BrainResearch,541 (1991) !34-138
134
Elsevier
BRES 24526
Exposure to heat restores sleep in cats with preopticianterior hypothalamic cell loss Ronald SzymusiaklT2, Janice Danowski’ and Dennis McGinty1,3 ‘Department of Anatomy and Cell Biology, School of Medicine and ‘Veterans Administration Medical Center, Sepulveda, CA (U.S.A.), ‘Department of Psychology, University of California, Los Angeles, CA (U.S. A.)
(Accepted 30 October 1990) Key words: Preoptic area; Anterior hypothalamus;
Sleep mechanism; Slow-wave sleep; Thermoregulation
Evidence suggests that thermosensitive neurons of the preoptic/anterior hypothalamus (POAH) influence sleep- and arousal-regulating mechanisms. We examined the effects of POAH cell loss, produced by microinjection of neurotoxin (N-methyl-or_-aspartic acid), on sleep and thermoregulation in cats. Cats with bilateral POAH cell loss did not defend their body temperatures in the heat as effectively as normals, and did not initiate panting until brain temperatures rose to abnormally high levels. During 14 h polygraphic recordings of sleep-waking state conducted at an ambient temperature (T,) of 23 “C, POAH-damaged cats exhibited reduced sleep. Amounts of deep slow-wave sleep (SWM) were significantly less than prelesion values through 7 weeks postlesion; significant REM sleep deficits persisted for 5 weeks. However, these sleep disturbances were dramatically attenuated when cats were exposed to high T,s. During 6 h recordings at T,s of 13, 23, or 33 “C, total sleep time was greatest at 33 “C at both 2 and 4 weeks postlesion. At 4 weeks, amounts of SWS2 at 33 “C were similar to maximal prelesion values. Increased sleep at 33 “C was associated with elevated brain temperatures. The finding that, after POAH damage, abnormally high brain temperatures were required to elicit both panting and normal amounts of SWS suggests that impaired hypothalamic sensitivity to heat was responsible for both deficits. These results support the hypothesis that thermosensitive neurons participate in the tonic regulation of sleep and arousal.
The preoptic/anterior hypothalamic area (POAH) is a major thermosensitive region of the mammalian brain*, 22. Local warming or cooling of the POAH evokes appropriate autonomic and behavioral thermoregulatory responses”,28. This region also participates in the regulation of sleep and arousal, and bilateral POAH damage causes severe and persistent reductions in sleep’6*18, 26,27,37.Several findings indicate that sleep-regulating and thermoregulating functions of the POAH are closely related (see refs. 9, 19, 20, 38 for review). Importantly, local warming of the POAH can have potent, acute sleep-enhancing effects 3,23-25 . This has led to the hypothesis that thermosensitive elements of the POAH modulate brain mechanisms of sleep and arousa19~“~i9. Hypothalamic warm sensitive neurons may facilitate sleeppromoting mechanisms36s38, or act primarily to permit sleep via effects on brainstem arousal systems’. Most studies of the sleep-enhancing actions of hypothalamic warming have demonstrated only acute effects, although in one report hypothalamic warming increased slow-wave sleep (SWS) for several hours25. The present study attempted to examine more tonic effects of hypothalamic thermoregulatory mechanisms on sleep and arousal, and to determine if sleep disturbance evoked by Correspondence:
R. Szymusiak, Neurophysiology
POAH cell loss could be related to thermoregulatory deficits. Six adult cats (4 female, 2 male) were surgically prepared for chronic polygraphic sleep-wake recordings under pentobarbital anesthesia, according to standard techniques39. Each cat also received a miniature thermocouple probe (Type T) placed in the region of the posterior hypothalamus (A 10.0, L 2.5, H -4.O), and 4 POAH guide cannula for subsequent neurotoxin microinjections. Cannulas were implanted bilaterally at two A-P planes (A 15.0-14.0 and A 13.0-12.0), 1.5 mm lateral to the midline. Before and after surgery, cats were housed on a 12:12 h light-dark schedule at an ambient temperature (r,) of 23 + 2 “C. Pre- and postlesion sleep-waking states were polygraphically evaluated during a 14 h period (18.0008.00 h) at a T, of 23 f 0.5 “C. Each 30 s epoch of polygraph recordings was categorized as either waking, light SWS (SWS l), deep SWS (SWS 2), or REM sleep according to established criteria39. Effects of T, on sleep-wake amounts were assessed with additional 6 h polygraphic recordings (09.00-15.00 h) at each of three T,‘s; 13, 23, and 33 + 0.5 “C. Heat-defense abilities were evaluated by exposing cats
Research, V.A. Medical Center, Sepulveda, CA 91343, U.S.A.
135 to gradually increasing T,s. They were placed in the chamber at 22 “C and allowed to acclimate for 1 h. T, was
dissolved
then increased
tized with pentobarbital
in 3 “C steps at 40 min intervals
until a
final T, of 43 “C was reached. Polygraph recordings were taken continuously, and hypothalamic temperature (Thy) was recorded at 10 min intervals. During the final 5 min at each T, > 31 “C, cats were examined for the presence of open-mouthed panting. Cold defense abilities were assessed by exposing cats to gradually decreasing T,s, ranging
from 22 to 1 “C. During
the final 5 min at each
T,, the animal and the polygraph recording of the nuchal electromyogram were examined for the presence of shivering. Lesions were produced by microinjections of Nmethyl-DL-aspartic acid (NMDA; Sigma, St. Louis),
A g
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PRELESION 2 WK POSTLESION
0
5 s 5
is
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,0-O
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,o-• P
p
36373 1
I 4
I 7
saline
at a concentration
a more
dorsal
location
each of 4 sites. At both the sites, 1 ,~l of NMDA solution location (H -4.O-4.5), and a was also made at each site at
(H -2.0-2.5).
