- Email: [email protected]
Activity of ventromedial hypothalamic neurons suppressing heart rate is associated with paradoxical sleep in the rat
Brain Research 797 Ž1998. 103–108
Research report
Activity of ventromedial hypothalamic neurons suppressing heart rate is associated with paradoxical sleep in the rat Michiru Hirasawa, Masugi Nishihara, Michio Takahashi
)
Department of Veterinary Physiology, Veterinary Medical Science, The UniÕersity of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan Accepted 24 March 1998
Abstract Cardiovascular change is one of the common features of paradoxical sleep. Our study offers evidence that one of the central areas regulating the circulation during sleep is the ventromedial nucleus of the hypothalamus ŽVMH.. We found a group of neurons in this hypothalamic nucleus of rats whose electrical activity was exclusively increased during paradoxical sleep, and was associated with a reduction in heart rate. The onset of this neural activity usually followed that of paradoxical sleep. The incidence and duration of paradoxical sleep was increased by means of microinjection of carbachol, a cholinergic agonist, into the pontine reticular formation, and the neural activity of the VMH still appeared in synchrony with carbachol-induced paradoxical sleep. These results suggest that the cholinergic paradoxical sleep-inducing mechanism in the pons facilitate the excitability of these neurons. We have previously shown that these VMH neurons suppress blood pressure and heart rate via inhibition of the vasomotor neurons in the medulla oblongata. Taken together, our findings suggest that a group of neurons in the VMH suppresses the circulatory system during paradoxical sleep. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Ventromedial nucleus of the hypothalamus; Paradoxical sleep; Cardiovascular regulation; Rat
1. Introduction The cardiovascular effects of sleep have been extensively studied. It has been shown that slow wave sleep is accompanied by hypotension, bradycardia, reduction in systemic vascular resistance and cardiac output, and therefore diminished workload on the heart w23x. In contrast, paradoxical sleep ŽPS. is known to be associated with disturbances in the normal integrative control over somatic and vegetative functions. During this stage of sleep, some characteristic changes in cardiovascular parameters are observed as a result of tonic sympathetic and phasic parasympathetic change and increased barosensitivity w2,5,19,24x. Resulting instability in cardiovascular system may upset the balance between the metabolic needs and the perfusion of vital organs, which is probably one of the reason why PS is a period of potential cardiovascular risk w25,26x. A better understanding of cardiovascular regulation by the central nervous system during sleep may serve to clarify why some cardiovascular pathologic events are related to this behavior. )
Corresponding author. Fax: [email protected]
q 81-3-3815-4266;
E-mail:
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 3 3 9 - 4
The ventromedial nucleus of the hypothalamus ŽVMH. has been recognized to play an important role in regulating the sympathetic nervous system w17,27,34,39x. In addition, it was shown that this nucleus is also involved in sleep regulation: VMH lesions result in a complex syndrome characterized by change in sleep pattern as well as disturbances of feeding behavior and endocrine function w8x, and PS was suppressed by serotonin Ž5-HT. receptor activation within the VMH by microinjection of selective inhibitor of 5-HT uptake w7x. These observations implicate this hypothalamic nucleus in autonomic regulation associated with sleep. Recently, we have reported that there exists a group of neurons in the VMH in the rat influencing blood pressure ŽBP. and heart rate ŽHR. w15,16x. These neurons show a spontaneous episodic activity of 1 to 4 min in duration at 15 to 30-min intervals in the light phase, but only seldom in the dark phase. This appears to be a true endogenous circadian rhythm, as the rhythm persists in the absence of light cues. These VMH neurons may serve to regulate the cardiovascular system during PS, because their activity pattern somewhat resembles the appearance pattern of PS episodes: the rat, being a nocturnal animal, sleeps mostly during the light phase, and PS replaces the slow wave
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sleep for several minutes at variable intervals Žabout 10 to 20 min in the rat.. We therefore hypothesized that the VMH is involved in cardiovascular regulation during PS, and attempted to determine whether this neural activity occurs in association with this specific stage of sleep.
2. Materials and methods 2.1. Animals All experiments were performed on adult male Wistar– Imamichi rats Ž400–490 g.. Animals were housed in a constant temperature Ž23 " 2C. animal room, illuminated between 0500 and 1900 h, and provided with rat chow and water ad libitum. 2.2. Electrode implantations All rats were chronically implanted with electrodes under sodium pentobarbital anaesthesia Ž5 mgr100 g b.wt, i.p.. for polygraphic recording of sleep–wake states, multiple unit activity ŽMUA. and HR. Electroencephalogram ŽEEG. was recorded by means of bipolar stainless steel screw electrodes, implanted to make contact with the dura mater in the frontal and occipital cortex of each hemisphere. Another screw electrode was fixed to serve as the ground. Two silver wire electrodes, insulated except for the tip formed into a sphere, were placed in one of the orbit for electrooculogram ŽEOG. recording w20x. Electromyogram ŽEMG. was monitored using steel wire electrodes positioned in both sides of the neck muscles.
