Brain structures and mechanisms involved in the generation of NREM sleep: focus on the preoptic hypothalamus

Sleep Medicine Reviews, Vol. 5, No. 4, pp 323–342, 2001 doi:10.1053/smrv.2001.0170, available online at http://www.idealibrary.com on

SLEEP MEDICINE reviews

PHYSIOLOGICAL REVIEW

Brain structures and mechanisms involved in the generation of NREM sleep: focus on the preoptic hypothalamus Dennis McGinty and Ronald Szymusiak Departments of Psychology and Medicine, UCLA, Veterans Administration, Greater Los Angeles Health System, USA KEYWORDS preoptic hypothalamus, thermoregulation, NREM sleep, c-Fos, arousal systems, staterelated neuronal activity

Summary Four lines of research have greatly increased our understanding of the hypothalamic preoptic area (POA) sleep-promoting system. First, sleep-active neurons within the POA have been identified using both electrophysiological recording and immediate early gene protein (c-Fos) staining methods. Segregated sleep-active neurons were found in ventrolateral and median POA (VLPO and MnPN). Additional sleep-active neurons may be intermixed with non-sleep specific neurons in other POA regions and the adjacent basal forebrain. Second, the putative sleep factors, adenosine and prostaglandin D2, were found to excite sleep-active neurons. Other sleep factors may also modulate these sleep-active populations. Third, many sleepactive neurons are warm-sensitive neurons (WSNs). WSNs are identified by excitatory responses to small increases in local POA temperature. The same local POA thermal stimuli strongly modulate sleep propensity and EEG delta activity within sleep. Interactions between sleep regulation and thermoregulation are consistent with studies of circadian sleep propensity, prolonged sleep deprivation in rats, and species differences in sleep amounts. Fourth, sleep-active neurons were found to co-localize the inhibitory neurotransmitter,
-aminobutyric acid and to have projections to arousal-related neuronal subgroups in the posterior hypothalamus and midbrain. Sleep-active and arousal-related neurons exhibit reciprocal changes in discharge across the wake–NREM–REM cycle, and activation of WSNs suppresses the neuronal activity of some arousal-related neuronal groups. These studies establish mechanisms by which POA hypnogenic neurons can inhibit EEG and behavioral arousal. In addition, there is evidence that arousal-related neurotransmitters inhibit VLPO sleep-active neurons. Mutually inhibitory interactions between sleep-promoting and the arousal system provide a substrate for a ‘‘sleep–wake switch’’.  2001 Harcourt Publishers Ltd

INTRODUCTION This review is concerned with our current understanding of the hypothalamic mechanisms that facili-

Correspondence should be addressed to: VAGLAHS, 16111 Plummer St. (151A3), North Hills, CA 91343, USA. Fax: (818)895-9575; E-mail: [email protected] 1087–0792/01/040323+20 $35.00/0

tate mammalian sleep, particularly NREM sleep. More than 70 years ago, von Economo [1] proposed that sleep is regulated by opposing wake-promoting and sleep-promoting mechanisms localized in the hypothalamus. Many critical features of this model have been confirmed and refined by recent studies. This evidence will be reviewed in detail below, with particular emphasis on the properties of the sleeppromoting or hypnogenic system located in the preoptic area (POA) of the hypothalamus.  2001 Harcourt Publishers Ltd

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The hypothalamus can be subdivided into a periventricular zone, including nuclear subgroups regulating anterior and posterior pituitary function, a medial zone with distinct nuclear groups including the medial preoptic nucleus within the POA, and a lateral POA zone with few discrete nuclei [2]. A magnocellular zone of the lateral POA is related to the adjacent basal forebrain (BF) by the presence of acetylcholine-containing neurons that are not found in the medial hypothalamus [3]. However, all of these regions contain multiple cell phenotypes and functional subgroups that are not associated with previously defined nuclei. In this category is a recently described sleep-promoting area in the ventrolateral preoptic area (VLPO) which was defined by the sleep-related expression of the immediate early gene, c-fos, a marker of neuronal activation [4]. This site is discussed further below. Current evidence suggests that the POA sleeppromoting neurons may not be confined to particular nuclear sites, although this is controversial. A focus of current research is the further identification of critical sleep-regulating cellular phenotypes within this complex and functionally diverse region, but this work is just beginning. In this review, we will refer to the POA or to specific nuclei within this region, bearing in mind that adjacent BF sites may also be important.