42
1 0
In one cat, this
dorsal injection was also 1.0 ,~l in volume. Every cat had extensive cell loss within
the medial
preoptic area and the anterior hypothalamic nucleus. The suprachiasmatic nucleus (SCN) was extensively, but not completely, damaged in two cats but this nucleus was largely spared in the remaining 4 cats. There was no relationship between extent of SCN damage and the severity of sleep or thermoregulatory disturbance. There was some loss of neurons within the supraoptic nucleus in all cats, but this occupied < 50% of the volume of the nucleus. Rostrally, 5 of 6 cats had some cell loss within the vertical limb of the diagonal bands of Broca. Caudally, 3 cats sustained partial cell loss in the dorsomedial nucleus. Tuberal portions of the periventricular nucleus and medial aspects of the ventromedial nucleus were damaged in 2 cats. The major thermoregulatory effect of POAH cell loss was an impaired heat defense response with an associated elevation in the Thy threshold for panting. Results of pre-
I I I , 1 I 1 , , , , , 10 13 16 19 22 25 26 31 34 37 40 43
TEMPERATURE
D
PRELESION 2 WKS POSTLESION
m
4 WKS POSTLESION
DWAKE
(‘C)
40
of 200
to 7.4. Cats were anesthe-
(30 mg/kg, i.p.). Microinjections
were placed bilaterally at rostra1 and caudal POAH was injected at a ventral second injection of 0.5 ~1
SWSl Ea sws2 I REM
m *
600
41
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-.+._o-
S
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0
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P
z 7
/
in sterile
nmol/,ul and the pH adjusted
5 z
400
2 F:
3oo 200
I
500
100
5 39
0
PRE
1ST 2ND
3RD
4TH
5TH
7TH
POSTLESIONWEEK SHIVERING THREHOLD
PANTING THRESHOLD
Fig. 1. A: pre- and postlesion effects of heat and cold exposure on brain temperature ( Tbr) and thresholds for shivering and panting in one cat. There was little change in the ability to regulate Tbr in the cold, or in the shivering threshold (onset of shivering indicated by S) at 2 weeks postlesion. In response to increasing ambient temperature (T,), Tbr rose more rapidly at 2 weeks postlesion, and the Tbr threshold for panting increased from 39.5 to 41.7 “C. B: mean (n = 6) pre- and postlesion Tbr thresholds for shivering and panting. There was no significant difference between prelesion and 2 week postlesion Tbr thresholds for shivering (paired r-test, P > 0.1). Thy thresholds for panting were significantly elevated above prelesion levels at both 2 and 4 weeks postlesion (paired I-tests; P < 0.011. ..
Fig. 2. Summary of pre- and postlesion amounts of waking and sleep from 14 h recordings at a T. of 23 + 0.5 “C. Repeated measures ANOVA indicated a significant effect of pre- and postlesion recording time on amounts of wakefulness (Fs,3,, = 7.80, P < O.Ol), with time spent awake being significantly greater than prelesion values from the 1st through the 5th postlesion week (stars indicate significantly different from corresponding prelesion value, Newman-Keuls test, P < 0.01). Amounts of SWSl did not vary with time of recording (F6,30 = 1.49, P > O.l), but amounts of SWS2 did vary significantly (F,,sO = 6.36, P < 0.01). Time spent in SWS2 remained significantly below prelesion values from the 1st through the 7th postlesion week. There was an overall effect on REM sleep time (Fs,30 = 6.68, P < O.Ol), with values at the first through the 5th postlesion week being significantly below prelesion levels.
136 and postlesion tests for thermoregulatory function in one cat are shown in Fig. 1A. In response to decreasing T,, pre- and postlesion Thys were maintained at comparable levels, and there was little change in the T,,,, at which shivering first occurred. However, upon exposure to increasing T,s, Thr rose more rapidly and to higher levels in the postlesion test, with the onset of panting occurring at a Thy of 41.7 “C, compared to a prelesion value of 39.5 “C. Fig. 1B demonstrates that the average Thy at which shivering occurred was unchanged at 2 weeks postlesion compared to prelesion values, while Thy associated with the initial onset of panting was significantly elevated at both 2 and 4 weeks postlesion. POAH cell loss resulted in significant and persistent reductions in deep SWS and REM sleep, in agreement with a previous report 26. Summaries of pre- and postlesion amounts of polygraphically defined sleep and waking states are shown in Fig. 2. Data are from 14 h continuous recordings conducted at 23 “C. Following POAH lesions, amounts of wakefulness were increased and amounts of REM sleep were significantly decreased from the 1st through the 5th postlesion week compared to correspond-
ing prelesion
values.
cantly reduced week.
