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Interrelationship of thermal and sleep-wakefulness changes elicited from the medial preoptic area in rats
EXPERIMENTAL
NEUROLOGY
loo,
40-50 ( 1988)
interrelationship of Thermal and Sleep-Wakefulness Changes Elicited from the Medial Preoptic Area in Rats S. DATTA, V. MOHAN KUMAR,
G. S. CHHINA,
AND B. SINGH’
Department ofPhysiology, All India Institute ofMedical Sciences, New Delhi I IO 029, India Received April 28, 1987; revision receivedAugust 12, 1987 The study investigated the possible interrelationship between changes in sleepwakefulness and body temperature, primarily induced by manipulation of the noradrenergic system in the medial preoptic area. Saline, norepinephrine, and its (Y-and j3blockers were injected in the medial preoptic area and in some control areas of rats, during their sleeping and active periods. 5-Hydroxytryptamine was injected in the medial preoptic area in another group of animals. Simultaneous changes in sleepwakefulness and the body temperature were continuously recorded. Norepinephrine produced hypothermia and arousal, whereas cu-adrenergic blockers induced hyperthermia and sleep. These changes in body temperature and in sleep-wakefulness did not follow an identical time course. 5-Hydroxytryptamine induced hyperthermia without affecting sleep-wakefulness. It is suggested that there are different neuronal mechanisms in the medial preoptic area that bring about the drug-induced changes in temperature and sleep-wakefulness. 0 1988 Academic press, IIIC.
INTRODUCTION Several reports in the literature suggest the existence of a close interrelationship between body temperature and sleep-wakefuIness (8, 18, 20, 2 1, 24). Alterations in sleep-awake states are accompanied by changes in body Abbreviations: mPOA-medial preoptic area, NE-norepinephrine, PBZ-phenoxybenzamine, PHENT-phentolamine, PROP-propranolol, 5-HT-5-hydroxytryptamine. ’ This work was supported by a research grant from the Indian Council of Medical Research. The authors acknowledge the help rendered by Mr. K. R. Sundaram, Associate Professor, Department of Biostatistics, in the statistical analysis of the data; Or. P. S. Rao, Department of Physiology, for critical reading of the manuscript; and CIBA-GEIGY, Ltd., Basel, Switzerland, for supplying phentolamine for this study. Correspondence may be addressed to Dr. V. Mohan Kumar.
40 00144886/88
$3.00
Gqyr@tO1988byAademicRss,Inc. AII rights of repn3dwtioa in any form resend.
BODY
TEMPERATURE
AND
SLEEP-WAKEFULNESS
41
temperature (17). Changes in ambient as well as local preoptic temperatures lead to alterations in sleep-awake states (5,18,20,2 I). Preoptic lesions affect both sleep-wakefulness and body temperature ( 13). Stimulation of adrenergic receptors of the medial preoptic area (mPOA) induces hypothermia (7). Adrenergic stimulation of the same area induces arousal ( 14). Thus we find that the thermal and sleep-wakefulness changes in the body are interrelated. Both these responses could be elicited from the mPOA with the same drugs (7, 14). These two responses need to be studied simultaneously for better understanding of the involvement of mPOA in these functions. In the present study, the thermal and sleep-wakefulness changes were simultaneously monitored after application of norepinephrine (NE) and its antagonists in the mPOA during the day and night. NE and its blockers were applied at regions around the mPOA to see whether or not the locations from where these responses could be elicited are anatomically identical. 5-Hydroxytryptamine (5-HT), which was shown to induce hyperthermia in some reports (6, 10, 16) and hypnogenesis in others (12, 25), was also applied in the mPOA, and simultaneous changes in the two variables were studied. METHODS Studies were undertaken on 72 male Wistar rats weighing between 200 and 300 g. The animals were housed separately for 4 weeks in an animal room with the temperature maintained at 26 f 1“C and light on period from 05.00 to 19.00 h, before they were operated on for implantation of cannulae and recording electrodes. Food and water were provided ad lib&m. All experiments were conducted at the ambient temperature of 26 f 1°C. Rectal temperature of the rats was monitored for at least 4 to 5 days preoperatively. The rats were anesthetized with pentobarbital sodium, (Nembutal, Abbott, India), at 35 mg/kg body wt., i.p. Bilateral guide cannulae assemblies consisting of 26-gauge stainless-steel tubes and styli were introduced aseptically toward the mPOA and some control regions adjoining the mPOA, as described elsewhere (2, 7, 14). EEG, EMG, and EOG electrodes were also chronically implanted as described ( 14,15). Four to five days after the operation, those animals which had a rectal temperature almost equal to that in the preoperative state were used for the study. Animals were placed in the cage in the recording room for at least 1 h before the experimental procedure for familiarization with the surroundings. Food and water were also provided in the recording cage. The experimental procedure included continuous recordings of EEG, EMG, EOG, and rectal temperature for 30 min before, and 60 to 120 min after, intracerebral injection of saline and drugs. Recordings were discontinued whenever the rats
42
DATTA
ET AL.
TABLE
1
Details of Drugs Injected into the Medial Preoptic Area (mPGA)
Number of animals
Substances injected
Site of injection
Normal saline
in Rats
Time of experiments
Amount of substance injected
mPGA
12-14 h 22-24 h
0.2 pl 0.2 PI
mPGA Control sites mPGA
12-14 h 12-14 h 22-24 h
3.0 pg in 0.2 d 3.0 pg in 0.2 ~1 3.0 pg in 0.2 ~1
Phenoxybenmmine (phenoxybenzarnine hydrochloride; Smith Kline and French, Philadelphia)
mPGA mPGA Control sites
12-14 h 22-24 h 22-24 h
1.O fig in 0.2 ~1 1.O pg in 0.2 ~1 I .O fig in 0.2 ~1
Phentolamine (phentolamine hydrochlorideRegitine; CIBA-GEIGY, Ltd., Switzerland)
mPGA
22-24 h
10.0 pg in 0.2 ,uI
6 6
mPOA mPGA
12-14 h 22-24 h
3.0 pg in 0.2 d 3.0 pg in 0.2 ~1
6
mPGA
22-24 h
Norcpinephrine (kuterenol Chemical Co.)