THE POA HYPNOGENIC AREA Von Economo [1] proposed the existence of a POA sleep-promoting area because patients with encephalitis and severe insomnia before death were found post mortem to have inflammatory lesions in this area. Other patients with hypersomnia before death had lesions in the vicinity of the posterior hypothalamus. On the basis of these observations, he proposed the concept of opposing hypothalamic sleep-promoting and wake-promoting systems. The existence of a sleep-promoting mechanism in the POA has been confirmed by variety of methods. Experimental lesions of this area result in insomnia. Bilateral 1–2-mm diameter lesions of the POA in the rats [5] and cats [6] induce partial sleep loss sometimes followed by partial recovery, and larger bilateral 3–5-mm lesions or transections which extend into the adjacent BF induce total or near total insomnia sometimes leading to death [7–9]. Current evidence suggests that the size of the lesion within this region determines the magnitude of the sleep

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deficit. Lesions encompassing VLPO, but extending dorsally into the lateral POA produced an approximately 40% reduction in NREM sleep (rats) [10], but more dorsal lesions of the lateral POA, sparing the VLPO, produced an equivalent NREM deficit (cats) [11]. These studies suggest that components of the POA hypnogenic mechanism are distributed throughout the POA. Cell-selective neurotoxic lesions induce insomnia [12,13], showing that loss of local neurons rather than fibers of passage is critical. After POA lesions that resulted in partial sleep loss, implantation of healthy fetal POA tissue into the lesion site promoted recovery of normal sleep amounts [14]. This finding also supports the hypothesis that the hypnogenic process originates in POA neurons. Electrical [15], neurochemical, or thermal stimulation (see below) of the POA will elicit or increase sleep. Many sleep-promoting substances act in the POA. Using the local micro-injection method, the POA was found to be an effective sleep-enhancing site for administration of growth hormone releasing hormone (GHRH) [16], triazolam [17], prostaglandin D2 (PGD2) [18] and, in one study, for adenosine agonists [19]. The adjacent magnocellular BF was an effective site for adenosine agonists in other studies [20,21]. The subarachnoid space under the anterior POA was found to be the most effective hypnogenic site for PGD2 [22]. PGD2 induced activation of ‘‘sleep-active’’ neurons (see below) in the VLPO as well as other specific POA sites [23]. After medial POA lesions, the sleepenhancing effect of PGD2 administered in the third ventricle was diminished [24]. Several additional neurochemical agents increase NREM sleep after administration into the adjacent 3rd or lateral ventricles. A partial list includes cytokines and related molecules (interleukin-1 (Il-1), tumor necrosis factor (TNF-), muramyl dipeptide) [25], other peptides such as delta-sleep-inducing peptide [26], cortistatin [27], as well as oxidized glutathione [28], desacetyl--melanocyte-stimulating hormone (an ACTH derivative found in brain) [29], insulin [30], and a lipid, oleamide [31]. The specific neuronal phenotypes that are the targets of these injected substances are not established (see regulatory inputs section, below). However, it is logical to hypothesize that the POA sleep-facilitating neurons lost in lesion studies are the targets of these neurochemical factors. This hypnogenic neuronal population might be the final common path for several

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mediators of NREM sleep. The further identification of the POA ‘‘hypnogenic’’ neuronal population is a focus of the next sections of this review.

CSNs, and has no effect on the discharge of most POA neurons, this study supports the hypothesis that these subsets of POA neurons are critical to the hypnogenic mechanism, at least within the 3-h time frame of this experiment. Mild to moderate ambient temperature (Ta) elevation also increases coincident sleep [36,40–42] as well as subsequent sleep [43–45]. Augmentation of sleep by ambient warming in kangaroo rats was prevented by coincident local POA cooling [36]. Since POA cooling would prevent activation of WSNs, this finding suggests that POA WSN activation mediates the sleep augmentation induced by ambient warming. It is established that sleep onset is coupled to heat loss processes. Most studies show an evoked fall in body temperature at sleep onset that is superimposed on the circadian temperature rhythm [46], although little temperature decline was found in women taking hormonal contraceptives [47]. The control of sleep relative to the circadian temperature rhythm is discussed further below. Depending on ambient conditions, sleep onset evokes heat loss effector processes such as cutaneous vasodilation and sweating [48]. In humans, sleep onset occurs closely in time after a discrete increase in vasodilation of the hands and feet [49] and, in rats, vasodilation of the tail is observed at sleep onset [50]. Vasodilation of these skin surfaces promotes heat loss in these species. In humans, if ‘‘lights-out’’ is scheduled, sleep latency is shorter if this vasodilation has occurred somewhat before ‘‘lights out’’ [49]. Self-selected human bedtimes are predicted by the maximum rate of decline of core body temperature [51]. These studies are compatible with a hypothesis that sleep onset is coupled to a process that induces body cooling. Although moderate ambient warming facilitates sleep (see above), higher ambient temperatures suppress sleep [52]. Sleep may be suppressed at high ambient temperatures because heat loss through skin and other body cooling mechanisms are ineffective. The best behavioral strategy would be escape from the hot environment rather than sleep [53].