MUA electrode assembly consisted of four 75 m m Teflon-insulated platinum Ž90%. –iridium Ž10%. wires encased in a stainless steel guide tube Ž650 m m in diameter., as previously described w28x. The DC resistance between stainless steel and each platinum–iridium electrode was 50–100 k V. The electrodes were implanted unilaterally to the left VMH Žcoordinates: A 6.0, L 0.8, D 1.5. w1x. All these electrodes were attached to a microconnector and mounted on the skull with dental cement. For monitoring of HR, two stainless steel wires for recording electrocardiogram, uninsulated at the tip, were implanted subcutaneously at the rostral and caudal end of sternum. The distal ends of the wires were tunnelled under the skin, exteriorized at the nape of the neck, and sutured to the skin.
2.3. Recording and identification of sleep–wake stages After a recovery period of 5 to 7 days, polygraphic recording was performed over 8 h during the light phase. MUA signals were amplified with low and high cutoff frequencies of 500 and 3000 Hz, respectively, and neural spikes were discriminated by their amplitude and the number of spikes was integrated for 1 s. Bursts of MUA ŽMUA volleys. were distinguished by 40% increase from the baseline value for more than 60 s. Those animals from which MUA volleys could be recorded were selected and used for further analysis. Assignment to sleep stages followed standard criteria: wakefulness was characterized by low-voltage fast EEG activity and high EMG amplitude;
Fig. 1. Representative sleep polygram and MUA of the VMH. MUA volleys Žhatched lines over MUA trace. always appeared in association with PS Žbars at the top of the trace.. Sleep was often observed to be interrupted by wakefulness after PS episodes.
M. Hirasawa et al.r Brain Research 797 (1998) 103–108
slow wave sleep was identified by high amplitude slow waves in the EEG; PS was characterized by low-voltage fast EEG, rapid eye movement which appeared as bursts of high amplitude EOG, and suppression of the EMG. 2.4. Microinjection of drug A guide cannula Ž23G. for microinjection of carbachol was implanted into the pontine reticular formation at the time of electrode implantations. After the recovery period, 8 h polygraphic recording was performed and only those rats from which MUA volleys could be recorded in the VMH were selected. More than a day later, control data was recorded for more than 1 h, then saline Ž0.1 m l; n s 3. or carbachol Žcarbamylcholine chloride, Sigma, 1 m gr0.1 m l; n s 5. was delivered over 1 min and the injection cannula was withdrawn after an additional 1 min. Signals were recorded for additional 1.5–3 h.
105
EEG desynchronization, accompanied or followed by rapid eye movement. Onset of MUA volleys usually delayed that of EEG desynchronization and rapid eye movement, and synchronized with or preceded that of decreases in HR during PS ŽFig. 2b.. The correlation between occurrence of MUA volleys in the VMH and PS episodes was further investigated by means of microinjection of a cholinergic receptor agonist carbachol into the pons, which is known to induce PS w11x. Carbachol increased the incidence of PS episodes, and total time of PS in the carbachol-treated group prolonged
2.5. Histological Õerification After completion of each experiment, the animal was anaesthetized with overdose of pentobarbital, anodal direct current Ž100 m A, 15 s. was passed through the MUA electrode tip to localize the recording site, and the animal was perfused with 10% formalin. The brain was removed and stored in 10% formalin for several days, then transferred to 10% formalin–10% sucrose solution and stored overnight or longer before cutting serial coronal sections of 50 m m. Sections were evaluated under a light microscope to localize the position of the MUA electrode tips and guide cannulas.