Propranolol London)
(+ propranolol;
bitartrate;
Sigma
ICI, Ltd.,
Serotonin (5-hydroxytryptamine sulfate; Sigma Chemical Co.)
creatine
10.0 pg in 0.2 rl
became restless and started biting the rectal probe and the recording cables. Those animals whose recordings were discontinued earlier than 60 min are not included in the analysis. Rats were observed continuously for activity, movements, and also postures assumed during inactivity. All drugs, made to a volume of 0.2 ~1, were administered slowly at 0.1 & min. The effectiveness of the drugs was studied in sleeping and awake animals. The details of the drugs injected and the grouping of the animals are indicated in Table 1. The rationale behind the selection of the drug dosage is mentioned elsewhere (15). No rat received more than one bilateral injection. Details of the injection procedure, recording of the rectal temperature, assessment of sleep-wakefulness, and analysis of the data are described in the earlier reports (7, 14, 15). In addition to manual analysis and tabulation for statistical analysis, in some animals the EEG was also fed to a pathfinder (Pathfinder-II, Nicolet Biomedical Instrument), for frequency analysis and rastor display. Records from all animals to 60 min after the injections and 30 min before the injections were analyzed statistically. The preinjection data on sleep-wakefulness from each group were first analyzed by nonparametric two-way analysis of variance (Friedman test) and those on temperature were analyzed by two-way analysis of variance (parametric). The data were further analyzed only after ensuring that there was no significant intragroup variation in the preinjection readings by the above tests. Statistical
BODY TEMPERATURE
AND SLEEP-WAKEFULNESS
43
FIG. 1. Histologic sections show phenoxybenzamine (PBZ) injection sites (shown by arrows) in the medial preoptic area (mPOA; A) which produced alteration, and injection sites outside the mPOA (B) which did not produce alteration in sleep-wakefulness and temperature.
significance of the saline group was determined on the basis of comparison with the preinjection record. In these, the preinjection mean readings were compared with each postinjection reading by a multiple range test (Friedman test) in the case of sleep-wakefulness and by Dunnet’s test in the case of temperature. Drug-induced changes, on the other hand, were compared with the saline-induced readings obtained at identical time points by the Wilcoxon nonparametric test for sleep-wakefulness and a simple t test for temperature. In most animals, it was not possible to have prolonged simultaneous recordings of changes in temperature and consciousness, as the rats usually did not tolerate the presence of the rectal probe for periods beyond 60 min after injection. Trends of changes after 60 min, observed in a few animals, are briefly mentioned. At the end of the experiment, the site of the injection (Fig. 1) and the spread of the administered substance were determined histologically by the ferric chloride method described elsewhere (3).
44
DATTA
ET AL.
RESULTS The preinjection basal rectal temperature of rats studied during the day was 37.8 + 0.03”C. They had spent 88 to 95% of the time in sleep during the 30 min of preinjection control recording. About 10 to 15% of this period was occupied by REM sleep, and the remainder by slow-wave sleep. During night, the preinjection basal temperature was 38.14 k 0.03”C. They were awake during most (95 to 100% of the time) of the control recording period. Control injection of saline in the mPOA in the awake rats (during the night) did not produce any significant change in rectal temperature and ongoing wakefulness. On the other hand, the sleeping rats, which were aroused because of handling during the administration of saline during the day, showed an increase in rectal temperature for 15 min. These changes in rectal temperature and the arousal were temporally correlated (Fig. 2). There was arousal and hypothermia after administration of NE in the mPOA in the sleeping animals (Fig. 2). The initial hypothermia for 45 min was followed by hyperthermia of low magnitude. The prolonged arousal induced by the drug persisted during the initial hypothermia and the second hyperthermia. Injection of NE in the mPOA during the night (in the awake rats) did not produce any behavioral or electrophysiologic changes. The initial hypothermia was not followed by hyperthermia in these animals. Changes in temperature and sleep-wakefulness were obtained only from injection sites in the mPOA. Injections in the medial part of the anterior amygdala region, the ventrolateral part of the lateral para-olfactory region, and the anterior end of the lateral hypothalamic region in six animals during the day did not produce any change in temperature and sleep-wakefulness. There was a short-lasting arousal and an increase in the body temperature immediately after the injection which were not significant compared with the saline-induced changes. There was a temporal coincidence in the onset of sleep and hyperthermia, induced by the injection of 1 pg of the a-adrenergic blocker phenoxybenzamine (PBZ) in the awake rats (Fig. 3). REM sleep was never observed after PBZ administration, except in one animal in which a short episode of lmin duration was obtained. The hyperthermia continued for a long period although the rats were aroused from induced sleep by about 55 min (Fig. 4). Injection of another cu-blocker, phentolamine (PHENT), in the mPOA during the night in the awake rats produced changes which were qualitatively identical to those induced by PBZ (Fig. 3). The magnitude of the rise in the rectal temperature was greater, and the appearance of REM sleep episodes was more frequent after injection of 10 pg PHENT as compared with 1 rg PBZ. REM sleep was obtained in all animals after PHENT injection. The
BODY TEMPERATURE DAV
SALINE
DAY
NE
45
AND SLEEP-WAKEFULNESS
I-
NIGHT
SALINE
7
NIGHT
NE
-iv ?RE
i i IRJ.
oi* I
, POST
IIIJ.
