL WITH OTHER DRUGS It has been pointed out in a number of general reviews (e.g. Borison and Clark, 1967; Freund, 1979; Bowman and Rand, 1980) that a great many drugs that affect the central

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nervous system also alter thermoregulatory function. Clark and Clark (1981) have compiled a monumental tabulation of body temperature effects of drugs of many different pharmacological categories, with detailed information on the species studied, the drug doses and routes of administration, ambient temperature and other pertinent data. It would be totally beyond the scope of this review to attempt a detailed analysis of the similarities and differences between ethanol and the other drugs that have been studied. Only a few specific comparisons are dealt with in this section, concerning mechanisms of action, and because of their relevance to the questions of tolerance and cross-tolerance (Sections 3.4 and 4 respectively). There is only a limited number of ways in which drugs can affect thermoregulation. These are set out in some detail by Borison and Clark (1967). In simpler form, Freund (1979) states that a drug may affect thermoregulation by (a) raising, lowering, broadening or abolishing the set-point, (b) impairing the functional capacity of thermoregulatory effector systems, including metabolic thermogenesis as well as heat loss and heat conservation reflexes, and (c) disturbing free or operant behaviors that contribute to temperature regulation. By comparing ethanol with a few drugs of other categories, and examining the circumstances under which cross-tolerance between them does and does not occur, one may perhaps gain additional insight into the mechanism of action of ethanol. 5.1. GENERALDEPRESSANTS It has long been accepted that alcohols and volatile general anesthetics have a similar mechanism of action, based on their lipid solubilities and their ability to fluidize cell membrane lipids (Seeman, 1972). Accordingly, it is not surprising that all of the general anesthetics can produce hypothermia and that, like ethanol, they also decrease panting and shivering responses, hypothalamic blood flow and POAH unit firing rates (Bligh, 1966; Hemingway, 1963). Halothane, in doses which produced hypothermia, raised DA levels and lowered 5-HT in the rat brain as ethanol did, and ethanol enhanced these effects of halothane (Nikki et al., 1971). In Tetrahymena pyriformis acute exposure to methoxyflurane apparently fluidized membrane lipids as ethanol does since the degree of membrane protein clumping, by exclusion from the lipid phase on chilling to 15°C, was diminished by the anesthetic agent (Nandini-Kishore et al., 1977). On prolonged incubation with methoxyflurane the cells showed a shift in membrane lipid composition in the direction of increased proportions of saturated fatty acids and a decrease in polyunsaturated acids, just as is observed with chronic ethanol exposure (Section 4.3.3). Fever produced in the rabbit by injection of E. coli pyrogen was reduced not only by ethanol but also by methanol, n-propanol and n-pentanol, with potencies inversely proportional to their respective chain lengths, i.e. directly proportional to their membrane:water partition coefficients (Genuit, 1940). Benzyl alcohol, like ethanol, produced a dosedependent hypothermia in the mouse, regardless of route of administration, at a T~ of 25°C (Freund, 1973). Injection of Ca 2+ i.c.v., which failed to reverse the hypothermic effect of ethanol in mice, also failed to reverse that of t-butanol and of chloral hydrate (Harris, 1979). These, and other similar observations, make it clear that the effects of ethanol on thermoregulation are by no means unique, but are probably only one facet of a general neuronal effect shared by many other CNS depressants. These similarities with ethanol extend also to non-volatile hypnotics. Early work (Grosse-Brockhoff and Schoedel, 1943) demonstrated that urethane-morphine hypnosis in dogs also suppressed shivering during immersion in cold water, and thus inhibited the rise in 02 consumption that otherwise occurred during the initial phase of cooling. In recent years there has been more attention devoted to comparisons of ethanol with non-volatile CNS depressants such as barbiturates and benzodiazepines. Like ethanol, pentobarbital causes a dose-dependent fall in rectal temperature in the rat (Lomax, 1966; Lin, 1981; Myers, 1981a; Commissaris et aL, 1982) and mouse (Madden and Hiestand, 1954; Ho, 1976; Ritzmann and Tabakoff, 1976), as does nitrazepam in the mouse (Chambers and Jefferson, 1977). The hypothermic effect of barbiturates was also increased at low Ta and