3. Results Seventy rats were implanted with the MUA recording electrodes within the VMH, and 23 Ž32.9%. of them showed the characteristic increases in MUA ŽMUA volleys.. Those with electrodes in the preoptic area Ž n s 29., the dorsomedial nucleus of the hypothalamus Ž n s 6. or the arcuate nucleus Ž n s 10. did not display any MUA volleys. PS episodes were characterized by EEG desynchronization, rapid eye movement and muscle atonia. MUA volleys in the VMH appeared 100% coincident with these PS episodes Ž154 episodesr23 rats. as shown in Fig. 1. Fig. 2a shows transitions of HR and MUA at the beginning and the end of PS: HR and MUA decreased and increased, respectively, in association with PS. HR decreased from 290.6 " 16.93 to 249.3 " 10.85 beatsrmin Žmean " S.E.. at the maximum change Ž p - 0.05, paired t-test.. It was considered onset of MUA volley when 40% increase in MUA was seen for 10 consecutive seconds. Onset of HR change was when HR started to decline and fluctuate. Beginning of PS episodes was characterized by
Fig. 2. Ža. Transition of HR and MUA during PS. HR and MUA were measured every 6 s during the transitions from slow wave sleep to PS Žonset of PS; time 0. and from PS to arousal Žend of PS; time 0., expressed as mean"S.E. for 17 PS episodes in five rats. Žb. Latencies of rapid eye movement ŽREM., MUA volley and HR change compared with onset of EEG desynchronization in PS. Solid symbols indicate individual data. Open symbols and bars show mean"S.E.
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Fig. 3. Representative drawing of MUA volleys during carbachol-induced PS. Solid and hatched bars at the top of the polygraphic traces and MUA trace indicate PS episodes and MUA volleys, respectively.
approximately 2.6 times as that in the saline injected control group. MUA volleys still appeared synchronously with carbachol-induced PS ŽFig. 3..
4. Discussion In the present study we have demonstrated for the first time the existence of a group of hypothalamic neurons which shows bursts of spontaneous electrical activity in association with PS. Our finding is interesting because the hypothalamic integrative activity has been known to be impaired during PS w9,10,30–32x. This neural activity was recordable only in 32.2% of the animals with MUA electrodes implanted within the VMH, which is probably because the VMH is not a uniform structure, but contains neurons of various functions w22,27,29,35x. Onset of MUA volleys was usually delayed compared with the onset of EEG desynchronization, suggesting that this neural activity of the VMH is not involved in initiation of PS. To test this further, microinjection of carbachol into the pons was performed. This procedure multipled the total duration of PS as previously reported w11x, and the appearance of MUA volleys was still correlated with PS episodes. These results suggest that the cholinergic mechanism in the pons which induces PS also facilitates the electrical activity of VMH neurons directly or indirectly. Thus, these MUA volleys are probably induced by a mechanism regulating PS in the brainstem. One possibility is disinhibition
from 5-HT neurons. The 5-HT system, which is known as one of the major sleep regulatory mechanisms, are active during waking, moderately active during slow wave sleep and silent during PS w3,4,14x. Also, the VMH is known to receive 5-HT innervation from the raphe nucleus w37,38x. Intracerebroventricular administration of 5-HT significantly suppressed MUA volleys, supporting this hypothesis Žunpublished observation.. Rapid eye movement was also observed in tight relation with MUA volleys: whether these phenomena have any causal relationships need further investigation to be elucidated. Furthermore, since the VMH was shown to be a brain structure which can affect the sleep pattern itself w7,8x, those neurons which show MUA volleys may in turn play some part in sleep regulation other than initiation of PS. On the other hand, the onset of MUA volleys was usually synchronized with or preceded that of decreases in HR during PS. Our previous study has shown that both HR and BP decreased when MUA volleys appeared and that stimulation of these neurons caused an identical decrease in BP w15x. These observations suggest that VMH neurons are responsible for the depression of the circulatory system at this stage of sleep. We have also found that this inhibitory control on the circulation by the VMH is mediated by the vasomotor neurons in the rostral ventrolateral medulla ŽRVL. w16x. Since the activity of vasomotor center in the RVL is critical for maintaining background sympathetic activity and normal levels of arterial pressure w6,12x, and vasomotor neurons in this area are known to synapse
M. Hirasawa et al.r Brain Research 797 (1998) 103–108
to preganglionic sympathetic neurons in the spinal cord w13,33x, it was suggested that the suppression of the circulatory system by the VMH is a result of inhibition of the vasomotor center and sympathetic activity. It has been reported that the rat shows HR decrease and BP increase during PS compared to those during slow wave sleep w19,21x. However, because hypotension during PS appears in baroreceptor denervated rat w19x, central influence on HR and BP are likely to be both inhibitory, but masked or even reversed by baroreceptor buffering. Although we did not measure BP in this study, MUA exhibiting neurons in the VMH may also have an influence on BP during PS, since these neurons were shown to lower BP under anaesthesia w15x. However, the cardiovascular influence of PS is more complex than a generalized inhibition of the sympathetic nervous system. During this stage of sleep, sympathetic drive decreases or increases depending on which peripheral vascular beds they project to w2,5,18,36x; which part the VMH is involved remains to be clarified. In conclusion, the present study found one of the inhibitory central areas regulating the circulatory system during PS, by showing the burst of activity of VMH neurons during PS. This neural activity in the VMH was suggested to be evoked by PS-inducing mechanism in the lower brainstem, which in turn suppresses the circulatory system by inhibiting the vasomotor center in the medulla. Our finding indicates that at least part of the impairment of sympathetic function observed during this phase of sleep is the result of active inhibition by the VMH.