Time
spent
in SWS2 was signifi-
from the first through
the 7th postlesion
Pre- and postlesion effects of 6 hours exposure to T,s of 13, 23, and 33 “C on sleep, waking and Thy are summarized in Fig. 3. In prelesion recordings, time spent awake was significantly less at 23 “C compared to values at the other T,s. However, at both 2 and 4 weeks postlesion, the lowest amounts of wakefulness were recorded at 33 “C. Prelesion amounts of SWS2 did not significantly vary with T,. In postlesion recordings, maxima1 amounts of SWM occurred at 33 “C; values at 4 weeks approached prelesion levels. Thus, at 4 weeks postlesion, the SWS2 deficit seen at the usual laboratory and vivarium T,, could be eliminated by raising the T, 10 “C. In prelesion recordings, REM sleep time was T,-dependent, with maximal amounts occurring at 23 “C. At 2 weeks postlesion, peak REM sleep time was recorded at 33 “C, while REM sleep time at 23 and 33 “C did not differ significantly from each other at 4 weeks postlesion. Improved sleep at 33 “C was associated with hyperthermia. At both 2 and 4 weeks postlesion, T,.+.sat
WAKING
PRE
2ND
PRE
4TH
POSTLESION WEEK
1
13%
..
PRE
m
23%
2ND
4TH
POSTLESION WEEK
BRAIN TEMPERATURE
REM SLEEP loo n
2ND
m
33%
4TH
POSTLESION WEEK
,)1 u
13Oc
PRE
m
23%
2ND
m
33%
4TH
POSTLESION WEEK
Fig. 3. Summary of effects of ambient temperature (T.) on waking, SWS2, REM sleep, and brain temperature (Tbr) before and after POAH neurotoxin lesions (n = 6). Minimal waking times shifted from 23 “C in prelesion recordings to 33 “C at 2 and 4 weeks postlesion. Amounts of SWS2 did not vary significantly with T, prior to POAH damage, but maximal amounts of this state were recorded at 33 “C during postlesion recordings. Peak REM sleep times occurred at 23 ‘“Cin prelesion recordings and, at 33 “C at 2 weeks postlesion. At 4 weeks, amounts of REM sleep at 23 and 33 “C did not differ significantly from each other. Prelesion Tbrs did not vary significantly with T,. At both 2 and 4 weeks postlesion, brain temperatures at 33 “C were significantly higher than values at the other conditions. * Indicates significantly different from all other values, Newman-Keuls; P < 0.01.
137 33 “C were significantly
higher than those at 13 or 23 “C,
and higher than prelesion
levels as well.
Our results agree with previous reports demonstrating that mechanical’s, electrolytic16,37, and neurotoxininduced26,27 lesions of the midline POAH cause severe and persistent reductions in sleep. In the present study, amounts of SWS and REM sleep were significantly reduced for 7 and 5 weeks, respectively. Insomnia was documented in 14 h continuous polygraphic recordings made at 23 “C. We emphasize that this was not an extreme temperature; the vivarium was maintained at 23 “C, and cats were well adapted to this T,. Also in agreement with previous reports, cats with POAH damage had thermoregulatory disturbances which were suggestive of a loss of warm sensitive neurons. In our study these consisted of a slightly higher than normal resting brain temperature, impaired ability to defend body temperature in the heat, and an associated elevation in the brain temperature at which panting initially occurred during progressive heat exposure. We were unable to demonstrate any cold defense impairment with the tests used here. Chronically elevated body temperatures have been reported following POAH damage in lesions of the several species 1,14,21,31,32,37.Electrolytic rostra1 midline hypothalamus in cats21 and dogsI can cause impaired heat defense ability with substantial sparing of the ability to defend body temperature in the cold. Elevated panting thresholds have been reported following such lesions in dogsi4. Warm-responsive neurons are the predominant thermosensitive cell type within the POAH4. Our results support the interpretation that these thermoregulatory disturbances reflect a lesioninduced impairment of POAH-mediated warm sensitivity, resulting in reduced thermal drive to heat loss effector mechanisms. What is new in the present study is the reversal of POAH lesion-induced insomnia by exposure to heat. Amounts of deep SWS and REM sleep increased significantly during 6 h exposures to a T, of 33 “C. Improved sleep was associated with elevated brain temperatures (Fig. 3). Thus, normal amounts of sleep could only be achieved in POAH-damaged cats when brain/body temperatures rose to higher than normal levels. This finding was similar to that seen in lesioninduced changes in heat loss effector mechanisms; i.e., higher than normal brain temperatures were required for activation of either panting or sleep. These results suggest that warm-sensitive elements of the midline 1 Andersson, B., Gale, C., Hokfelt, B. and Larsson, B., Acute and chronic effects of preoptic lesions, Actu Physiol. Stand., 65 (1965) 45-58. 2 Aschoff, J., Circadian control of body temperature, J. Therm. Biol., 8 (1983) 143-147.