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diminished at high T, in mice, rats and dogs (Madden and Hiestand, 1954; Setnikar and Temelcou, 1962; Lomax, 1966; George et al., 1981; Lin, 1981; Myers, 1981a). Indeed, pentobarbital caused hyperthermia in the rat at a T, of 36°C, as ethanol had done (Myers, 1981a) and in the dog at a Ta of 30°C (Setnikar and Temelcou, 1962). Pentobarbital induced hypothermia in the rat at 8°C and 22°C, like that caused by ethanol, was characterized by elevation of tail and foot skin temperatures and decrease in metabolic rate (Lomax, 1966; Lin, 1981); these findings again suggest a downward shift in set-point of the POAH. On the basis of such evidence it was concluded that pentobarbital and ethanol act on the same thermoregulatory mechanism in the brain (Myers, 1981a). This conclusion appears to be supported by the finding that chronic administration of ethanol results in cross-tolerance to the hypothermic effect of pentobarbital (Ritzmann and Tabakoff, 1976; Khanna et al., 1980a) and the spontaneous loss of tolerance to ethanol during very prolonged ethanol treatment (Section 4.1.4) was accompanied by loss of cross-tolerance to pentobarbital (Khanna et al., 1980a). Further support for the identity of ethanol and barbiturate effects on thermoregulation is provided by studies of the site and mechanism of action. Hypothermia was produced by i.c.v, injection of very small doses of pentobarbital in the mouse (Ho, 1976) and rat (Lin, 1981), though, surprisingly, injection directly into the POAH of the rat had no effect (Lomax, 1966). Indomethacin pretreatment reduced the hypothermic effect of pentobarbital, as well as that of ethanol (George et al., 1981) and i.c.v, injection of Ca 2÷ failed to reverse the hypothermia caused by either drug (Harris, 1979). Depletion of DA by i.c.v. injection of 6-OHDA, which did not alter the initial hypothermic response to ethanol, also had no effect on phenobarbital induced hypothermia in the mouse (Tabakoff et al., 1978b). Depletion of rat brain 5-HT by pretreatment with 5,7-DHT or pCPA, which reduced the intial hypothermic response to ethanol (Frankel et al., 1978), did the same with pentobarbital (Lin, 1981). Withdrawal of chronic barbiturate treatment in mice resulted in a decreased sensitivity of DA receptors, as shown by decreased DA stimulation of adenylate cyclase activity, just as in ethanol withdrawal (Seeber and Kuschinsky, 1976; Hoffman and Tabakoff, 1977). Finally, the facilitation of hypothermia tolerance by conditional stimuli in the environment in which ethanol is habitually given (Section 4.1.2) also applies to tolerance to pentobarbital and cross-tolerance to ethanol in pentobarbital treated rats (Cappell et al., 1981). Since barbiturates, like ethanol and volatile anesthetics, are generally believed to act through non-specific physical effects on cell membranes these similarities were expected. Cannabinoids, which have very high membrane:water partition coefficients and are not known to have specific receptors might be expected to behave in the same way. There is a considerable amount of literature on this subject which is generally in accord with this expectation (for a review, see Rosenkrantz, 1983). Ag-Tetrahydrocannabinol (THC), l l-hydroxy-THC, and the synthetic cannabinoid DMHP, produced a dose-dependent hypothermia in monkey, mouse (Haavik and Hardman, 1973) and rat (Pryor et al., 1976) at normal Ta or at 10°C, but a slight hyperthermia at 35°C (Haavik and Hardman, 1973), especially with small doses (Rosenkrantz, 1983). Very similar effects were seen in humans smoking cannabis, i.e. a slight transient hypothermia at 23.5°C and hyperthermia at 40°C (Jones et al., 1980). This pattern resembles that produced by ethanol and the effect of THC was increased (though less than additively) by concurrent administration of ethanol (Pryor et al., 1976). Chronic administration of cannabis results in tolerance to the hypothermic effect (Jones, 1983), just as is the case with ethanol. The one point of disagreement is that cannabinoid-induced hypothermia in the rat and human was accompanied by reduced skin temperature (Haavik, 1977; Jones et al., 1980), and the hypothermia was therefore attributed to decreased heat production rather than to increased heat loss (Haavik, 1977). This was tentatively explained by reduced activity of microsomal (Mg2-/Ca 2÷)-ATPase; however, the evidence in support of this mechanism is derived from studies on mouse brain microsomes (Haavik, 1977) rather than muscle sarcolemma or tubules, so that the connection with thermogenesis is rather tenuous. Other investigators, however, have reported that cannabis induced hypothermia involves both