THERMOREGULATION AND CONTROL OF SLEEP We have been fascinated by the potent influences of POA thermal stimuli on sleep. The POA was recognized as a thermoregulatory control site on the basis of effects of local warming and cooling, lesions, micro-injection, and neuronal unit recording studies. For example, local POA warming (using a chronically implanted water-perfused ‘‘thermode’’) induces heat loss responses such as panting [32]. Local POA warming was also shown to trigger NREM sleep or EEG slow wave activity in cats [33], rabbits [34] and rats [35]. Sakaguchi et al. [36] found that NREM could be tonically increased during several hours with sustained POA warming in kangaroo rats. The effects of POA thermal stimuli must be mediated by the response of temperature-sensitive neurons that have been localized within this region. Many studies have confirmed that POA contains populations of warm-sensitive and cold-sensitive neurons (WSNs and CSNs) both in vivo and in vitro [37]. These neurons are identified by changes in neuronal discharge in response to locally applied mild thermal stimuli (±>2°C). These changes exceed those that might be expected on the basis of temperature-dependent metabolic effects. In most studies, WSNs and CSNs constitute 20–25% of neurons in these areas in vivo, a finding consistent with the concept that the POA has multiple functions in addition to thermoregulation, with partially overlapping localization. In cats, POA warming increased EEG slow wave activity (delta EEG frequency range) within sustained NREM [38]. Enhanced EEG slow-wave activity was not due to changes in sleep continuity, but was like that induced following sleep deprivation. Since the relative proportion of EEG slow wave activity within sleep is considered to be a measure of sleep depth, this study supports a hypothesis that sleep depth as well as sleep induction is controlled by POA thermosensitive neurons. In contrast, mild POA cooling (−1.0 to −1.8°C) strongly suppressed both NREM and REM sleep for 3 h early in the light phase of the light–dark cycle, when rats normally sleep almost continuously [39]. Since mild local cooling selectively inhibits only WSNs and excites only

NEURONAL BASIS OF POA THERMOREGULATORY CONTROL OF SLEEP We have carried out several studies of the relationships between sleep–wake states and POA/

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Figure 1 POA Temperature Sensitive Neurons and Sleep/Wake States. A. Warm-sensitive neuron (WSN) identified by increased discharge rate during transient elevation of local temperature. B. Polygraphic record with compressed EEG activity showing increases in neuronal spike unit discharge (SU) of a WSN during emergence of synchronized EEG activity at sleep onset. C. Polygraphic record showing WSN discharge suppression during a transient arousal from sleep. D. Using standard criteria, WSNs and CSNs were classified as sleep-active, wake-active, or state-indifferent. About 60% of WSNs were sleep-active neurons. About 70% CSNs were wake-active neurons. Similar findings were obtained in cats and rats. E. An analysis of WSN and CSN discharge changes during sleep onset transitions defined by the first EEG spindle-like events. Sleeprelated WSNs showed increased discharge and wake-related CSNs showed reduced discharge in the transition. Thus, these neuronal discharge changes preceded EEG changes by a few seconds. (Reproduced from Alam et al. [54] with permission).

BF neuronal unit activity as measured in freely moving awake and sleeping cats and rats. The thermosensitivity of neurons was tested by manipulating local POA temperature (measured at the recording site) with a water-perfused thermode located adjacent to the microelectrode path. Thermosensitivity was quantified using three established criteria. These studies showed that most WSNs are sleep-active, that is, they exhibit increased discharge during NREM sleep compared to waking. Most CSNs are wake-active [54,55]. Sleep-active WSNs increased discharge by an average of 48% during NREM compared to waking.

Wake-active CSNs decreased discharge 50% in NREM. Increases in WSN discharge and decreases in CSN discharge were found to anticipate EEG changes at sleep onset by several seconds in both species (see Fig. 1). In our studies, WSNs and CSNs exhibited mirror-image changes in rate and thermosensitivity in NREM compared to waking [54]. This is consistent with the idea of inhibitory interactions between WSNs and CSNs within the POA, as was proposed previously [56]. This local circuitry provides a mechanism by which both excitatory and inhibitory input to the POA can modulate sleep–wake states.

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Practice Points 1. Activation of POA WSNs and/or deactivation of CSNs by local warming are necessary and sufficient to induce and sustain NREM sleep, and 2. The same discharge changes, activation of WSNs and deactivation of CSNs, occur spontaneously at the natural sleep onset transitions. 3. Therefore, it is necessary to conclude that these neuronal events play a central role in the control of spontaneous NREM sleep. 4. Since body temperature is falling at sleep onset, the activation of POA WSNs and deactivation of CSNs is not a response to temperature. Although brain temperature changes can modulate sleep, spontaneous sleep must, instead, be a response to some form of intrinsic neuromodulatory input to sleep-promoting neurons. 5. Sleep-facilitating neurochemical factors (‘‘sleep factors’’) appear to act in POA, but the specific neuronal phenotypes mediating their hypnogenic responses are not yet established.