Acknowledgements Authors are grateful to Dr. Q.J. Pittman for a critical evaluation of the manuscript. We also acknowledge Dr. H. Sei for helpful suggestions. M. Hirasawa is a recipient of JSPS Fellowships for Japanese Junior Scientists. This research was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan, and ‘Research for the Future’ Program, JSPS Ž97L00904..
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w5x T. Cianci, G. Zoccoli, P. Lenzi, C. Franzini, Loss of integrative control of peripheral circulation during desynchronized sleep, Am. J. Physiol. 261 Ž1991. R373–R377. w6x R.A.L. Dampney, Functional organization of central pathways regulating the cardiovascular system, Physiol. Rev. 74 Ž1994. 323–364. w7x J. Danguir, J.-L. Elghozi, Superfusion of clomipramine within the ventromedial hypothalamus selectively suppresses paradoxical sleep in freely moving rats, Brain Res. Bull. 15 Ž1985. 1–4. w8x J. Danguir, S. Nicolaidis, Sleep and feeding patterns in the ventromedial hypothalamic lesioned rat, Physiol. Behav. 21 Ž1978. 769– 777. w9x S.F. Glotzbach, H.C. Heller, Central nervous regulation of body temperature during sleep, Science 194 Ž1976. 537–539. w10x S.F. Glotzbach, H.C. Heller, Changes in thermal characteristics of hypothalamic neurons during sleep and wakefulness, Brain Res. 309 Ž1984. 17–26. w11x J.W. Gnadt, G. Vernon Pegram, Cholinergic brainstem mechanisms of REM sleep in the rat, Brain Res. 384 Ž1986. 29–41. w12x P.G. Guyenet, Role of the ventral medulla oblongata in blood pressure regulation, in: A.D. Loewy, K.M. Spyer ŽEds.., Role of the Ventral Medulla Oblongata in Blood Pressure Regulation, Oxford Univ. Press, New York, 1990, pp. 145–167. w13x J.R. Hansel, P.G. Guyenet, Electrophysiological characterization of putative C1 adrenergic neurons in the rat, Neuroscience 30 Ž1989. 199–214. w14x K.-D.S. Hilaire, H. Merica, J.M. Gaillard, The effects of indalpine —a selective inhibitor of 5-HT uptake-on rat paradoxical sleep, Eur. J. Pharmacol. 98 Ž1984. 413–418. w15x M. Hirasawa, M. Nishihara, M. Takahashi, Neural activity in the VMH associated with suppression of the circulatory system in rats, Physiol. Behav. 59 Ž1996. 1017–1023. w16x M. Hirasawa, M. Nishihara, M. Takahashi, The rostral ventrolateral medulla mediates suppression of the circulatory system in rats, Brain Res. 724 Ž1996. 186–190. w17x K. Itoi, N. Jost, E. Badoer, C. Tschope, C. Culman, T. Unger, Localization of the substance P-induced cardiovascular responses in the rat hypothalamus, Brain Res. 558 Ž1991. 123–126. w18x Y. Iwamura, S. Uchino, S. Ozawa, S. Torii, Somatosympathetic reflexes and paradoxical sleep, Brain Res. 3 Ž1966. 381–383. w19x L.F. Junquiera, E.M. Krieger, Blood pressure and sleep in the rat in normotension and neurogenic hypertension, J. Physiol. London 259 Ž1976. 725–735. w20x H. Kametani, H. Kawamura, Alterations in acetylcholine release in the rat hippocampus during sleep–wakefulness detected by intracerebral dialysis, Life Sci. 47 Ž1990. 421–426. w21x J. Lacombe, A. Nosjean, J.M. Meunier, R. Laguzzi, Computer analysis of cardiovascular changes during sleep–wake cycle in Sprague–Dawley rats, Am. J. Physiol. 254 Ž1988. H217–H222. w22x J. Ma, S. Aou, T. Hori, Ventromedial hypothalamus mediates stress-induced hypocalcemia via gastric vagus in rats, Brain Res. Bull. 34 Ž1994. 41–45. w23x G. Mancia, Autonomic modulation of the cardiovascular system during sleep, New Engl. J. Med. 328 Ž1993. 347–349. w24x G. Mancia, A. Zanchetti, Cardiovascular regulation during sleep, in: J. Orem, C.D. Barns, ŽEds.., Cardiovascular Regulation During Sleep, Academic Press, New York, 1980, pp. 2–50. w25x J. Marshal, Diurnal variation in occurrence of strokes, Stroke 8 Ž1977. 230–231. w26x J.E. Muller, G.H. Tofler, P.H. Ston, Circadian variation and triggers of onset of acute cardiovascular disease, Circulation 79 Ž1989. 733–743. w27x K. Narita, M. Nishihara, M. Takahashi, Concomitant regulation of running activity and metabolic change by the ventromedial nucleus of the hypothalamus, Brain Res. 642 Ž1994. 290–296. w28x M. Nishihara, Y. Mori, M.-J. Yoo, M. Takahashi, In vivo electrophysiological monitoring of the GnRH pulse generator in rats and goats, Methods Neurosci. 20 Ž1994. 114–126.