FIG. 2. The graph shows the rectal temperature (mean k SD) and sleep/awake periods (mean f SE) simultaneously recorded from the groups of rats Before and after injection of saline (0.2 ~1) and norepinephrine (NE) (3.0 fig in 0.2 gl) in the mPOA during the day and night. Hatched area shows the postinjection alteration in rectal temperature. Shaded and unshaded areas represent awake and sleep periods, respectively. *P < 0.05, **P < 0.0 1, and ***P -C0.00 1, levels of significance of the change compared with values during the identical time points of control saline injection.
total duration of REM sleep within the postinjection recordings ranged from 2 min to 6 min. Injection of PBZ in sleeping rats during the day also produced an increase in the rectal temperature. The SWS:REM ratio in these
46
DATTA NIGHT
PIlENT
NIGHT
PI2
ET AL.
FIG. 3. The graph shows the rectal temperature (mean + SD) and sleep/awake periods (mean f SE) simultaneously recorded from the groups of rats before and atIer injection of PBZ ( 1.O ccg in 0.2 d) during the day and night. Phentolamine (PHENT, 10.0 a in 0.2 ~1) and 5-hydroxytryp tamine (5-HT, 10.0 pg in 0.2 rJ) were injected during the night. The hatched area shows the postinjection alteration in rectal temperature; shaded and unshaded are.as represent awake and sleep periods, respectively. *P-c 0.05,“cP < 0.0 l,and **+P < 0.001 levels of signiiicance of the change compared with the values obtained during identical time points of control saline injection.
animals was not much altered even after PBZ injection in the mPOA during their normal sleeping periods. Injection of PBZ in the anterior amygdala region and lateral to the mPOA in five animals, and in the posterior end of
BODY TEMPERATURE
AND SLEEP-WAKEFULNESS NIGHT
40.0
47
PBZ
I 36.0
-I
Y ‘“0 z 3bO2 t t 37.036.0
-
5’ 4
0
15
39
a
iy
io
05,
&
7&
Ss I
IPRE INJ.
POST INJ.
FIG. 4. The graph shows the rectal temperature and sleep/awake periods simultaneously recorded after injection of PBZ ( 1.Osg in 0.2 ~1) in one rat. Hatched area shows the postinjection alteration in rectal temperature compared with the mean preinjection reading. Shaded and unshaded areas represent awake and sleep periods, respectively.
the mPOA in one animal, did not produce any changesin body temperature and sleep-wakefulness. Injection of the &blocker proprandol (PROP) in the mPOA did not produce any significant changes in temperature and sleep-wakefulness. Injection-bound increases in rectal temperature and arousal, produced in the sleeping rats, were not found to be significant compared with saline-induced changes. Injection of 5-HT in the mPOA during the night produced a rise in rectal temperature which was similar in nature to the change induced after administration of PBZ and PHENT (Fig. 3). Unlike the a-adrenergic blockers, the 5-HT injection did not produce sleep. Although the electrophysiologic re-
48
DATTA
ET AL.
cordings showed arousal, the rats’ behavior showed inactivity. postures similar to those of the sleeping rats.
They assumed
DISCUSSION Application of NE in the mPOA induced hypothermia and arousal, whereas a-blockers produced hyperthermia and sleep. Changes in sleepwakefulness obtained on application of NE and its blockers would not have contributed toward the thermal alterations. A decrease in body temperature is not associated with arousal nor is an increase associated with sleep (8, 9). Similarly the induced arousal and sleep are not likely to be totally dependent on thermal changes. Reports available in the literature indicate that reduction in environmental or preoptic-anterior hypothalamic temperature can bring about reduction in sleep (17,2 1,24). A local increase in preoptic-anterior hypothalamic temperature has a sleep-inducing effect (5, 18, 20). It might be argued that the arousal observed after NE application and the sleep after a-blockers were the results of hypothermia and hyperthermia, respectively, which were induced simultaneously. But the arousal obtained in this study after application of NE during the day was observed not only during the initial hypothermia but also during the second hyperthermia of low magnitude. Similarly, the rats were aroused from sleep induced by a-blockers, even when the hyperthermia was persisting. It has been reported that REM sleep time is markedly altered by small alterations of environmental temperature (22). But in the present study we found that the SWS:REM ratio was not altered even during the hyperthermia produced by PBZ administered during the day. Alterations in local temperature also probably did not contribute to the observed changes in sleep-wakefulness. Applications of NE in the mPOA may produce an increase in local temperature (18). If we consider the increase in local temperature produced by NE as causing the changes in sleepwakefulness, the rats should have gone to sleep ($1820) rather than becoming aroused. Our findings suggest that the thermal and sleep-wakefulness responses were primarily elicited by independent mechanisms, though some contribution of the former to the latter cannot be ruled out. These conclusions were further supported by the observations obtained after 5-HT injection. The hyperthermia induced by 5-HT injection in the mPOA was not accompanied by any change in true sleep-wakefulness. Although the two responses elicited by NE and its a-blockers may not be interrelated, there is striking identity in the sites where they were elicited. Injection of NE and an a-blocker outside the mPOA did not produce any significant alterations in sleep-wakefulness and rectal temperature.