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decreased thermogenesis and increased heat loss (Rosenkrantz, 1983); this is suggestive of a lowered set-point. In humans, moreover, the hyperthermia caused by smoking cannabis at 40°C was accompanied by reduced skin temperature and reduced sweating. These findings are suggestive of an elevation of set-point. It may well be that cannabinoids have two independent mechanisms of action on thermoregulation, one linked to a nonspecific membrane effect and one to a more selective action on neurotransmitters. Since the membrane fluidization hypothesis treats ethanol essentially as an uncharged local anesthetic, it is interesting to compare its effects with those of a typical charged local anesthetic. Vuorinen et al. (1976) reported that lidocaine (50mg/kg i.v.) also produced hypothermia in the rat at 22°C, but not at 32°C. A dose of ethanol (3 g/kg gavage) that did not produce hypothermia by itself also did not modify the effect of lidocaine, though it prolonged the narcosis caused by lidocaine. These data are insufficient to permit any conclusion, but they are at least compatible with the idea that nonspecific neuronal membrane disturbances, however produced, can result in a common set of impairments of thermoregulatory effector systems. 5.2. OPIATES In contrast to the general depressants, the opiates and opioids are known to act through well-defined stereospecific receptors, with direct links to intracellular 'second messengers' such as the prostaglandin stimulated adenylate cyclase. It might therefore be anticipated that the thermoregulatory effects of opiates would differ in various respects from those of ethanol. As noted in Section 3.4.4.1, a clear difference is that the most characteristic effect of opiates is hyperthermia, caused by upward displacement of the set-point, and that hypothermia occurs only with larger doses that impair the effector systems. Moreover, the SS and LS mouse strains, selected genetically for their low and high sensitivities respectively to ethanol, were reported to show the expected order of sensitivity to ethanol induced hypothermia (LS > SS), but the opposite order for morphine hypothermia (SS > LS), and naloxone reversed ethanol hypothermia only in the SS strain (Brick and Horowitz, 1982). However, these are generalizations that overlook some puzzling differences among opioids and perhaps among species. For example, large doses of morphine or methadone produced hypothermia in the rat at 21°C, but pethidine (meperidine) did not (Oka, 1977). This might appear merely to reflect a lower potency of pethidine, since all three produced hypothermia at 12°C that was blockable by naloxone. However, in mice, pretreatment with phenelzine (which increases principally the brain 5-HT content in mice) diminished the hypothermic effect of morphine injected either i.p. or i.c.v., but increased that of pethidine (Botting et al., 1978). This effect with pethidine was reversed by methysergide and the inference was drawn that 5-HT neurons may mediate the hypothermic effect of pethidine (Botting et al., 1978). One would have to draw the conclusion, also, that morphine hypothermia is not mediated by 5-HT. Indeed, morphine induced hyperthermia in the cat was increased by pretreatment with either a DA blocker (pimozide) or an inhibitor of 5-HT reuptake (fluoxetine), so that an increased ratio of 5-HT:DA activity at their respective receptors appeared to cause hyperthermia (French et al., 1978a). Yet in the rat, hypothermia following morphine, methadone or pethidine was blocked by pretreatment with p CPA and restored by 5-hydroxytryptophan (Oka, 1977). These apparent contradictions are reminiscent of the conflicting evidence concerning the mediation of ethanol hypothermia by 5-HT (Section 3.4.2.3). Therefore it is difficult to draw a firm conclusion about the resemblances or differences between the thermoregulatory effects of opiates and those of ethanol. There are nevertheless some unexpected similarities. Morphine tolerance in the rat, produced by s.c. implantation of a morphine pellet, produced hypersensitivity to the hypothermic effect of icv injection of DA (Lomax et al., 1978). This suggests that the continued action of morphine depressed the release of endogenous DA, just as several groups had observed during chronic ethanol administration (Section 4.3.1.1). Rats treated chronically with ethanol in a liquid diet developed tolerance to ethanol induced hypother-