THE THERMOREGULATORY SET POINT A central concept in thermoregulation is the set point. This is a critical threshold temperature (Tset) for triggering thermoeffector responses such as shivering (heat production) or panting (heat loss), and is analogous to the critical temperature on a home thermostat for starting a heating or airconditioning system. This threshold temperature can be determined either for body temperature or for the POA. Tset is normally identified by gradually warming or cooling the site until the threshold for initiation of a thermoeffector response is found. Using this approach, Sakaguchi et al. [36] measured the POA threshold temperature (Tset) for increased metabolic heat production at different ambient temperatures. Effects on sleep of exposure to various combinations of ambient and POA temperatures were also measured. As noted above, sleep was increased in the ambient and POA warming conditions. In an analysis of all conditions, total sleep time was proportional to Thypo−Tset, where

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Thypo was the baseline hypothalamic temperature. In this study, the increase in metabolic rate induced by POA/BF cooling was also linearly proportional to the difference, Thypo−Tset. The difference between the set point and hypothalamic temperature is the central ‘‘error signal’’ that activates thermoeffector processes. This is known as proportional control of thermoregulation. Since sleep time was also linearly related to Thypo−Tset, these data suggest that sleep, like thermoregulatory responses, is governed by proportional control. In this analysis, Tset was determined for heat production but, on the basis of the evidence summarized above, sleep may be more related to heat loss. Each thermoeffector process has a different Tset. Further analysis of the relationship of sleep regulation to thermoregulatory set points is needed. The thermoregulatory threshold (Tset) is lowered at NREM sleep onset [57]. If Tset is lowered, Thypo will be then above the set point, yielding the error signal Thypo−Tset. Under these conditions, heat loss processes will be activated, in agreement with observed heat loss processes at sleep onset. Inferences about the error signal, Thypo−Tset, can be derived from observations of peripheral heat exchange processes such as vasodilation, which is produced by activation of heat loss processes. The discrete increase in hand and foot skin vasodilation in humans [49], and tail skin vasodilation in the rat [50] at sleep onset could reflect an increase in Thypo−Tset. In our studies in chronic animals we did not define a feature of temperature-sensitive neurons that might correspond to a set point. However, in an in-vitro study [27], we showed that a majority of WSNs exhibit two discrete linear segments with low and high thermosensitivities in the temperature response function which meet in an inflection point (Fig. 2). The inflection point in thermosensitivity of a hypnogenic WSN could provide a mechanistic basis for a functional set point, Tset-sleep. In the hypothalamic tissue slice bathed in artificial cerebrospinal fluid, the inflection points were distributed narrowly around the normal body temperature of the awake rat. Our study also showed that the inflection point could be displaced by synaptic inputs. In this example, shown in Figure 2, synaptic blockade (SB) induced a discrete displacement of the inflection point to a higher temperature. Displacement of the inflection point to the left by synaptic input, while hypothalamic temperature is hardly changed, would result in an activation of

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Figure 2 WSN Recorded in the Diagonal Band in the in-vitro slice preparation. Most WSNs recorded in vitro exhibited an inflection point in the thermoresponse function. Synaptic blockade achieved by altering the slice media induced a displacement of the inflection point to the right. These inflection points could also be displaced to the left or right with agonists (muscimol) and antagonists of the inhibitory neurotransmitter, -aminobutyric acid (GABA). The thermosensitivity (slope) of the response function could also be shifted. (Reproduced from Hays et al. with permission [104]). WSNs in agreement with our observation in chronic animals (Fig. 3). Thus, we hypothesize that the onset of sleep is induced by the displacement of the inflection point of WSNs to a lower temperature in response to synaptic input. We propose to refer to this process as regulation of Tset-sleep. We also found that exposing the slice to GABAergic agonists or antagonists, could displace the inflection points of WSNs. Individual WSNs showed a wide range of responses to GABAergic modulation, including both ‘‘left’’ and ‘‘right’’ shifts in inflection points. Thus, GABAergic regulation of WSNs appears to be heterogeneous, and may include both wakerelated and sleep-related influences [58].