BODY TEMPERATURE
AND SLEEP-WAKEFULNESS
49
There were two instances of temperature changes of small magnitude which could be attributed to alterations in sleep-wakefulness. The small increase in body temperature immediately after application of saline, and that observed at a later stage after NE in sleeping rats, may be related to the induced arousal and activity. Earlier reports claimed an increase in body temperature after application of saline in the mPOA (1, 11, 23). The present study suggests that the increase in temperature might have resulted from arousal and activity induced by handling at the time of injection in the sleeping rats. There was no change in temperature in the rats which received saline injection during their normal waking period. Simultaneous recording of the two variables during the day and night also demonstrated that a pure hypothermia is the effect of administration of NE in the mPOA and not a biphasic response as reported (4,7, 19). The delayed increase in temperature of small magnitude was observed only in the day experiments. It is likely that the thermal and sleep-wakefulness responses elicited by NE were mediated through the q postsynaptic receptors (15). While interpreting the results obtained on application of adrenergic blockers, one must bear in mind the side effects of these drugs, although small doses were used in this study. The differences in the magnitude of the hypothermia SWS:REM ratio after administration of PBZ and PIIENT may be attributed to the differences in the dosage of the drug. REFERENCES 1. AVERY, D. D. 197 1. Intrahypothalamic adrenergic and cholinergic injection effects on temperature and ingestive behaviour in the rat. Neuropharmacology 10: 753-163. 2. BAGGA, N., G. S. CHHINA, V. MOHAN KUMAR, AND B. SINGH. 198 1. Mechanism of participation of medial preoptic area in the hippocampal inhibition of ovulation. Brain Res. 216: 444-448. 3. BAGGA, N., G. S. CHHINA, V. MOHAN KUMAR, AND B. SINGH. 1984. Chohnergic activation of medial preoptic area by amygdala for ovulation in rat. Physiol. Behav. 32: 45-48. 4. BECKMAN, A. L. 1970. Effect of i&a-hypothalamic norepinephrine on thermoregulatory responses in the rat. Am. J. Physiol. 218: 1596- 1604. 5. BENEDEK, G., F. OB~, JR., I;. SZEKERES, AND F. O&L. 1976. Cortical synchronization induced by thermal stimulation of the preoptic area in immobilized rats. Acta Physiol. Acad. Sci. Hung. 48: 65-12. 6. CRAWSUAW, L. I. 1972. Effects of intracerebral5-hydroxytryptamine injection on thermoregulation in rat. Physiol. Behav. 9: 133-140. 7. DATTA, S., V. MOHAN KUMAR, G. S. CHHINA, AND B. SINGH. 1985. Tonic activity of medial preoptic norepinephrine mechanism for body temperature maintenance in sleep ing and awake rats. Brain Rex Bull. 15: 447-45 1. 8. HELLER, H. C., AND S. F. GLQTZBACK. 1977. Thermoregulation during sleep and hibernation. Int. Rev. Physiol. 15: 147-188. 9. KLEITMAN, N., AND E. KLEXTMAN. 1953. Effect of non-twenty-four-hour routines of living on oral temperature and heart rate. J. Appl. Physiol. 6: 283-29 1,
50
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ET AL.
10. KUBIKOWSW, P., AND W. RE~ERSKI. 1969. Biogenic amines and body temperature regulation in the rats. Diss. Pharrn. Pharmacol. 21: 207-2 12. 11. LOMAX, P., R. S. FOSTER, AND W. E. KIRKPATRICK. 1969. Cholinergic and adrenergic interactions in the thermoregulatory centres of the rat. Brain Res. 15: 43 l-438. 12. MARLEY, E., AND J. F. WHELAN. 1975. Some central effects of 5-hydroxytryptamine in young chickens at and below thermoneutrality. Br. J. Pharmacol. 53: 37-41. 13. MCGINT~, D. J., AND M. B. STERMAN. 1968. Sleep suppression after basal forebrain lesions in the cat. Science 160: 1253-1255. 14. MOHAN KUMAR, V., S. DATTA, G. S. CHHINA, N. GANDHI, AND B. SINGH. 1984. Sleepawake responses elicited from medial preoptic area on application of norepinephrine and phenoxybenzamine in free moving rats. Brain Rex 322: 322-325. 15. MOHAN KUMAR, V., S. DAI-~A, G. S. CHHINA, AND B. SINGH. 1986. Alpha adrenergic system in medial preoptic area involved in sleep-wakefulness in rats. Brain Res. Bull. 16: 463-468. 16. MYERS, R. D. 1974. Temperature regulation. Pages 237-285 in R. D. MYERS, Ed., Handbook of Drug and Chemical Stimulation of the Brain. Von Nostrand-Reinhold, New York. 17. PARMEGGIANI, P. L. 1980. Temperature regulation during sleep: a study in homeostasis. Pages 97-143 in J. OREM AND C. D. BARNES, Eds. Physiology in Sleep. Academic Press, New York. 18. PARMEGGIANI, P. L., G. ZAMBONI, T. CIANCI, L. F. AGNATI, AND C. RICCI. 1974. Influence of anterior hypothalamic heating on the duration of fast wave sleep episodes. Electroencephalogr. Clin. Neurophysiol. 36: 465-470. 19. POOLE, J., AND J. D. STEPHENSON.1979. Effects of noradrenaline and carbachol on temperature regulation of rats. Br. J. Pharmacol. 65: 43-5 1. 20. ROBERTS, W. W., AND T. C. L. ROBINSON. 1969. Relaxation and sleep induced by warming of preoptic region and anterior hypothalamus in cats. Exp. Neural. 25: 282-294. 21. SCHMIDEK, W. R., K. HOSHINO, M. SCHMIDEK, AND C. TIMO-IARIA. 1972. Influence of environmental temperature on the sleep-wakefulness cycle in the rat. Physiol. Behav. 8: 363-371. 22. SZYMUSJAK, R., AND E. SATINO~. 1981. Maximal REM sleep time defines a narrower thermoneutral zone than does minimal metabolic rate. Physiol. Behav. 26: 687-690. 23. TSOUCARIS-KUPFFER, D., AND H. SCHMITT. 1972. Hypothermic effect of or-sympathomimetic agents and their antagonism by adrenergic and cholinergic blocking drugs. Neuropharmacologyll: 625-635. 24. VALATX, J. L., B. ROUSSEL, AND M. CURE. 1973. Sommeil et temperature c&brale du rat au course de l’exposition chronique en ambiance chaude. Brain Res. 55: 107-l 22. 25. YAMAGUCHI, N., T. J. MARCZYNSKI, AND G. M. LING. 1963. The effects of electrical and chemical stimulation of the preoptic region and some non-specific thalamic nuclei in unrestrained, waking animals. Electroencephalogr. Clin. Neurophysiol. 15: 154.