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mia, and cross-tolerance to the hypothermic effect of large doses of morphine, but not to the hyperthermic effect of small doses (Khanna et al., 1979a). Conversely, rats receiving morphine (30 mg/kg) i.p. daily for three days become tolerant to morphine hypothermia, and cross-tolerant to ethanol hypothermia. These findings would be consistent with the hypothesis that tolerance is a response to the drug induced functional disturbance rather than to the drug p e r se (Section 4.1.2). Thus, hypothermia, however produced, should give rise to tolerance to the hypothermic effects of diverse drugs regardless of pharmacological class, and also of cold exposure (Section 4.3.3). In the morphine dependent rat naloxone precipitates a withdrawal hypothermia that is proportional to the morphine maintenance dose (French et al., 1978b). Ethanol (0. 5-2 g/kg) i.p. decreased certain of the naloxone precipitated withdrawal symptoms, including diarrhea, wet-dog shakes and jumping, but increased the withdrawal hypothermia (Ho et al., 1979). This is not in conflict with the cross-tolerance mentioned above, since withdrawal hypothermia and acute drug induced hypothermia are quite different phenomena. But one might have expected that the morphine pellet implantation used by Ho et al. (1979) would produce cross-tolerance to the hypothermic effect of ethanol, so that ethanol would not have increased the withdrawal hypothermia. Since ethanol was given by i.p. injection and the morphine by s.c. pellet, perhaps cross-tolerance was minimized by the absence of conditioned stimuli associated with the drug administration (Section 4.1.2). When the two drugs were given by the same route and in the same environment, cross-tolerance to their hypothermic effects was found (Mansfield and Woods, 1981). 5.3. CHLORPROMAZINE AND HYDRALAZINE

Chlorpromazine represents a class of drugs that produce poikilothermia by yet another mechanism, viz. the direct blockade of post-synaptic monoamine receptors (Bowman and Rand, 1980). Many of its effects on thermoregulation are similar to those of ethanol. In both dog and human it suppresses shivering and reduces the metabolic thermogenic response to cold (Hemingway, 1963). In keeping with this effect it reduced the tissue temperature measured in the rat by needle thermoelectrodes in liver and skeletal muscle, just as did ethanol (Molenda and Obrzut, 1967). Chlorpromazine at first also causes peripheral vasodilatation (Kollias and Bullard, 1964), as ethanol does (Section 3.3.1.1), but patients on long-term high dose chlorpromazine therapy apparently develop tolerance to this action, as shown by normal resting blood flow in the hand and a normal vasoconstriction response to cold water (Downey and Frewin, 1970). Since tolerance also develops to the effects of ethanol, it is noteworthy that rats consuming ethanol for seven weeks showed some cross-tolerance to the hypothermic effect of i.p. injection of chlorpromazine at a Td of 20°C (Ratcliffe, 1972). These similarities perhaps reflect common central effects of chlorpromazine and ethanol in the POAH, ethanol possibly acting to reduce release of monoamine neurotransmitters, and chlorpromazine to block their postsynaptic receptors. However, some of the actions of chlorpromazine are exerted peripherally, and these may lead to differences from ethanol. Chlorpromazine (but not ethanol) diminished the rise in liver temperature in the rat after injection of epinephrine, while ethanol (but not chlorpromazine) exaggerated the fall in muscle temperature after NE (Molenda and Obrzut, 1967). Similarly, hydralazine causes a dose-dependent hypothermia in rats and mice at 21 °C to which tolerance develops rapidly, but without cross-tolerance to ethanol (Rigter et al., 1980b). Since tolerance to hydralazine induced hypothermia was demonstrable only after i.p. and not after icv injection, the tolerance may have been mediated by peripheral mechanisms not shared with ethanol. 5.4. COMMENT The three classes of dugs noted above----central depressants, opiates, and neuroleptics-act by quite different molecular mechanisms, and on different groups of neurons, yet there is extensive overlap of their acute effects on thermoregulation and an unexpected degree