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w29x Y. Oomura, S. Aou, Y. Koyama, I. Fujita, H. Yoshimatsu, Central control of sexual behavior, Brain Res. Bull. 20 Ž1988. 863–870. w30x P.L. Parmeggiani, Behavioral phenomenology of sleep, Experientia 36 Ž1980. 6–11. w31x P.L. Parmeggiani, M. Calasso, T. Cianci, Respiratory effects of preoptic–anterior hypothalamic electrical stimulation during sleep in cats, Sleep NY 4 Ž1981. 71–82. w32x P.L. Parmeggiani, A. Azzaroni, D. Cevolani, G. Ferrari, Responses of anterior hypothalamic–preoptic neurons to direct thermal stimulation during wakefulness and sleep, Brain Res. 269 Ž1983. 382–385. w33x C.A. Ross, D.A. Ruggiero, T.H. Joh, D.H. Park, D.J. Reis, Rostral ventrolateral medulla: selective projections to the thoracic autonomic cell column from the region containing C1 adrenaline neurons, J. Comp. Neurol. 228 Ž1984. 168–185. w34x M. Saito, Y. Minokoshi, T. Shimazu, Accelerated norepinephrine turnover in peripheral tissues after ventromedial hypothalamic stimulation in rats, Brain Res. 481 Ž1989. 298–303. w35x N. Shimizu, Y. Oomura, C.R. Plata-Salaman, M. Morimoto, Hyperphagia and obesity in rats with bilateral ibotenic acid-induced le-
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sions of the ventromedial hypothalamic nucleus, Brain Res. 416 Ž1987. 153–156. V.K. Somers, M.E. Dyken, A.L. Mark, F.M. Abboud, Sympatheticnerve activity during sleep in normal subjects, New Engl. J. Med. 328 Ž1993. 303–307. H.W.M. Steinbusch, Distribution of serotonin-immunoreactivity in the central nervous system of the rat—cell bodies and terminals, Neuroscience 6 Ž1981. 557–618. H.W.M. Steinbusch, Localization of serotonin-like immunoreactivity in the central nervous system of the rat, with special references to the innervation of the hypothalamus, in: B. Haber, S. Gabay, M.R. Issidorides, S.G.A. Alivisatos ŽEds.., Localization of Serotonin-like Immunoreactivity in the Central Nervous System of the Rat, With Special References to the Innervation of the Hypothalamus, Plenum, New York, 1981, pp. 7–35. H. Yoshimatsu, M. Egawa, G.A. Bray, Sympathetic nerve activity after discrete hypothalamic injections of L-glutamate, Brain Res. 601 Ž1993. 121–128.