NEUROLOGY
loo,
40-50 ( 1988)
interrelationship of Thermal and Sleep-Wakefulness Changes Elicited from the Medial Preoptic Area in Rats S. DATTA, V. MOHAN KUMAR,
G. S. CHHINA,
AND B. SINGH’
Department ofPhysiology, All India Institute ofMedical Sciences, New Delhi I IO 029, India Received April 28, 1987; revision receivedAugust 12, 1987 The study investigated the possible interrelationship between changes in sleepwakefulness and body temperature, primarily induced by manipulation of the noradrenergic system in the medial preoptic area. Saline, norepinephrine, and its (Y-and j3blockers were injected in the medial preoptic area and in some control areas of rats, during their sleeping and active periods. 5-Hydroxytryptamine was injected in the medial preoptic area in another group of animals. Simultaneous changes in sleepwakefulness and the body temperature were continuously recorded. Norepinephrine produced hypothermia and arousal, whereas cu-adrenergic blockers induced hyperthermia and sleep. These changes in body temperature and in sleep-wakefulness did not follow an identical time course. 5-Hydroxytryptamine induced hyperthermia without affecting sleep-wakefulness. It is suggested that there are different neuronal mechanisms in the medial preoptic area that bring about the drug-induced changes in temperature and sleep-wakefulness. 0 1988 Academic press, IIIC.
INTRODUCTION Several reports in the literature suggest the existence of a close interrelationship between body temperature and sleep-wakefuIness (8, 18, 20, 2 1, 24). Alterations in sleep-awake states are accompanied by changes in body Abbreviations: mPOA-medial preoptic area, NE-norepinephrine, PBZ-phenoxybenzamine, PHENT-phentolamine, PROP-propranolol, 5-HT-5-hydroxytryptamine. ’ This work was supported by a research grant from the Indian Council of Medical Research. The authors acknowledge the help rendered by Mr. K. R. Sundaram, Associate Professor, Department of Biostatistics, in the statistical analysis of the data; Or. P. S. Rao, Department of Physiology, for critical reading of the manuscript; and CIBA-GEIGY, Ltd., Basel, Switzerland, for supplying phentolamine for this study. Correspondence may be addressed to Dr. V. Mohan Kumar.
40 00144886/88
$3.00
Gqyr@tO1988byAademicRss,Inc. AII rights of repn3dwtioa in any form resend.
BODY
TEMPERATURE
AND
SLEEP-WAKEFULNESS
41
temperature (17). Changes in ambient as well as local preoptic temperatures lead to alterations in sleep-awake states (5,18,20,2 I). Preoptic lesions affect both sleep-wakefulness and body temperature ( 13). Stimulation of adrenergic receptors of the medial preoptic area (mPOA) induces hypothermia (7). Adrenergic stimulation of the same area induces arousal ( 14). Thus we find that the thermal and sleep-wakefulness changes in the body are interrelated. Both these responses could be elicited from the mPOA with the same drugs (7, 14). These two responses need to be studied simultaneously for better understanding of the involvement of mPOA in these functions. In the present study, the thermal and sleep-wakefulness changes were simultaneously monitored after application of norepinephrine (NE) and its antagonists in the mPOA during the day and night. NE and its blockers were applied at regions around the mPOA to see whether or not the locations from where these responses could be elicited are anatomically identical. 5-Hydroxytryptamine (5-HT), which was shown to induce hyperthermia in some reports (6, 10, 16) and hypnogenesis in others (12, 25), was also applied in the mPOA, and simultaneous changes in the two variables were studied. METHODS Studies were undertaken on 72 male Wistar rats weighing between 200 and 300 g. The animals were housed separately for 4 weeks in an animal room with the temperature maintained at 26 f 1“C and light on period from 05.00 to 19.00 h, before they were operated on for implantation of cannulae and recording electrodes. Food and water were provided ad lib&m. All experiments were conducted at the ambient temperature of 26 f 1°C. Rectal temperature of the rats was monitored for at least 4 to 5 days preoperatively. The rats were anesthetized with pentobarbital sodium, (Nembutal, Abbott, India), at 35 mg/kg body wt., i.p. Bilateral guide cannulae assemblies consisting of 26-gauge stainless-steel tubes and styli were introduced aseptically toward the mPOA and some control regions adjoining the mPOA, as described elsewhere (2, 7, 14). EEG, EMG, and EOG electrodes were also chronically implanted as described ( 14,15). Four to five days after the operation, those animals which had a rectal temperature almost equal to that in the preoperative state were used for the study. Animals were placed in the cage in the recording room for at least 1 h before the experimental procedure for familiarization with the surroundings. Food and water were also provided in the recording cage. The experimental procedure included continuous recordings of EEG, EMG, EOG, and rectal temperature for 30 min before, and 60 to 120 min after, intracerebral injection of saline and drugs. Recordings were discontinued whenever the rats
42
DATTA
ET AL.
TABLE
1
Details of Drugs Injected into the Medial Preoptic Area (mPGA)
Number of animals
Substances injected
Site of injection
Normal saline
in Rats
Time of experiments
Amount of substance injected
mPGA
12-14 h 22-24 h
0.2 pl 0.2 PI
mPGA Control sites mPGA
12-14 h 12-14 h 22-24 h
3.0 pg in 0.2 d 3.0 pg in 0.2 ~1 3.0 pg in 0.2 ~1
Phenoxybenmmine (phenoxybenzarnine hydrochloride; Smith Kline and French, Philadelphia)
mPGA mPGA Control sites
12-14 h 22-24 h 22-24 h
1.O fig in 0.2 ~1 1.O pg in 0.2 ~1 I .O fig in 0.2 ~1
Phentolamine (phentolamine hydrochlorideRegitine; CIBA-GEIGY, Ltd., Switzerland)
mPGA
22-24 h
10.0 pg in 0.2 ,uI
6 6
mPOA mPGA
12-14 h 22-24 h
3.0 pg in 0.2 d 3.0 pg in 0.2 ~1
6
mPGA
22-24 h
Norcpinephrine (kuterenol Chemical Co.)