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of cross-tolerance among them. This tends to support the notion that common end effects on thermoregulatory function constitute the stimulus to tolerance, regardless of the molecular mechanism by which the effects are initiated. At the same time there is a similar conflict of evidence concerning the roles of catecholamines, 5-HT, and other transmitters and their respective receptors in the thermoregulatory effects of all these drugs. This raises a legitimate suspicion that the observed transmitter changes may be reflections of altered thermoregulatory neuron function, rather than primary mechanisms. There is only a very limited number of ways in which neurotransmitter systems can respond to alterations in stimulus input, but an enormous complexity of patterns of simultaneous alteration in different pathways. Therefore systemic or icv injection of drugs, neurotransmitters, or their respective blockers is a relatively crude method that has probably yielded most of the insights of which it is capable. Probably significant future progress in the understanding of thermoregulation will require the development of micromethods for the simultaneous recording and measurement of st,.'mulus input, effector response, and quantitative drug-transmitter interactions on these, in identified neuronal sequences.

6. BIOLOGICAL SIGNIFICANCE OF ETHANOL EFFECTS ON THERMOREGULATION While the study of ethanol induced thermoregulatory changes is interesting because of the light it may shed on thermoregulatory processes themselves and on the mechanisms of ethanol action and tolerance on these processes, it is also important because it may help to separate direct from indirect effects of ethanol in the periphery. Many of the intermediary metabolic effects of ethanol, for example, are due in whole or in part to ethanol-induced temperature changes. When hypothermia was prevented by a Ta of 37°C, ethanol no longer retarded the uptake of ~-aminoisobutyrate into mouse brain (Freund, 1973). Reduced incorporation of [14C]valine into tissue proteins in various regions of the rat brain and other viscera, that occurred after administration of ethanol, was largely prevented by a T, of 38°C, except at very high blood ethanol levels (Henderson et al., 1980). Raised Ta, which prevented hypothermia, also attenuated the ethanol induced rise in plasma corticosterone and free fatty acid levels in the rat (Pohorecky and Rizek, 1981). The dose-dependent increase in SGOT, SGPT and serum aldolase produced by ethanol in the rat coincided in time with the maximum fall in body temperature (Altland et al., 1970); the same investigators had found earlier that the same changes in serum enzymes could be produced by hypothermia resulting from cold exposure without ethanol. There have been numerous demonstrations that a fall in body temperature slows the elimination of ethanol itself in the dog, rat, cat and rabbit (Mufioz, 1937a; Dybing, 1945; Jaulmes et al., 1956; Larsen, 1971; Krarup and Larsen, 1972). One report that the ethanol elimination rate in the rat was unaffected by the prevention of hypothermia (Ferko and Bobyock, 1978) and another that the rate of fall of blood ethanol level was actually higher in hypothermic than in normothermic dogs (MacGregor et al., 1965) may be reflections of distribution artifacts. In the latter study the same intravenous infusion of ethanol produced higher blood alcohol levels in the hypothermic dogs, because of greatly slowed equilibration in a smaller volume of distribution. Probably the latter was due to extensive reduction of peripheral circulation and slower diffusion across capillary walls at low temperature. If the actual rate of ethanol metabolism in the liver was reduced less than the volume of distribution this would result in a more rapid fall of concentration in the reduced volume in which turnover was still occurring. Certainly, studies in the cat indicate that hypothermia produced by external cooling reduces the hepatic clearance of indocyanin green and the bile secretion rate to the same extent as it decreases the rate of ethanol clearance (Larsen, 1971; Krarup and Larsen, 1972). These reductions occur suddenly when the rectal temperature falls below 37.5°C suggesting a sudden deviation of blood flow away from the sinusoids and into the internal shunts.