CIRCADIAN MODULATION OF SLEEP One input to the WSNs may originate in the GABAergic neurons of circadian pacemaker in the suprachiasmatic nucleus (SCN) which sends afferents to the adjacent POA [59]. SCN neurons are more active in the light phase of the light–dark cycle [60]. If the SCN GABAergic input to WSNs lowers the set point, as suggested in Figure 3, this could account for the facilitation of sleep during

the light phase of the circadian cycle in the rat. There are circadian rhythms in the ‘‘set points’’ or thresholds of thermoeffector functions [61], supporting a hypothesis that the circadian pacemaker directly modulates thermoregulatory mechanisms. One advantage of the Tset-sleep hypothesis is that it provides a mechanism for rapid changes in sleep propensity. For example, this hypothesis is congruent with evidence for opening of a sleep ‘‘gate’’ in the late evening, as suggested by the work of Lavie [62]. In humans, within the circadian cycle, sleep and a decline of body temperature are normally coincident, but this does not prove that the two processes are coupled; that is, circadian sleep modulation and the circadian rhythm of body temperature could be two independent and separable outputs of the SCN. Under certain experimental conditions, including short sleep–wake cycles [62], internal desynchronization [63], and forced desynchronization [46], sleep onset may occur at all phases of the circadian temperature rhythm, and the interactions of the temperature rhythm and sleep propensity can be studied and partially isolated from effects of prior waking. Although sleep may occur at any circadian phase, these studies show that sleep propensity is strongly increased primarily on the late descending phase of the circadian temperature rhythm and is highest when temperature is low. Awakenings tend to occur as temperature increases, even if sleep time is short. There is an evening ‘‘wake-maintenance zone’’. Under entrained conditions sleep onset occurs at the time of maximum rate of fall of core temperature [51]. Sleep onset evokes a further decrease in temperature, even during continuous bed rest [46]. It is possible that the critical circadianrelated variable is not temperature, but some correlated variable such as melatonin secretion. However, melatonin is a weak hypnogen at the usual time of sleep onset and is not applicable to nocturnal species [64]. The most evident hypothesis is that sleep propensity is directly coupled to the mechanism that reduces body temperature. This coupling would be mediated by hypnogenic WSNs.

USING C-FOS EXPRESSION TO MAP POA SLEEP-ACTIVE NEURONS The further identification of the sleep-active neurons is a key problem. Expression of the proto-

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Figure 3 Theoretical model of how a shift of the inflection point of a WSN can increase the hypnogenic output without a change in brain temperature. After a shift in the inflection point to the left, hypothalamic temperature is higher than the inflection point and intercepts the steep portion of the thermoresponse function. Even with a small decrease of temperature the hypnogenic output could be maintained. This function can be expressed as follows: WSN discharge (hypnogenic output)=Thypo+(Thypo−Tset-sleep)+C where  and  are the slopes or proportionality of the hypnogenic drive above and below the set point Tsetsleep. C is a fixed basal discharge rate. T+C is the basal neuronal discharge rate in the absence of an error signal, assuming Thypo−Tset-sleep cannot be a negative number.

oncogene, c-fos, has been shown to be a marker of neuronal activation in most brain sites [65]. C-fos is identified as an immediate early gene (IEG) whose message was shown to be induced within a few minutes of neuronal activation. The c-Fos protein can be measured within 30 min following neuronal activation. This protein dimerizes with the product of another IEG, c-jun, and binds to the nuclear AP1 site to regulate the expression of other genes. The presence of the c-Fos protein in the nucleus can be readily measured with immunostaining methods. A discrete cluster of neurons that exhibit c-Fos protein immunostaining following sustained sleep, but not waking, was described by Sherin et al. in the VLPO of rats [4]. Subsequently, using electrophysiological recordings, we found that the VLPO also contains a high proportion of neurons that exhibit sleep-active neuronal discharge [66]. The latter study confirms that the c-Fos method can be used to identify sleep-active neurons in this region. We used the c-Fos immunostaining method to obtain additional information about the anatomical distribution and numbers of POA sleep-active neurons, as such mapping is difficult to achieve using electrophysiological methods, alone. A second goal was to determine whether this method could be

used to identify POA sites where thermal influences on sleep might be expressed. In this study [67], we examined the distribution of c-Fos protein immunoreactive neurons (Fos IRNs) during sleep and waking at a standard rat laboratory Ta (22°C) and a mildly warm Ta compatible with sleep in the rat (31.5°C). Three sites, the rostral median preoptic nucleus (MnPN), caudal MnPN, and VLPO exhibited pronounced and localized increases in the number of Fos IRNs in sleeping compared to waking animals at 22°C (Fig. 4). The rostral MnPN includes a midline cell group that widens to form a ‘‘cap’’ around the rostral end of the third ventricle and extends ventrally to the organum vasculosum of the lamina terminalis (OVLT) just anterior to the decussation of the anterior commissure. In the caudal MnPN, Fos IRNs were found in the midline immediately above the third ventricle and included sites both dorsal and ventral to the decussation of the anterior commissure. This is the first evidence that the MnPN is a hypnogenic site. The VLPO region containing Fos IRNs was lateral to the optic chiasm, extending caudally from behind the OVLT to near the emergence of the supraoptic nucleus (SON) and extending dorsally from the base of the brain into the lateral preoptic area without a distinct border.