POAH
normally
have facilitatory
anisms which function
to promote
which regulate heat loss. Several kinds of evidence
effects on brain mechsleep as well as those
support
the hypothesis
that
hypothalamic and/or extrahypothalamic thermosensitive neurons participate in the regulation of sleep (see refs. 9, 17, 19, 20, 38 for review). These include sleep-related reductions in body temperature which are independent of circadian temperature rhythms2**, activation of heat loss mechanisms and lowering of the thermal set point during and sleep 10,12,29, and the ability of whole-body6,‘3 hypothalamic3*23-25 warming to acutely promote sleep. Our current results further suggest that hypothalamic warm sensing elements exert tonic modulatory effects on sleep and arousal mechanisms, as POAH lesion-induced reductions in warm sensitivity were associated with persistent insomnia. Sleep was restored by exposure to heat, when increases in brain/body temperature could be expected to promote enhanced discharge in residual POAH warm-sensitive neurons, and/or activate extrahypothalamic thermosensing elements. While the evidence for a hypothalamic thermosensitive modulation of sleep and arousal is compelling, the anatomical and neurophysiological details of such a mechanism are not well characterized. Some evidence suggests that hypothalamic mechanisms function primarily to suppress activity in brainstem ascending activating systems. Electrical stimulation of the midline POAH can suppress discharge in neurons of the paramedian midbrain reticular formation15*30,34, as can local POAH warming’. In cats exhibiting insomnia after POAH neurotoxin lesions, microinjection of the GABAergic agonist muscimol into the rostra1 midbrain/posterior hypothalamus results in acute restoration of sleep”, suggesting a tonic disinhibition of these activating structures in the POAH-damaged cat. Another possibility is that POAH warm-sensing neurons exert relatively direct facilitatory effects on sleep promoting mechanisms. Preliminary evidence from our laboratory indicates that local POAH warming can cause increased waking discharge in SWS-active neurons of the lateral-ventral basal forebrain33,35,36. The mechanistic details of the interactions among hypothalamic and extrahypothalamic thermoregulatory systems, and those brain regions regulating sleep and arousal are important topics for future research.
Supported by the Veterans Administration
and USHHS NS22127.
3 Benedek, G., Obal, F., Jr., Lelkes, Z. and Obal, F., Thermal and chemical stimulations of the hypothalamic heat detectors: the effects on the EEG, Acta. Physiol. Acad. Sci. Hung., 60 (1982) 27-35. 4 Boulant, J.A.
and Dean, J.B., Temperature
receptors
in the
138 central nervous system, Annu. Rev. Physiol., 48 (1986) 639-654. F., Preoptic hypnogenic area and reticular activating system, Arch. Ital. Biol., 111 (1973) 85-111. Bunnell, D.E., Agnew, J.A., Horvath, SM., Jopson, L. and Wills, M., Passive body heating and sleep: influence of proximity to sleep, Sleep, 11 (1988) 210-219. DeArmond, S.J. and Fusco, M.M., The effect of preoptic warming on the arousal system of the mesencephalic reticular formation, Exp. Neurol., 33 (1971) 653-670. Gillberg, M. and Akerstedt, T., Body temperature and sleep at different times of day, Sleep, 5 (1982) 378-388. Glotzbach, S. and Heller, H.C., Thermoregulation. In M.H. Kryger, T. Roth and W.C. Dement (Eds.), Principles and Practice of Sleep Medicine, W.B. Saunders, Philadelphia, 1990, pp. 300-309. Glotzbach, S.F. and Heller, H.C., Central nervous regulation of body temperature during sleep, Science, 194 (1976) 537-539. Hammel, H.T., Hardy, J.D. and Fusco, M.M., Thermoregulatory responses to hypothalamic cooling in unanesthetized dogs,
local warming of the preoptic
5 Bremer,
6
7
8 9
10 11
Am. J. Physioi.,
Psycho/.,
W.W. and Robinson, T.C.L., Relaxation and sleep induced by warming of the preoptic region and anterior hypothalamus in cats, Exp. Neural., 25 (1969) 282-294. 25 Sakaguchi, S., Glotzbach, S.F. and Heller, H.C., Influence of hypothalamic and ambient temperatures on sleep in kangaroo rats, Am. J. Physiol., 237 (1979) RSO-R88. 26 Sallanon, M., Kitahama, K., Denoyer, M., Gay, N. and Jouvet, M., Insomnie de longue duree aprbs lesions des perykarions de I’aire preoptique paramediane chez le chat, C.R. Acad. Sci. Paris, 303 (1986) 403-409. 27 Sallanon, M., Sakai, K., Denoyer,
28 29
12 Heller,
perature:
15
16 17
18 19
20
21
Its Measurement
and Control
in Science and Industry,
Reinhold, New York, 1963, pp. 181-215. Lineberry, G.C. and Siegel, J., EEG synchronized, behavioral inhibition, and mesencephalic unit effects produced by stimulation of orbital frontal cortex, basal forebrain and caudate nucleus, Brain Research, 34 (1971) 143-161. McGinty, D. and Sterman, M.B., Sleep suppression after basal forebrain lesions in the cat, Science, 160 (1968) 1253-1255. McGinty, D. and Szymusiak, R., Keeping cool: a hypothesis about the mechanisms and functions of slow wave sleep, Trends Neurosci., in press.. Nauta, W.J.H., Hypothalamic regulation of sleep in rats. An experimental study, J. Neurophysiol., 9 (1946) 285-316. Obal, F., Jr., Thermoregulation during sleep, Exp. Brain. Res., Suppl. 8 (1984) 157-172. Parmeggiani, P.L., Temperature regulation during sleep: a study in homeostasis. In J. Orem and C.D. Barnes (Eds.), Physiology in Sleep, Academic Press, New York, 1980, pp. 97-143. Ranson, S.W., Regulation of body temperature, Proc. Sot.