Research report
Activity of ventromedial hypothalamic neurons suppressing heart rate is associated with paradoxical sleep in the rat Michiru Hirasawa, Masugi Nishihara, Michio Takahashi
)
Department of Veterinary Physiology, Veterinary Medical Science, The UniÕersity of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan Accepted 24 March 1998
Abstract Cardiovascular change is one of the common features of paradoxical sleep. Our study offers evidence that one of the central areas regulating the circulation during sleep is the ventromedial nucleus of the hypothalamus ŽVMH.. We found a group of neurons in this hypothalamic nucleus of rats whose electrical activity was exclusively increased during paradoxical sleep, and was associated with a reduction in heart rate. The onset of this neural activity usually followed that of paradoxical sleep. The incidence and duration of paradoxical sleep was increased by means of microinjection of carbachol, a cholinergic agonist, into the pontine reticular formation, and the neural activity of the VMH still appeared in synchrony with carbachol-induced paradoxical sleep. These results suggest that the cholinergic paradoxical sleep-inducing mechanism in the pons facilitate the excitability of these neurons. We have previously shown that these VMH neurons suppress blood pressure and heart rate via inhibition of the vasomotor neurons in the medulla oblongata. Taken together, our findings suggest that a group of neurons in the VMH suppresses the circulatory system during paradoxical sleep. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Ventromedial nucleus of the hypothalamus; Paradoxical sleep; Cardiovascular regulation; Rat
1. Introduction The cardiovascular effects of sleep have been extensively studied. It has been shown that slow wave sleep is accompanied by hypotension, bradycardia, reduction in systemic vascular resistance and cardiac output, and therefore diminished workload on the heart w23x. In contrast, paradoxical sleep ŽPS. is known to be associated with disturbances in the normal integrative control over somatic and vegetative functions. During this stage of sleep, some characteristic changes in cardiovascular parameters are observed as a result of tonic sympathetic and phasic parasympathetic change and increased barosensitivity w2,5,19,24x. Resulting instability in cardiovascular system may upset the balance between the metabolic needs and the perfusion of vital organs, which is probably one of the reason why PS is a period of potential cardiovascular risk w25,26x. A better understanding of cardiovascular regulation by the central nervous system during sleep may serve to clarify why some cardiovascular pathologic events are related to this behavior. )
Corresponding author. Fax: [email protected]
q 81-3-3815-4266;
E-mail:
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 3 3 9 - 4
The ventromedial nucleus of the hypothalamus ŽVMH. has been recognized to play an important role in regulating the sympathetic nervous system w17,27,34,39x. In addition, it was shown that this nucleus is also involved in sleep regulation: VMH lesions result in a complex syndrome characterized by change in sleep pattern as well as disturbances of feeding behavior and endocrine function w8x, and PS was suppressed by serotonin Ž5-HT. receptor activation within the VMH by microinjection of selective inhibitor of 5-HT uptake w7x. These observations implicate this hypothalamic nucleus in autonomic regulation associated with sleep. Recently, we have reported that there exists a group of neurons in the VMH in the rat influencing blood pressure ŽBP. and heart rate ŽHR. w15,16x. These neurons show a spontaneous episodic activity of 1 to 4 min in duration at 15 to 30-min intervals in the light phase, but only seldom in the dark phase. This appears to be a true endogenous circadian rhythm, as the rhythm persists in the absence of light cues. These VMH neurons may serve to regulate the cardiovascular system during PS, because their activity pattern somewhat resembles the appearance pattern of PS episodes: the rat, being a nocturnal animal, sleeps mostly during the light phase, and PS replaces the slow wave
104
M. Hirasawa et al.r Brain Research 797 (1998) 103–108
sleep for several minutes at variable intervals Žabout 10 to 20 min in the rat.. We therefore hypothesized that the VMH is involved in cardiovascular regulation during PS, and attempted to determine whether this neural activity occurs in association with this specific stage of sleep.
2. Materials and methods 2.1. Animals All experiments were performed on adult male Wistar– Imamichi rats Ž400–490 g.. Animals were housed in a constant temperature Ž23 " 2C. animal room, illuminated between 0500 and 1900 h, and provided with rat chow and water ad libitum. 2.2. Electrode implantations All rats were chronically implanted with electrodes under sodium pentobarbital anaesthesia Ž5 mgr100 g b.wt, i.p.. for polygraphic recording of sleep–wake states, multiple unit activity ŽMUA. and HR. Electroencephalogram ŽEEG. was recorded by means of bipolar stainless steel screw electrodes, implanted to make contact with the dura mater in the frontal and occipital cortex of each hemisphere. Another screw electrode was fixed to serve as the ground. Two silver wire electrodes, insulated except for the tip formed into a sphere, were placed in one of the orbit for electrooculogram ŽEOG. recording w20x. Electromyogram ŽEMG. was monitored using steel wire electrodes positioned in both sides of the neck muscles.
MUA electrode assembly consisted of four 75 m m Teflon-insulated platinum Ž90%. –iridium Ž10%. wires encased in a stainless steel guide tube Ž650 m m in diameter., as previously described w28x. The DC resistance between stainless steel and each platinum–iridium electrode was 50–100 k V. The electrodes were implanted unilaterally to the left VMH Žcoordinates: A 6.0, L 0.8, D 1.5. w1x. All these electrodes were attached to a microconnector and mounted on the skull with dental cement. For monitoring of HR, two stainless steel wires for recording electrocardiogram, uninsulated at the tip, were implanted subcutaneously at the rostral and caudal end of sternum. The distal ends of the wires were tunnelled under the skin, exteriorized at the nape of the neck, and sutured to the skin.