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Figure 4 Plots of c-fos expression after waking (left) or sleep (right) during the light phase at three levels of the preoptic hypothalamus. Segregated sleep-related c-fos expression was found in the rostral and caudal MnPN (see midline sites above the third ventrical) and VLPO regions (see cluster on the base of the brain lateral to the optic chiasm). C-Fos counts were made within the standardized rectangular grids placed over each of these sites as shown. Other POA regions also showed c-fos expression after sleep, but this expression was not segregated from wake-related expression, and cannot be related specifically to sleep. (Reproduced from Gong et al. with permission [67]).

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Figure 5 Correlations between c-Fos counts in the MnPN and VLPO regions and sleep amounts from individual animals at both the control and mildly elevated ambient temperatures (Ta). At the control Ta there were significant correlations in all sites. At the elevated Ta, c-Fos counts and correlations with sleep amounts increased in the MnPN sites, but in VLPO, c-Fos counts were no longer significantly above awake levels. (Reproduced from Gong et al. with permission [67]). Squares: control sleep (CS); diamonds: control wake (CW); triangles: heat sleep (HS); circles: heat wake (HW); solid line: CS versus CW; dotted line: HS versus HW. Numbers of c-Fos IRNs were counted within standard rectangular grids placed in relation to anatomical landmarks to allow between animal comparisons. The correlations between c-Fos counts and sleep amounts are shown in Figure 5. We found

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in the rostral MnPN that the number of sleeprelated Fos IRNs was increased during exposure to 31.5°C (heat-sleep) compared to 22°C (controlsleep). We have differentiated rostral and caudal MnPN because of differential effects of ambient warming on c-fos expression in these two areas. In the VLPO area, the number of Fos IRNs was significantly reduced in the heat-sleep condition compared to control-sleep. Thus, mild ambient warming increased the sleep-related c-fos expression in the rostral MnPN, but decreased sleeprelated expression in VLPO. Although sleep amounts were slightly reduced in some rats at the higher ambient temperature, three rats exhibited more than 75% sleep, well above the threshold amount reported to be required for sleep-related strong c-Fos immunostaining in VLPO. The MnPN and VLPO sleep-active neuronal sites were identified because they did not show expression during waking; that is, they are sites where sleepactive neurons are segregated from other functional groups. Other sites within the POA and BF also exhibited sleep-related c-fos expression, but the same sites showed greater numbers of immunostained cells following waking. At this time we cannot tell whether the cells expressing c-fos following sleep in these widespread POA sites correspond to sleepactive neurons or if they are activated by some other process that is present during both sleep and waking. Future studies will attempt to use double-labeling techniques to distinguish sleep-active and wake-active discharge in these other POA sites. Since sleepactive WSNs were found diffusely in the POA and lesions that do not include the VLPO or MnPN suppress sleep [11], we can hypothesize that some of the diffuse sleep-related c-fos expression will also correspond to sleep-specific activation. C-fos expression and subsequent nuclear binding may have downstream effects on gene expression that could play a role in the long term adaptations associated with sleep, including sleep homeostasis. There is preliminary evidence that POA c-fos expression plays a role in sleep control. Cirelli et al. [68] applied the anti-sense method, which is based on micro-injection of a oligonucleotide sequence complimentary to portions of a target mRNA, in this case the mRNA for c-fos. The complementary message is thought to bind to and inactivate the target mRNA. After c-fos anti-sense was micro-injected into the POA, sleep on the next day was reduced by 26%. This finding suggests that the inactivation of cfos interfered with the hypnogenic process. C-fos

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‘‘knock-out’’ mice (mice genetically altered so as not to have the c-fos gene) also had reduced sleep [69], although these mice had several developmental abnormalities that might affect sleep and therefore, make the study difficult to interpret with certainty. More work is needed to understand the sleepregulatory role of POA c-fos. It is important to note that sleep-active neurons identified by c-Fos are not necessary WSNs. Many sleep-active neurons in the POA [54] and VLPO [66] identified by electrophysiological methods did not respond to local warming. However, a preliminary study showed that ambient warming induced many c-Fos positive neurons during waking in both VLPO and MnPN [70]. These may be identical to sleepactive neurons in these sites or they may be a distinct neuronal population that is co-localized with sleepactive neurons. Studies to resolve this issue are planned.