Assoc. Res. Nerv. Ment. Dis., 20 (1940) 342-372. 22 Reaves, T.A., Jr. and Hayward, J.N., Hypothalamic
30
31
M. and Jouvet, M., Long lasting insomnia induced by preoptic neuron lesions and its transient reversal by muscimol injection into the posterior hypothalamus, J. Neurosci., 32 (1989) 669-683. Satinoff, E., Behavioral thermoregulation in response to local cooling of the rat brain, Am. J. Physiol., 206 (1964) 1389-1394. Sewitch, D.E., Dinges, D.F., Kribbs, N.B., Powell, J. W., Orne, E.C. and Orne, M.T., Sleep is used as a primary physiological process for circadian heat loss, Sieep Res., 19 (1990) 406. Siegel, J. and Wang, R.Y., Electroencephalographic, behavioral, and single unit effects produced by stimulation of forebrain inhibitory structures in cats, Exp. Neural., 42 (1974) 28-50. Squires, R.D. and Jacobson, F.H., Chronic deficits of temperature regulation produced in cats by preoptic lesions, Am. J.
Physioi., 214 (1968) 549-560. 32 Szymusiak, R., DeMory, A., Kittrell, E.M.W. and Satinoff, E.,
Diurnal changes in thermoregulatory behavior in rats medial preoptic lesions, Am. J. Physiol., 249 (1985) R219-R227. 33 Szymusiak, R. and McGinty, D., Sleep-related neuronal discharge in the basal forebrain of cats, Brain Research, 370 (1986) 82-92. 34 Szymusiak,
R. and McGinty, D., Effects of basal forebrain stimulation on the waking discharge of neurons in the midbrain reticular formation of cats, Brain Research, 498 (1989a) pp.
355-359. 35 Szymusiak,
36
37
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and centers. In P. Lomax and
extrahypothalamic thermoregulatory E. Schenbaum (Eds.), Body Temperature: Regulation. Drug Effects, and Therapeutic Implications, Marcel Dekker, New York, 1979, pp. 39-70. 23 Roberts, W.W., Bergquist, E.H. and Robinsan, T.C.L., Thermoregulatory grooming and sleep-like relaxation induced by
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H.C., Glotzbach, S., Grahn, D. and Radeke, C., Sleep-dependent changes in the thermoregulatory system. In R. Lydic and J.F. Biebuyck (Eds.), Clinical Physiology of Sleep, Am. Physiol. Sot., Bethesda, 1988, pp. 145-158. 13 Horne, J.A. and Staff, L.E.H., Exercise and sleep: body heating effects, Sleep, 6 (1983) 36-46. 14 Keller, A.D., Temperature regulation disturbances in dogs following hypothalamic ablations. In J.D. Hardy (Ed.), Tem-
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39
R. and McGinty, D., Sleep-waking discharge of basal forebrain projection neurons in cats, Brain Res. Bull., 22 (1989b) 423-430. Szymusiak, R. and McGinty, D., State-dependent neurophysiology of the basal forebrain: relationship to sleep, arousal, and thermoregulatory function. In M. Mancia and G. Marini (Eds.), The Diencephalon and Sleep, Raven, New York, in press.. Szymusiak, R. and Satinoff, E., Ambient temperature-dependence of sleep disturbances produced by basal forebrain damage in rats, Brain Res. Bull., 12 (1984) 295-305. Szymusiak, R. and Satinoff, E., Thermal influences on basal forebrain hypnogenic mechanisms. In D.J. McGinty, R. Drucker-Cohn, A. Morrison, and P.L. Parmeggiani (Eds.), Brain Mechanisms of Sleep, Raven, New York, 1985, pp. 301-320. Ursin, R. and Sterman, M.B., A Manual for Standard Scoring of Sleep and Waking States in the Adult Cat, UCLA Publications Services Dept., Los Angeles, 1981.
134
Elsevier
BRES 24526
Exposure to heat restores sleep in cats with preopticianterior hypothalamic cell loss Ronald SzymusiaklT2, Janice Danowski’ and Dennis McGinty1,3 ‘Department of Anatomy and Cell Biology, School of Medicine and ‘Veterans Administration Medical Center, Sepulveda, CA (U.S.A.), ‘Department of Psychology, University of California, Los Angeles, CA (U.S. A.)
(Accepted 30 October 1990) Key words: Preoptic area; Anterior hypothalamus;
Sleep mechanism; Slow-wave sleep; Thermoregulation
Evidence suggests that thermosensitive neurons of the preoptic/anterior hypothalamus (POAH) influence sleep- and arousal-regulating mechanisms. We examined the effects of POAH cell loss, produced by microinjection of neurotoxin (N-methyl-or_-aspartic acid), on sleep and thermoregulation in cats. Cats with bilateral POAH cell loss did not defend their body temperatures in the heat as effectively as normals, and did not initiate panting until brain temperatures rose to abnormally high levels. During 14 h polygraphic recordings of sleep-waking state conducted at an ambient temperature (T,) of 23 “C, POAH-damaged cats exhibited reduced sleep. Amounts of deep slow-wave sleep (SWM) were significantly less than prelesion values through 7 weeks postlesion; significant REM sleep deficits persisted for 5 weeks. However, these sleep disturbances were dramatically attenuated when cats were exposed to high T,s. During 6 h recordings at T,s of 13, 23, or 33 “C, total sleep time was greatest at 33 “C at both 2 and 4 weeks postlesion. At 4 weeks, amounts of SWS2 at 33 “C were similar to maximal prelesion values. Increased sleep at 33 “C was associated with elevated brain temperatures. The finding that, after POAH damage, abnormally high brain temperatures were required to elicit both panting and normal amounts of SWS suggests that impaired hypothalamic sensitivity to heat was responsible for both deficits. These results support the hypothesis that thermosensitive neurons participate in the tonic regulation of sleep and arousal.