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Some of the effects of ethanol on brain function are markedly affected by changes in body temperature. In mice, low doses of ethanol that cause hyperthermia also produce increased locomotor activity, while after larger doses that produce hypothermia the animals are sedated and huddled together (Papanicolaou and Fennessy, 1980). However, it is difficult to be sure that these are causally linked since ethanol induced narcosis (loss of righting reflex) in mice and rats is prolonged by a Ta of 30-35°C, which prevents hypothermia (George et al., 1981; Pohorecky and Rizek, 1981). The picture is actually somewhat complicated. As Ta is reduced from 38 to 32°C, the duration of narcosis is shortened and the blood alcohol level at the time of awakening is raised, indicating increasing resistance to ethanol narcosis (Malcolm and Alkana, 1981). As the body temperature continues to drop below 29°C, by progressive reduction of T.~ down to 12°C, the duration of ethanol narcosis increases progressively. These changes are consistent with a critical phase change in neuronal membranes at a temperature of 29-30°C. A similar explanation is advanced for the sudden decrease in cerebral glucose utilization (2-deoxyglucose uptake) when the temperature is reduced from 32 to 28°C (Towell and Erwin, 1982). The sudden decrease is consistent with the membrane transfer of glucose becoming rate-limiting at 29-30°C. At 37°C, ethanol decreased the rate of glucose utilization, possibly by an effect on intracellular enzymes, while at 28°C it increased the rate, possibly by fluidizing the membrane and facilitating the transfer. Hyperbaric conditions, which reverse ethanol narcosis in the mouse at room temperature, also did so at a Ta of 33.5-34.5°C, which prevented hypothermia (Malcolm and Aikana, 1982). Since hyperbaric conditions would tend to over-ride membrane phase changes resulting from body temperature variation or from ethanol, this finding serves to emphasize the importance of such changes at normal pressure. Effects of ethanol on psychomotor performance of various types are also influenced by temperature. In the rat, a conditioned avoidance response is more impaired by ethanol at a Ta of 4°C (at which hypothermia probably occurred) than at 20°C (Molenda, 1967). Retrograde amnesia for a learned passive avoidance behavior, produced by lowering of body temperature to 21°C, could be overcome by re-cooling the animal shortly before a recall test, but not if the recooling was followed immediately by 5 min or more of re-warming (Mactutus et al., 1980). This suggests that internal cues produced by hypothermia served as 'contextual cues' or for state dependent learning. Since ethanol has been shown to produce state dependent learning (Weingartner and Murphy, 1977; Cicero, 1978) it is possible that temperature changes form part of the ethanol state. Other forms of ethanol related behavior also vary with body temperature. Not all effects of ethanol on body temperature are deleterious. Several groups have shown, in rats, dogs and humans, that ethanol not only facilitates the production of hypothermia by external cooling, but enables the subject to be cooled to a lower temperature before ventricular fibrillation or cardiac arrest ensues, and facilitates defibrillation during rewarming (Bertho et al., 1964; White and Nowell, 1965; MacGregor et aL, 1966; Duthie and White, 1977). n-Propanol and n-butanol were also effective (MacGregor et al., 1966). These beneficial effects may reflect decreased sensitivity of myocardial catecholamine receptors. Also, under hypobaric conditions mice survived longer after a dose of ethanol that produced profound hypothermia (Flacke et al., 1953). This is probably due to reduced metabolic activity in the brain and other tissues, with consequent reduction of oxygen requirement. In summary, many effects of ethanol are probably mediated by body temperature changes. If suitable control of T,~ is used, to keep the animals normothermic, then such secondary effects of ethanol should be eliminated. This might make it much easier to identify the primary effects, and to focus our attention more clearly on the basic mechanisms of action. 7. SUMMARY AND CONCLUSIONS Clinical reports of accidental hypothermia in alcohol intoxicated individuals exposed to low ambient temperature (Paton, 1983) have generally been borne out by experimental