Propranolol London)
(+ propranolol;
bitartrate;
Sigma
ICI, Ltd.,
Serotonin (5-hydroxytryptamine sulfate; Sigma Chemical Co.)
creatine
10.0 pg in 0.2 rl
became restless and started biting the rectal probe and the recording cables. Those animals whose recordings were discontinued earlier than 60 min are not included in the analysis. Rats were observed continuously for activity, movements, and also postures assumed during inactivity. All drugs, made to a volume of 0.2 ~1, were administered slowly at 0.1 & min. The effectiveness of the drugs was studied in sleeping and awake animals. The details of the drugs injected and the grouping of the animals are indicated in Table 1. The rationale behind the selection of the drug dosage is mentioned elsewhere (15). No rat received more than one bilateral injection. Details of the injection procedure, recording of the rectal temperature, assessment of sleep-wakefulness, and analysis of the data are described in the earlier reports (7, 14, 15). In addition to manual analysis and tabulation for statistical analysis, in some animals the EEG was also fed to a pathfinder (Pathfinder-II, Nicolet Biomedical Instrument), for frequency analysis and rastor display. Records from all animals to 60 min after the injections and 30 min before the injections were analyzed statistically. The preinjection data on sleep-wakefulness from each group were first analyzed by nonparametric two-way analysis of variance (Friedman test) and those on temperature were analyzed by two-way analysis of variance (parametric). The data were further analyzed only after ensuring that there was no significant intragroup variation in the preinjection readings by the above tests. Statistical
BODY TEMPERATURE
AND SLEEP-WAKEFULNESS
43
FIG. 1. Histologic sections show phenoxybenzamine (PBZ) injection sites (shown by arrows) in the medial preoptic area (mPOA; A) which produced alteration, and injection sites outside the mPOA (B) which did not produce alteration in sleep-wakefulness and temperature.
significance of the saline group was determined on the basis of comparison with the preinjection record. In these, the preinjection mean readings were compared with each postinjection reading by a multiple range test (Friedman test) in the case of sleep-wakefulness and by Dunnet’s test in the case of temperature. Drug-induced changes, on the other hand, were compared with the saline-induced readings obtained at identical time points by the Wilcoxon nonparametric test for sleep-wakefulness and a simple t test for temperature. In most animals, it was not possible to have prolonged simultaneous recordings of changes in temperature and consciousness, as the rats usually did not tolerate the presence of the rectal probe for periods beyond 60 min after injection. Trends of changes after 60 min, observed in a few animals, are briefly mentioned. At the end of the experiment, the site of the injection (Fig. 1) and the spread of the administered substance were determined histologically by the ferric chloride method described elsewhere (3).
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RESULTS The preinjection basal rectal temperature of rats studied during the day was 37.8 + 0.03”C. They had spent 88 to 95% of the time in sleep during the 30 min of preinjection control recording. About 10 to 15% of this period was occupied by REM sleep, and the remainder by slow-wave sleep. During night, the preinjection basal temperature was 38.14 k 0.03”C. They were awake during most (95 to 100% of the time) of the control recording period. Control injection of saline in the mPOA in the awake rats (during the night) did not produce any significant change in rectal temperature and ongoing wakefulness. On the other hand, the sleeping rats, which were aroused because of handling during the administration of saline during the day, showed an increase in rectal temperature for 15 min. These changes in rectal temperature and the arousal were temporally correlated (Fig. 2). There was arousal and hypothermia after administration of NE in the mPOA in the sleeping animals (Fig. 2). The initial hypothermia for 45 min was followed by hyperthermia of low magnitude. The prolonged arousal induced by the drug persisted during the initial hypothermia and the second hyperthermia. Injection of NE in the mPOA during the night (in the awake rats) did not produce any behavioral or electrophysiologic changes. The initial hypothermia was not followed by hyperthermia in these animals. Changes in temperature and sleep-wakefulness were obtained only from injection sites in the mPOA. Injections in the medial part of the anterior amygdala region, the ventrolateral part of the lateral para-olfactory region, and the anterior end of the lateral hypothalamic region in six animals during the day did not produce any change in temperature and sleep-wakefulness. There was a short-lasting arousal and an increase in the body temperature immediately after the injection which were not significant compared with the saline-induced changes. There was a temporal coincidence in the onset of sleep and hyperthermia, induced by the injection of 1 pg of the a-adrenergic blocker phenoxybenzamine (PBZ) in the awake rats (Fig. 3). REM sleep was never observed after PBZ administration, except in one animal in which a short episode of lmin duration was obtained. The hyperthermia continued for a long period although the rats were aroused from induced sleep by about 55 min (Fig. 4). Injection of another cu-blocker, phentolamine (PHENT), in the mPOA during the night in the awake rats produced changes which were qualitatively identical to those induced by PBZ (Fig. 3). The magnitude of the rise in the rectal temperature was greater, and the appearance of REM sleep episodes was more frequent after injection of 10 pg PHENT as compared with 1 rg PBZ. REM sleep was obtained in all animals after PHENT injection. The
BODY TEMPERATURE DAV
SALINE
DAY
NE
45
AND SLEEP-WAKEFULNESS
I-
NIGHT
SALINE
7
NIGHT
NE
-iv ?RE
i i IRJ.
oi* I
, POST
IIIJ.