2.3. Recording and identification of sleep–wake stages After a recovery period of 5 to 7 days, polygraphic recording was performed over 8 h during the light phase. MUA signals were amplified with low and high cutoff frequencies of 500 and 3000 Hz, respectively, and neural spikes were discriminated by their amplitude and the number of spikes was integrated for 1 s. Bursts of MUA ŽMUA volleys. were distinguished by 40% increase from the baseline value for more than 60 s. Those animals from which MUA volleys could be recorded were selected and used for further analysis. Assignment to sleep stages followed standard criteria: wakefulness was characterized by low-voltage fast EEG activity and high EMG amplitude;
Fig. 1. Representative sleep polygram and MUA of the VMH. MUA volleys Žhatched lines over MUA trace. always appeared in association with PS Žbars at the top of the trace.. Sleep was often observed to be interrupted by wakefulness after PS episodes.
M. Hirasawa et al.r Brain Research 797 (1998) 103–108
slow wave sleep was identified by high amplitude slow waves in the EEG; PS was characterized by low-voltage fast EEG, rapid eye movement which appeared as bursts of high amplitude EOG, and suppression of the EMG. 2.4. Microinjection of drug A guide cannula Ž23G. for microinjection of carbachol was implanted into the pontine reticular formation at the time of electrode implantations. After the recovery period, 8 h polygraphic recording was performed and only those rats from which MUA volleys could be recorded in the VMH were selected. More than a day later, control data was recorded for more than 1 h, then saline Ž0.1 m l; n s 3. or carbachol Žcarbamylcholine chloride, Sigma, 1 m gr0.1 m l; n s 5. was delivered over 1 min and the injection cannula was withdrawn after an additional 1 min. Signals were recorded for additional 1.5–3 h.
105
EEG desynchronization, accompanied or followed by rapid eye movement. Onset of MUA volleys usually delayed that of EEG desynchronization and rapid eye movement, and synchronized with or preceded that of decreases in HR during PS ŽFig. 2b.. The correlation between occurrence of MUA volleys in the VMH and PS episodes was further investigated by means of microinjection of a cholinergic receptor agonist carbachol into the pons, which is known to induce PS w11x. Carbachol increased the incidence of PS episodes, and total time of PS in the carbachol-treated group prolonged
2.5. Histological Õerification After completion of each experiment, the animal was anaesthetized with overdose of pentobarbital, anodal direct current Ž100 m A, 15 s. was passed through the MUA electrode tip to localize the recording site, and the animal was perfused with 10% formalin. The brain was removed and stored in 10% formalin for several days, then transferred to 10% formalin–10% sucrose solution and stored overnight or longer before cutting serial coronal sections of 50 m m. Sections were evaluated under a light microscope to localize the position of the MUA electrode tips and guide cannulas.
3. Results Seventy rats were implanted with the MUA recording electrodes within the VMH, and 23 Ž32.9%. of them showed the characteristic increases in MUA ŽMUA volleys.. Those with electrodes in the preoptic area Ž n s 29., the dorsomedial nucleus of the hypothalamus Ž n s 6. or the arcuate nucleus Ž n s 10. did not display any MUA volleys. PS episodes were characterized by EEG desynchronization, rapid eye movement and muscle atonia. MUA volleys in the VMH appeared 100% coincident with these PS episodes Ž154 episodesr23 rats. as shown in Fig. 1. Fig. 2a shows transitions of HR and MUA at the beginning and the end of PS: HR and MUA decreased and increased, respectively, in association with PS. HR decreased from 290.6 " 16.93 to 249.3 " 10.85 beatsrmin Žmean " S.E.. at the maximum change Ž p - 0.05, paired t-test.. It was considered onset of MUA volley when 40% increase in MUA was seen for 10 consecutive seconds. Onset of HR change was when HR started to decline and fluctuate. Beginning of PS episodes was characterized by
Fig. 2. Ža. Transition of HR and MUA during PS. HR and MUA were measured every 6 s during the transitions from slow wave sleep to PS Žonset of PS; time 0. and from PS to arousal Žend of PS; time 0., expressed as mean"S.E. for 17 PS episodes in five rats. Žb. Latencies of rapid eye movement ŽREM., MUA volley and HR change compared with onset of EEG desynchronization in PS. Solid symbols indicate individual data. Open symbols and bars show mean"S.E.