REGULATORY INPUTS TO THE HYPNOGENIC SYSTEM A central question in sleep regulation concerns the biological basis of sleep homeostasis. Several sleeppromoting factors that act in our near the POA or BF have also been hypothesized to play a role in sleep homeostasis (see POA hypnogenic area section). We hypothesized that the putative sleep factor, adenosine, would act on the sleep-active or wake-active neurons we had identified previously. Our study [71] implemented the method of chronic in vivo neuronal unit recording adjacent to a microdialysis membrane. Drugs are perfused inside this fine cylindrical membrane (300 m diameter) and can diffuse 500 m or more to sites of cells being studied by micro-wires. A typical experiment consisted of a 30–45 min baseline including a complete sleep–wake cycle, a 10–15 min period of drug delivery, and a 45–60 min washout and recovery period. Clear neuronal responses to drug administration were normally evident within 2–3 min of drug delivery and reversal of effects was evident within 4–8 min of onset of washout. Drug doses were adjusted to near threshold levels for neuronal responses, in order to examine the likely effects of small changes in endogenous adenosine which are most likely to be involved in state regulation. This study examined primarily wake-related neurons in the BF, as these are the focus of the adenosine hypothesis, but some sleep-active neurons were

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also studied. We found that administration of an adenosine transport inhibitor, which raises extracellular levels of adenosine of endogenous origin, suppressed activity of all wake-related neurons and increased the NREM/wake discharge ratio of a small sample of sleep-related neurons. In contrast, the adenosinergic A1 receptor antagonist, cyclopentyldimethylxanthine (CPDX), induced increased discharge of wake-related neurons. Thus, it is likely that endogenous adenosine has a tonic effect on neuronal regulation in vivo. Our results support the hypothesis that adenosine contributes to the tonic regulatory input to these neurons during waking and sleep and that this substance could promote sleep through inhibition of wake-related neurons and excitation of sleep-active neurons. It is important to point out that during each continuous adenosinergic drug infusion, neurons continued to show clear staterelated discharge changes at state transitions. Thus, changes in neuronal discharge at transitions between states did not appear to be modulated by adenosinergic inputs, so that additional controls of sleepwake transitions are required. The targets for the sleep factor, PGD2, have been examined using the c-Fos method. Administration of PGD2 into the subarachnoid space induced sleep and c-Fos immunostaining in the VLPO, MnPN, and bed nucleus of the stria terminalis, and other sites [23]. This result is consistent with the hypothesis that this sleep factor induces sleep by activation of identified sleep-active neurons, although this study did not separate the effects of PGD2 versus sleep, itself, on c-Fos expression. Possibly several putative sleep factors contribute to tonic regulatory input to the hypnogenic neuronal system identified by cFos and electrophysiological methods.

PATHWAYS MEDIATING THE HYPNOGENIC OUTPUT OF THE POA The sleep process clearly involves widespread changes in physiological function, including EEG, motor, endocrine, metabolic, and autonomic processes. Following identification of the sleep-active neuronal population, it was important to describe the output pathways that may mediate the orchestration of the physiological changes at sleep onset. In particular, how can hypnogenic processes

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regulate wake-promoting neuronal groups that have been identified in the BF, posterior hypothalamus and brainstem. Again, the segregated sleep-active neurons of the VLPO provided a strategic advantage, since in this site sleep-active neurons could be identified by c-fos staining. Sherin et al. [72] and Steininger et al. [73] described descending pathways from VLPO to neuronal populations that have been found to promote wakefulness, including the serotonergic neurons of the dorsal raphe nucleus (DRN), noradrenergic cell groups in the locus coeruleus (LC), the histaminergic populations in the tuberomammallary nucleus (TM) of the posterior hypothalamus (PH), as well as to additional PH regions. The wake-promoting roles of the DRN, LC, PH, and TM cell groups as well as the PH have been demonstrated by many methods [74]. Immunostaining methods were used to show that the VLPO neurons with descending projections to the wake-promoting populations contained the inhibitory neurotransmitters GABA and galanin [72]. In MnPN and VLPO sleep-active neurons identified by c-Fos also co-localize glutamic acid decarboxylase (GAD), the enzymatic marker of GABAergic neurons [75]. MnPN neurons, although not identified as sleep-active, also project to brainstem monoaminergic cell groups [76]. Indeed, GABAergic neurons throughout the POA and BF project to the posterior hypothalamus, although these were not identified as sleep-active neurons [77]. Activation of VLPO, MnPN, or other POA neurons could, through their descending pathways, induce GABAergic inhibition of the wake-promoting neuronal cells in the posterior hypothalamus and midbrain. In support of this model, wake-promoting neurons exhibit progressive reductions in activity across sleep stages as VLPO neurons exhibit progressive activation (Fig. 6). Since WSNs are an important component of the POA hypnogenic neuronal population, these neurons must contribute to the descending pathways described above. We hypothesized that WSN activation would suppress activity of arousal-related systems. In our studies, animals were prepared with thermodes to induce local warming in the POA and electrodes for neuronal unit recording in other sites. We found that POA warming suppressed discharge of putative wake-promoting neurons recorded in the posterior lateral hypothalamus [78] and the magnocellular basal forebrain [79]. A recent study of this type focused on additional