FIG. 2. The graph shows the rectal temperature (mean k SD) and sleep/awake periods (mean f SE) simultaneously recorded from the groups of rats Before and after injection of saline (0.2 ~1) and norepinephrine (NE) (3.0 fig in 0.2 gl) in the mPOA during the day and night. Hatched area shows the postinjection alteration in rectal temperature. Shaded and unshaded areas represent awake and sleep periods, respectively. *P < 0.05, **P < 0.0 1, and ***P -C0.00 1, levels of significance of the change compared with values during the identical time points of control saline injection.
total duration of REM sleep within the postinjection recordings ranged from 2 min to 6 min. Injection of PBZ in sleeping rats during the day also produced an increase in the rectal temperature. The SWS:REM ratio in these
46
DATTA NIGHT
PIlENT
NIGHT
PI2
ET AL.
FIG. 3. The graph shows the rectal temperature (mean + SD) and sleep/awake periods (mean f SE) simultaneously recorded from the groups of rats before and atIer injection of PBZ ( 1.O ccg in 0.2 d) during the day and night. Phentolamine (PHENT, 10.0 a in 0.2 ~1) and 5-hydroxytryp tamine (5-HT, 10.0 pg in 0.2 rJ) were injected during the night. The hatched area shows the postinjection alteration in rectal temperature; shaded and unshaded are.as represent awake and sleep periods, respectively. *P-c 0.05,“cP < 0.0 l,and **+P < 0.001 levels of signiiicance of the change compared with the values obtained during identical time points of control saline injection.
animals was not much altered even after PBZ injection in the mPOA during their normal sleeping periods. Injection of PBZ in the anterior amygdala region and lateral to the mPOA in five animals, and in the posterior end of
BODY TEMPERATURE
AND SLEEP-WAKEFULNESS NIGHT
40.0
47
PBZ
I 36.0
-I
Y ‘“0 z 3bO2 t t 37.036.0
-
5’ 4
0
15
39
a
iy
io
05,
&
7&
Ss I
IPRE INJ.
POST INJ.
FIG. 4. The graph shows the rectal temperature and sleep/awake periods simultaneously recorded after injection of PBZ ( 1.Osg in 0.2 ~1) in one rat. Hatched area shows the postinjection alteration in rectal temperature compared with the mean preinjection reading. Shaded and unshaded areas represent awake and sleep periods, respectively.
the mPOA in one animal, did not produce any changesin body temperature and sleep-wakefulness. Injection of the &blocker proprandol (PROP) in the mPOA did not produce any significant changes in temperature and sleep-wakefulness. Injection-bound increases in rectal temperature and arousal, produced in the sleeping rats, were not found to be significant compared with saline-induced changes. Injection of 5-HT in the mPOA during the night produced a rise in rectal temperature which was similar in nature to the change induced after administration of PBZ and PHENT (Fig. 3). Unlike the a-adrenergic blockers, the 5-HT injection did not produce sleep. Although the electrophysiologic re-
48
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ET AL.
cordings showed arousal, the rats’ behavior showed inactivity. postures similar to those of the sleeping rats.
They assumed
DISCUSSION Application of NE in the mPOA induced hypothermia and arousal, whereas a-blockers produced hyperthermia and sleep. Changes in sleepwakefulness obtained on application of NE and its blockers would not have contributed toward the thermal alterations. A decrease in body temperature is not associated with arousal nor is an increase associated with sleep (8, 9). Similarly the induced arousal and sleep are not likely to be totally dependent on thermal changes. Reports available in the literature indicate that reduction in environmental or preoptic-anterior hypothalamic temperature can bring about reduction in sleep (17,2 1,24). A local increase in preoptic-anterior hypothalamic temperature has a sleep-inducing effect (5, 18, 20). It might be argued that the arousal observed after NE application and the sleep after a-blockers were the results of hypothermia and hyperthermia, respectively, which were induced simultaneously. But the arousal obtained in this study after application of NE during the day was observed not only during the initial hypothermia but also during the second hyperthermia of low magnitude. Similarly, the rats were aroused from sleep induced by a-blockers, even when the hyperthermia was persisting. It has been reported that REM sleep time is markedly altered by small alterations of environmental temperature (22). But in the present study we found that the SWS:REM ratio was not altered even during the hyperthermia produced by PBZ administered during the day. Alterations in local temperature also probably did not contribute to the observed changes in sleep-wakefulness. Applications of NE in the mPOA may produce an increase in local temperature (18). If we consider the increase in local temperature produced by NE as causing the changes in sleepwakefulness, the rats should have gone to sleep ($1820) rather than becoming aroused. Our findings suggest that the thermal and sleep-wakefulness responses were primarily elicited by independent mechanisms, though some contribution of the former to the latter cannot be ruled out. These conclusions were further supported by the observations obtained after 5-HT injection. The hyperthermia induced by 5-HT injection in the mPOA was not accompanied by any change in true sleep-wakefulness. Although the two responses elicited by NE and its a-blockers may not be interrelated, there is striking identity in the sites where they were elicited. Injection of NE and an a-blocker outside the mPOA did not produce any significant alterations in sleep-wakefulness and rectal temperature.