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Fig. 3. Representative drawing of MUA volleys during carbachol-induced PS. Solid and hatched bars at the top of the polygraphic traces and MUA trace indicate PS episodes and MUA volleys, respectively.
approximately 2.6 times as that in the saline injected control group. MUA volleys still appeared synchronously with carbachol-induced PS ŽFig. 3..
4. Discussion In the present study we have demonstrated for the first time the existence of a group of hypothalamic neurons which shows bursts of spontaneous electrical activity in association with PS. Our finding is interesting because the hypothalamic integrative activity has been known to be impaired during PS w9,10,30–32x. This neural activity was recordable only in 32.2% of the animals with MUA electrodes implanted within the VMH, which is probably because the VMH is not a uniform structure, but contains neurons of various functions w22,27,29,35x. Onset of MUA volleys was usually delayed compared with the onset of EEG desynchronization, suggesting that this neural activity of the VMH is not involved in initiation of PS. To test this further, microinjection of carbachol into the pons was performed. This procedure multipled the total duration of PS as previously reported w11x, and the appearance of MUA volleys was still correlated with PS episodes. These results suggest that the cholinergic mechanism in the pons which induces PS also facilitates the electrical activity of VMH neurons directly or indirectly. Thus, these MUA volleys are probably induced by a mechanism regulating PS in the brainstem. One possibility is disinhibition
from 5-HT neurons. The 5-HT system, which is known as one of the major sleep regulatory mechanisms, are active during waking, moderately active during slow wave sleep and silent during PS w3,4,14x. Also, the VMH is known to receive 5-HT innervation from the raphe nucleus w37,38x. Intracerebroventricular administration of 5-HT significantly suppressed MUA volleys, supporting this hypothesis Žunpublished observation.. Rapid eye movement was also observed in tight relation with MUA volleys: whether these phenomena have any causal relationships need further investigation to be elucidated. Furthermore, since the VMH was shown to be a brain structure which can affect the sleep pattern itself w7,8x, those neurons which show MUA volleys may in turn play some part in sleep regulation other than initiation of PS. On the other hand, the onset of MUA volleys was usually synchronized with or preceded that of decreases in HR during PS. Our previous study has shown that both HR and BP decreased when MUA volleys appeared and that stimulation of these neurons caused an identical decrease in BP w15x. These observations suggest that VMH neurons are responsible for the depression of the circulatory system at this stage of sleep. We have also found that this inhibitory control on the circulation by the VMH is mediated by the vasomotor neurons in the rostral ventrolateral medulla ŽRVL. w16x. Since the activity of vasomotor center in the RVL is critical for maintaining background sympathetic activity and normal levels of arterial pressure w6,12x, and vasomotor neurons in this area are known to synapse
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to preganglionic sympathetic neurons in the spinal cord w13,33x, it was suggested that the suppression of the circulatory system by the VMH is a result of inhibition of the vasomotor center and sympathetic activity. It has been reported that the rat shows HR decrease and BP increase during PS compared to those during slow wave sleep w19,21x. However, because hypotension during PS appears in baroreceptor denervated rat w19x, central influence on HR and BP are likely to be both inhibitory, but masked or even reversed by baroreceptor buffering. Although we did not measure BP in this study, MUA exhibiting neurons in the VMH may also have an influence on BP during PS, since these neurons were shown to lower BP under anaesthesia w15x. However, the cardiovascular influence of PS is more complex than a generalized inhibition of the sympathetic nervous system. During this stage of sleep, sympathetic drive decreases or increases depending on which peripheral vascular beds they project to w2,5,18,36x; which part the VMH is involved remains to be clarified. In conclusion, the present study found one of the inhibitory central areas regulating the circulatory system during PS, by showing the burst of activity of VMH neurons during PS. This neural activity in the VMH was suggested to be evoked by PS-inducing mechanism in the lower brainstem, which in turn suppresses the circulatory system by inhibiting the vasomotor center in the medulla. Our finding indicates that at least part of the impairment of sympathetic function observed during this phase of sleep is the result of active inhibition by the VMH.
Acknowledgements Authors are grateful to Dr. Q.J. Pittman for a critical evaluation of the manuscript. We also acknowledge Dr. H. Sei for helpful suggestions. M. Hirasawa is a recipient of JSPS Fellowships for Japanese Junior Scientists. This research was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan, and ‘Research for the Future’ Program, JSPS Ž97L00904..
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