The preoptic/anterior hypothalamic area (POAH) is a major thermosensitive region of the mammalian brain*, 22. Local warming or cooling of the POAH evokes appropriate autonomic and behavioral thermoregulatory responses”,28. This region also participates in the regulation of sleep and arousal, and bilateral POAH damage causes severe and persistent reductions in sleep’6*18, 26,27,37.Several findings indicate that sleep-regulating and thermoregulating functions of the POAH are closely related (see refs. 9, 19, 20, 38 for review). Importantly, local warming of the POAH can have potent, acute sleep-enhancing effects 3,23-25 . This has led to the hypothesis that thermosensitive elements of the POAH modulate brain mechanisms of sleep and arousa19~“~i9. Hypothalamic warm sensitive neurons may facilitate sleeppromoting mechanisms36s38, or act primarily to permit sleep via effects on brainstem arousal systems’. Most studies of the sleep-enhancing actions of hypothalamic warming have demonstrated only acute effects, although in one report hypothalamic warming increased slow-wave sleep (SWS) for several hours25. The present study attempted to examine more tonic effects of hypothalamic thermoregulatory mechanisms on sleep and arousal, and to determine if sleep disturbance evoked by Correspondence:
R. Szymusiak, Neurophysiology
POAH cell loss could be related to thermoregulatory deficits. Six adult cats (4 female, 2 male) were surgically prepared for chronic polygraphic sleep-wake recordings under pentobarbital anesthesia, according to standard techniques39. Each cat also received a miniature thermocouple probe (Type T) placed in the region of the posterior hypothalamus (A 10.0, L 2.5, H -4.O), and 4 POAH guide cannula for subsequent neurotoxin microinjections. Cannulas were implanted bilaterally at two A-P planes (A 15.0-14.0 and A 13.0-12.0), 1.5 mm lateral to the midline. Before and after surgery, cats were housed on a 12:12 h light-dark schedule at an ambient temperature (r,) of 23 + 2 “C. Pre- and postlesion sleep-waking states were polygraphically evaluated during a 14 h period (18.0008.00 h) at a T, of 23 f 0.5 “C. Each 30 s epoch of polygraph recordings was categorized as either waking, light SWS (SWS l), deep SWS (SWS 2), or REM sleep according to established criteria39. Effects of T, on sleep-wake amounts were assessed with additional 6 h polygraphic recordings (09.00-15.00 h) at each of three T,‘s; 13, 23, and 33 + 0.5 “C. Heat-defense abilities were evaluated by exposing cats
Research, V.A. Medical Center, Sepulveda, CA 91343, U.S.A.
135 to gradually increasing T,s. They were placed in the chamber at 22 “C and allowed to acclimate for 1 h. T, was
dissolved
then increased
tized with pentobarbital
in 3 “C steps at 40 min intervals
until a
final T, of 43 “C was reached. Polygraph recordings were taken continuously, and hypothalamic temperature (Thy) was recorded at 10 min intervals. During the final 5 min at each T, > 31 “C, cats were examined for the presence of open-mouthed panting. Cold defense abilities were assessed by exposing cats to gradually decreasing T,s, ranging
from 22 to 1 “C. During
the final 5 min at each
T,, the animal and the polygraph recording of the nuchal electromyogram were examined for the presence of shivering. Lesions were produced by microinjections of Nmethyl-DL-aspartic acid (NMDA; Sigma, St. Louis),
A g
42-
V k
41 -
0-O O-O
P
PRELESION 2 WK POSTLESION
0
5 s 5
is
40-
,0-O
c~~o-o_~_o_o~o-o~O 39”-o~~-o-o-o~~
,o-• P
p
36373 1
I 4
I 7
saline
at a concentration
a more
dorsal
location
each of 4 sites. At both the sites, 1 ,~l of NMDA solution location (H -4.O-4.5), and a was also made at each site at
(H -2.0-2.5).