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studies in healthy volunteers. Small doses of ethanol, given to human subjects at normal ambient temperature (Ta), have very little effect on body temperature but a combination of large dose, low Ta and vasodilatation provoked by strenuous exercise, causes a sharp fall in rectal temperature. In experimental animals, the use of relatively larger doses of alcohol and more extreme temperatures, both above and below the thermoneutral zone, has shown that the effect of ethanol is essentially poikilothermic, i.e. an impairment of adaptation to both heat and cold. This effect has been studied in greater detail, in relation to each of the basic thermoregulatory processes. Though small doses of alcohol may increase the metabolic rate under some circumstances, the most common effect at low Ta is inhibition of shivering and therefore reduction of thermogenesis. At the same time it tends to cause increased heat loss by cutaneous vasodilatation. This makes for a greater feeling of comfort in the cold exposed subjects but increases the rate of fall of core temperature. The combination of decreased thermogenesis and increased heat loss, despite falling body temperature, is suggestive of a lowering of the set-point of the thermoregulatory control mechanisms. Consistent with this is a slight increase in ventilatory heat loss after low doses of ethanol but larger doses cause respiratory depression, so that heat loss through the lungs is minor. However, at high Ta ethanol causes hyperthermia in experimental animals and shows enhanced lethality, so that impairment of thermoregulatory effector mechanisms seems to be at least as important as change in set-point. Studies of the effects of ethanol on electrophysiological activity of single neurons in the pre-optic area and anterior hypothalamus (POAH), biochemical activities of neuronal membranes, hypothalamic blood flow, conventional neurotransmitters, amino acid putative neurotransmitters, neuropeptides, prostaglandins and inorganic ions have all failed so far to yield a clear comprehensive picture of the mechanisms by which ethanol affects thermoregulation. In each case, contradictory evidence has been obtained concerning the consequences of ethanol administration, whether by oral, intraperitoneal, intravenous, intracerebroventricular, or direct local (POAH) route. Behavior directed towards maintenance of normothermia, by selection of an appropriate Ta, in animals at a low T, is consistent with an ethanol-induced reduction of set-point, but this has not been verified by appropriate tests at high Ta. Chronic administration of ethanol leads to tolerance to its hypothermic effects and alcohol withdrawal may under certain circumstances be accompanied by an apparent rebound hyperthermia. The more characteristic withdrawal effect, however, is hypothermia and this is accompanied by a behavioral preference for a raised Ta. There are clear genetic influences; not only on initial sensitivity to ethanol hypothermia, but also on the ability to develop tolerance to it. Once more, however, detailed examination of the changes in central thermoregulatory mechanisms during and after chronic ethanol exposure has failed to yield consistent patterns able to explain the tolerance. Comparison of the effects of ethanol with those of barbiturates and other central depressants, cannabinoids, opiates, phenothiazines and hydralazine has shown a surprising number of similarities and even unexpected cross-tolerance between ethanol and morphine or chlorpromazine. The most credible hypothesis to date is that such cross-tolerance represents physiological and behavioral (including Pavlovian conditioned) adaptive responses to a common end result, hypothermia, even though this end result is initiated by different molecular mechanisms of the various drugs. Regardless of the means by which they are produced, the thermoregulatory effects of ethanol play a very important role in the mediation of many of the physiological, biochemical and possibly behavioral effects of ethanol. Identification of these indirect, temperature mediated effects and prevention of them by appropriate manipulation of Ta may help greatly in future research to identify the direct primary actions of ethanol. This approach, together with a filling-in of the major gaps that have been identified in our knowledge of alcohol effects under certain specified conditions (particularly at higher Ta), should result in a clearer and more comprehensive explanation of alcohol actions on thermoregulation.

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Acknowledgements--We are greatly indebted to Dr. J. M. Khanna for valuable discussions during the organization of this review, and to Mrs. V. Cabral for her excellent work in the preparation of the manuscript.

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