BODY TEMPERATURE
AND SLEEP-WAKEFULNESS
49
There were two instances of temperature changes of small magnitude which could be attributed to alterations in sleep-wakefulness. The small increase in body temperature immediately after application of saline, and that observed at a later stage after NE in sleeping rats, may be related to the induced arousal and activity. Earlier reports claimed an increase in body temperature after application of saline in the mPOA (1, 11, 23). The present study suggests that the increase in temperature might have resulted from arousal and activity induced by handling at the time of injection in the sleeping rats. There was no change in temperature in the rats which received saline injection during their normal waking period. Simultaneous recording of the two variables during the day and night also demonstrated that a pure hypothermia is the effect of administration of NE in the mPOA and not a biphasic response as reported (4,7, 19). The delayed increase in temperature of small magnitude was observed only in the day experiments. It is likely that the thermal and sleep-wakefulness responses elicited by NE were mediated through the q postsynaptic receptors (15). While interpreting the results obtained on application of adrenergic blockers, one must bear in mind the side effects of these drugs, although small doses were used in this study. The differences in the magnitude of the hypothermia SWS:REM ratio after administration of PBZ and PIIENT may be attributed to the differences in the dosage of the drug. REFERENCES 1. AVERY, D. D. 197 1. Intrahypothalamic adrenergic and cholinergic injection effects on temperature and ingestive behaviour in the rat. Neuropharmacology 10: 753-163. 2. BAGGA, N., G. S. CHHINA, V. MOHAN KUMAR, AND B. SINGH. 198 1. Mechanism of participation of medial preoptic area in the hippocampal inhibition of ovulation. Brain Res. 216: 444-448. 3. BAGGA, N., G. S. CHHINA, V. MOHAN KUMAR, AND B. SINGH. 1984. Chohnergic activation of medial preoptic area by amygdala for ovulation in rat. Physiol. Behav. 32: 45-48. 4. BECKMAN, A. L. 1970. Effect of i&a-hypothalamic norepinephrine on thermoregulatory responses in the rat. Am. J. Physiol. 218: 1596- 1604. 5. BENEDEK, G., F. OB~, JR., I;. SZEKERES, AND F. O&L. 1976. Cortical synchronization induced by thermal stimulation of the preoptic area in immobilized rats. Acta Physiol. Acad. Sci. Hung. 48: 65-12. 6. CRAWSUAW, L. I. 1972. Effects of intracerebral5-hydroxytryptamine injection on thermoregulation in rat. Physiol. Behav. 9: 133-140. 7. DATTA, S., V. MOHAN KUMAR, G. S. CHHINA, AND B. SINGH. 1985. Tonic activity of medial preoptic norepinephrine mechanism for body temperature maintenance in sleep ing and awake rats. Brain Rex Bull. 15: 447-45 1. 8. HELLER, H. C., AND S. F. GLQTZBACK. 1977. Thermoregulation during sleep and hibernation. Int. Rev. Physiol. 15: 147-188. 9. KLEITMAN, N., AND E. KLEXTMAN. 1953. Effect of non-twenty-four-hour routines of living on oral temperature and heart rate. J. Appl. Physiol. 6: 283-29 1,
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10. KUBIKOWSW, P., AND W. RE~ERSKI. 1969. Biogenic amines and body temperature regulation in the rats. Diss. Pharrn. Pharmacol. 21: 207-2 12. 11. LOMAX, P., R. S. FOSTER, AND W. E. KIRKPATRICK. 1969. Cholinergic and adrenergic interactions in the thermoregulatory centres of the rat. Brain Res. 15: 43 l-438. 12. MARLEY, E., AND J. F. WHELAN. 1975. Some central effects of 5-hydroxytryptamine in young chickens at and below thermoneutrality. Br. J. Pharmacol. 53: 37-41. 13. MCGINT~, D. J., AND M. B. STERMAN. 1968. Sleep suppression after basal forebrain lesions in the cat. Science 160: 1253-1255. 14. MOHAN KUMAR, V., S. DATTA, G. S. CHHINA, N. GANDHI, AND B. SINGH. 1984. Sleepawake responses elicited from medial preoptic area on application of norepinephrine and phenoxybenzamine in free moving rats. Brain Rex 322: 322-325. 15. MOHAN KUMAR, V., S. DAI-~A, G. S. CHHINA, AND B. SINGH. 1986. Alpha adrenergic system in medial preoptic area involved in sleep-wakefulness in rats. Brain Res. Bull. 16: 463-468. 16. MYERS, R. D. 1974. Temperature regulation. Pages 237-285 in R. D. MYERS, Ed., Handbook of Drug and Chemical Stimulation of the Brain. Von Nostrand-Reinhold, New York. 17. PARMEGGIANI, P. L. 1980. Temperature regulation during sleep: a study in homeostasis. Pages 97-143 in J. OREM AND C. D. BARNES, Eds. Physiology in Sleep. Academic Press, New York. 18. PARMEGGIANI, P. L., G. ZAMBONI, T. CIANCI, L. F. AGNATI, AND C. RICCI. 1974. Influence of anterior hypothalamic heating on the duration of fast wave sleep episodes. Electroencephalogr. Clin. Neurophysiol. 36: 465-470. 19. POOLE, J., AND J. D. STEPHENSON.1979. Effects of noradrenaline and carbachol on temperature regulation of rats. Br. J. Pharmacol. 65: 43-5 1. 20. ROBERTS, W. W., AND T. C. L. ROBINSON. 1969. Relaxation and sleep induced by warming of preoptic region and anterior hypothalamus in cats. Exp. Neural. 25: 282-294. 21. SCHMIDEK, W. R., K. HOSHINO, M. SCHMIDEK, AND C. TIMO-IARIA. 1972. Influence of environmental temperature on the sleep-wakefulness cycle in the rat. Physiol. Behav. 8: 363-371. 22. SZYMUSJAK, R., AND E. SATINO~. 1981. Maximal REM sleep time defines a narrower thermoneutral zone than does minimal metabolic rate. Physiol. Behav. 26: 687-690. 23. TSOUCARIS-KUPFFER, D., AND H. SCHMITT. 1972. Hypothermic effect of or-sympathomimetic agents and their antagonism by adrenergic and cholinergic blocking drugs. Neuropharmacologyll: 625-635. 24. VALATX, J. L., B. ROUSSEL, AND M. CURE. 1973. Sommeil et temperature c&brale du rat au course de l’exposition chronique en ambiance chaude. Brain Res. 55: 107-l 22. 25. YAMAGUCHI, N., T. J. MARCZYNSKI, AND G. M. LING. 1963. The effects of electrical and chemical stimulation of the preoptic region and some non-specific thalamic nuclei in unrestrained, waking animals. Electroencephalogr. Clin. Neurophysiol. 15: 154.