42
1 0
In one cat, this
dorsal injection was also 1.0 ,~l in volume. Every cat had extensive cell loss within
the medial
preoptic area and the anterior hypothalamic nucleus. The suprachiasmatic nucleus (SCN) was extensively, but not completely, damaged in two cats but this nucleus was largely spared in the remaining 4 cats. There was no relationship between extent of SCN damage and the severity of sleep or thermoregulatory disturbance. There was some loss of neurons within the supraoptic nucleus in all cats, but this occupied < 50% of the volume of the nucleus. Rostrally, 5 of 6 cats had some cell loss within the vertical limb of the diagonal bands of Broca. Caudally, 3 cats sustained partial cell loss in the dorsomedial nucleus. Tuberal portions of the periventricular nucleus and medial aspects of the ventromedial nucleus were damaged in 2 cats. The major thermoregulatory effect of POAH cell loss was an impaired heat defense response with an associated elevation in the Thy threshold for panting. Results of pre-
I I I , 1 I 1 , , , , , 10 13 16 19 22 25 26 31 34 37 40 43
TEMPERATURE
D
PRELESION 2 WKS POSTLESION
m
4 WKS POSTLESION
DWAKE
(‘C)
40
of 200
to 7.4. Cats were anesthe-
(30 mg/kg, i.p.). Microinjections
were placed bilaterally at rostra1 and caudal POAH was injected at a ventral second injection of 0.5 ~1
SWSl Ea sws2 I REM
m *
600
41
oG ;
/O’
-.+._o-
S
AMBIENT
B
0
0’
P
z 7
/
in sterile
nmol/,ul and the pH adjusted
5 z
400
2 F:
3oo 200
I
500
100
5 39
0
PRE
1ST 2ND
3RD
4TH
5TH
7TH
POSTLESIONWEEK SHIVERING THREHOLD
PANTING THRESHOLD
Fig. 1. A: pre- and postlesion effects of heat and cold exposure on brain temperature ( Tbr) and thresholds for shivering and panting in one cat. There was little change in the ability to regulate Tbr in the cold, or in the shivering threshold (onset of shivering indicated by S) at 2 weeks postlesion. In response to increasing ambient temperature (T,), Tbr rose more rapidly at 2 weeks postlesion, and the Tbr threshold for panting increased from 39.5 to 41.7 “C. B: mean (n = 6) pre- and postlesion Tbr thresholds for shivering and panting. There was no significant difference between prelesion and 2 week postlesion Tbr thresholds for shivering (paired r-test, P > 0.1). Thy thresholds for panting were significantly elevated above prelesion levels at both 2 and 4 weeks postlesion (paired I-tests; P < 0.011. ..
Fig. 2. Summary of pre- and postlesion amounts of waking and sleep from 14 h recordings at a T. of 23 + 0.5 “C. Repeated measures ANOVA indicated a significant effect of pre- and postlesion recording time on amounts of wakefulness (Fs,3,, = 7.80, P < O.Ol), with time spent awake being significantly greater than prelesion values from the 1st through the 5th postlesion week (stars indicate significantly different from corresponding prelesion value, Newman-Keuls test, P < 0.01). Amounts of SWSl did not vary with time of recording (F6,30 = 1.49, P > O.l), but amounts of SWS2 did vary significantly (F,,sO = 6.36, P < 0.01). Time spent in SWS2 remained significantly below prelesion values from the 1st through the 7th postlesion week. There was an overall effect on REM sleep time (Fs,30 = 6.68, P < O.Ol), with values at the first through the 5th postlesion week being significantly below prelesion levels.
136 and postlesion tests for thermoregulatory function in one cat are shown in Fig. 1A. In response to decreasing T,, pre- and postlesion Thys were maintained at comparable levels, and there was little change in the T,,,, at which shivering first occurred. However, upon exposure to increasing T,s, Thr rose more rapidly and to higher levels in the postlesion test, with the onset of panting occurring at a Thy of 41.7 “C, compared to a prelesion value of 39.5 “C. Fig. 1B demonstrates that the average Thy at which shivering occurred was unchanged at 2 weeks postlesion compared to prelesion values, while Thy associated with the initial onset of panting was significantly elevated at both 2 and 4 weeks postlesion. POAH cell loss resulted in significant and persistent reductions in deep SWS and REM sleep, in agreement with a previous report 26. Summaries of pre- and postlesion amounts of polygraphically defined sleep and waking states are shown in Fig. 2. Data are from 14 h continuous recordings conducted at 23 “C. Following POAH lesions, amounts of wakefulness were increased and amounts of REM sleep were significantly decreased from the 1st through the 5th postlesion week compared to correspond-
ing prelesion
values.
cantly reduced week.
Time
spent
in SWS2 was signifi-
from the first through
the 7th postlesion
Pre- and postlesion effects of 6 hours exposure to T,s of 13, 23, and 33 “C on sleep, waking and Thy are summarized in Fig. 3. In prelesion recordings, time spent awake was significantly less at 23 “C compared to values at the other T,s. However, at both 2 and 4 weeks postlesion, the lowest amounts of wakefulness were recorded at 33 “C. Prelesion amounts of SWS2 did not significantly vary with T,. In postlesion recordings, maxima1 amounts of SWM occurred at 33 “C; values at 4 weeks approached prelesion levels. Thus, at 4 weeks postlesion, the SWS2 deficit seen at the usual laboratory and vivarium T,, could be eliminated by raising the T, 10 “C. In prelesion recordings, REM sleep time was T,-dependent, with maximal amounts occurring at 23 “C. At 2 weeks postlesion, peak REM sleep time was recorded at 33 “C, while REM sleep time at 23 and 33 “C did not differ significantly from each other at 4 weeks postlesion. Improved sleep at 33 “C was associated with hyperthermia. At both 2 and 4 weeks postlesion, T,.+.sat
WAKING
PRE
2ND
PRE
4TH
POSTLESION WEEK
1
13%
..
PRE
m
23%
2ND
4TH
POSTLESION WEEK
BRAIN TEMPERATURE
REM SLEEP loo n
2ND
m
33%
4TH
POSTLESION WEEK
,)1 u
13Oc
PRE
m
23%
2ND
m
33%
4TH
POSTLESION WEEK
Fig. 3. Summary of effects of ambient temperature (T.) on waking, SWS2, REM sleep, and brain temperature (Tbr) before and after POAH neurotoxin lesions (n = 6). Minimal waking times shifted from 23 “C in prelesion recordings to 33 “C at 2 and 4 weeks postlesion. Amounts of SWS2 did not vary significantly with T, prior to POAH damage, but maximal amounts of this state were recorded at 33 “C during postlesion recordings. Peak REM sleep times occurred at 23 ‘“Cin prelesion recordings and, at 33 “C at 2 weeks postlesion. At 4 weeks, amounts of REM sleep at 23 and 33 “C did not differ significantly from each other. Prelesion Tbrs did not vary significantly with T,. At both 2 and 4 weeks postlesion, brain temperatures at 33 “C were significantly higher than values at the other conditions. * Indicates significantly different from all other values, Newman-Keuls; P < 0.01.
137 33 “C were significantly
higher than those at 13 or 23 “C,
and higher than prelesion
levels as well.
Our results agree with previous reports demonstrating that mechanical’s, electrolytic16,37, and neurotoxininduced26,27 lesions of the midline POAH cause severe and persistent reductions in sleep. In the present study, amounts of SWS and REM sleep were significantly reduced for 7 and 5 weeks, respectively. Insomnia was documented in 14 h continuous polygraphic recordings made at 23 “C. We emphasize that this was not an extreme temperature; the vivarium was maintained at 23 “C, and cats were well adapted to this T,. Also in agreement with previous reports, cats with POAH damage had thermoregulatory disturbances which were suggestive of a loss of warm sensitive neurons. In our study these consisted of a slightly higher than normal resting brain temperature, impaired ability to defend body temperature in the heat, and an associated elevation in the brain temperature at which panting initially occurred during progressive heat exposure. We were unable to demonstrate any cold defense impairment with the tests used here. Chronically elevated body temperatures have been reported following POAH damage in lesions of the several species 1,14,21,31,32,37.Electrolytic rostra1 midline hypothalamus in cats21 and dogsI can cause impaired heat defense ability with substantial sparing of the ability to defend body temperature in the cold. Elevated panting thresholds have been reported following such lesions in dogsi4. Warm-responsive neurons are the predominant thermosensitive cell type within the POAH4. Our results support the interpretation that these thermoregulatory disturbances reflect a lesioninduced impairment of POAH-mediated warm sensitivity, resulting in reduced thermal drive to heat loss effector mechanisms. What is new in the present study is the reversal of POAH lesion-induced insomnia by exposure to heat. Amounts of deep SWS and REM sleep increased significantly during 6 h exposures to a T, of 33 “C. Improved sleep was associated with elevated brain temperatures (Fig. 3). Thus, normal amounts of sleep could only be achieved in POAH-damaged cats when brain/body temperatures rose to higher than normal levels. This finding was similar to that seen in lesioninduced changes in heat loss effector mechanisms; i.e., higher than normal brain temperatures were required for activation of either panting or sleep. These results suggest that warm-sensitive elements of the midline 1 Andersson, B., Gale, C., Hokfelt, B. and Larsson, B., Acute and chronic effects of preoptic lesions, Actu Physiol. Stand., 65 (1965) 45-58. 2 Aschoff, J., Circadian control of body temperature, J. Therm. Biol., 8 (1983) 143-147.
POAH
normally
have facilitatory
anisms which function
to promote
which regulate heat loss. Several kinds of evidence
effects on brain mechsleep as well as those
support
the hypothesis
that
hypothalamic and/or extrahypothalamic thermosensitive neurons participate in the regulation of sleep (see refs. 9, 17, 19, 20, 38 for review). These include sleep-related reductions in body temperature which are independent of circadian temperature rhythms2**, activation of heat loss mechanisms and lowering of the thermal set point during and sleep 10,12,29, and the ability of whole-body6,‘3 hypothalamic3*23-25 warming to acutely promote sleep. Our current results further suggest that hypothalamic warm sensing elements exert tonic modulatory effects on sleep and arousal mechanisms, as POAH lesion-induced reductions in warm sensitivity were associated with persistent insomnia. Sleep was restored by exposure to heat, when increases in brain/body temperature could be expected to promote enhanced discharge in residual POAH warm-sensitive neurons, and/or activate extrahypothalamic thermosensing elements. While the evidence for a hypothalamic thermosensitive modulation of sleep and arousal is compelling, the anatomical and neurophysiological details of such a mechanism are not well characterized. Some evidence suggests that hypothalamic mechanisms function primarily to suppress activity in brainstem ascending activating systems. Electrical stimulation of the midline POAH can suppress discharge in neurons of the paramedian midbrain reticular formation15*30,34, as can local POAH warming’. In cats exhibiting insomnia after POAH neurotoxin lesions, microinjection of the GABAergic agonist muscimol into the rostra1 midbrain/posterior hypothalamus results in acute restoration of sleep”, suggesting a tonic disinhibition of these activating structures in the POAH-damaged cat. Another possibility is that POAH warm-sensing neurons exert relatively direct facilitatory effects on sleep promoting mechanisms. Preliminary evidence from our laboratory indicates that local POAH warming can cause increased waking discharge in SWS-active neurons of the lateral-ventral basal forebrain33,35,36. The mechanistic details of the interactions among hypothalamic and extrahypothalamic thermoregulatory systems, and those brain regions regulating sleep and arousal are important topics for future research.
Supported by the Veterans Administration
and USHHS NS22127.
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and Dean, J.B., Temperature
receptors
in the
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