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Figure 6 Reciprocal discharge rate changes across the wake-sleep cycle in sleep-active neurons, in this case from the VLPO, and putative histaminergic arousal-related neurons recorded from the tuberomammillary nucleus. Similar reciprocal patterns are found with DRN neurons. These data support a hypothesis that sleep-active neurons inhibit arousal systems, and vice versa. Adapted from McGinty and Szymusiak [82].

arousal-promoting system, the serotonergic neurons of the DRN [80]. We first showed that a subgroup of rat DRN neurons exhibit slow regular discharge during waking, progressive reduction in discharge at NREM onset and during NREM sleep, and near cessation of discharge during REM, as was described previously in putative serotonergic neurons in the cat. These neurons also exhibited other distinctive features of putative serotonergic neurons. Activation of WSNs by unilateral POA warming induced an approximately 25% reduction per decgree centigrade in discharge of most DRN neurons during waking, including the subgroup of presumed serotonergic neurons (Fig. 7). The increases in discharge of POA WSNs that normally occur prior to and during NREM (equivalent to a 2–3°C activation) should therefore contribute to the coincident suppression of DRN discharge. Taken together, these studies suggest that the POA warmsensitive hypnogenic system induces sleep by a coordinated inhibition of multiple arousal systems. We have not yet shown that WSNs contribute to the descending pathway from the POA. POA WSNs

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Figure 7 Effects of mild POA warming on the discharge of a putative serotonin-containing neuron of the dorsal raphe nucleus (DRN) in the awake rat. Discharge was consistently suppressed during warming trials, without any change in behavioral state. Other arousal systems exhibited similar responses to POA warming (see text). Anatomical pathways from the POA to the DRN that may mediate this inhibitory process have been described. (Reproduced from Guzman-Marin et al. with permission [80]).

may induce inhibition of arousal systems through either the direct projections noted above or through multisynaptic descending pathways that may include interneurons within the POA.

HYPNOGENIC AND AROUSAL SYSTEM INTERACTIONS: THE SLEEP–-WAKE SWITCH MODEL A recent study by Gallopin et al. [81] provided evidence for the hypothesis that brainstem wakepromoting neurons feedback on VLPO sleep-promoting neurons, establishing what seems to be a reciprocal network. This study also took advantage of the segregated sleep-active population in the VLPO. These investigators used intracellular electrodes to study VLPO neurons in the in-vitro slice preparation. They identified a predominant subgroup of VLPO neurons with a distinctive electrophysiological properties and distinctive morphology.

They then applied wake-promoting neurotransmitters, serotonin, noradrenaline, and acetlycholine in the media that supports the slice preparation. This study showed that each of these substances induced direct inhibition of the predominant type of VLPO neuron. They additionally identified these neurons as GABAergic. A depiction of the system hypothesized by von Economo 70 years ago, and developed in recent studies, is shown in Figure 8. The picture which emerges is that (1) GABAergic neurons in VLPO, MnPN and possibly other POA sites give rise to descending projections that inhibit histaminergic, serotonergic, noradrenergic and other arousal-related neurons and (2) collaterals from some arousalrelated neurons inhibit VLPO neurons and probably other POA neurons. Thus, a sleep process starting with VLPO or POA hypnogenic neuronal activation would inhibit wake-promoting systems, which, in turn, would remove inhibition from VLPO neurons, facilitating the sleep onset process. In contrast, an

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ARE THERE MULTIPLE COMPONENTS OF THE HYPNOGENIC SYSTEM Arousal 5HT

Arousal Homeostatic control Physiological and WSN hormonal control other CSN

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Figure 8 Simplified anatomical-physiological model which summarizes the current understanding of the control of NREM sleep from the POA. Sleep-active WSNs have descending inhibitory projections to multiple arousal systems in posterior hypothalamus (PH), including histaminergic neurons (hist.), and to the midbrain/pons, including to serotonergic (5HT) and noradrenergic (NE) neurons. The arousal systems project to the thalamus and forebrain to control brain activation and also back to the POA, where they participate in the ‘‘sleep–wake switch’’ (see text). Circadian, homeostatic, and physiological influences on sleep may be mediated by modulatory neurochemical inputs to WSNs and CSNs, or to other sleep-active neurons (SAN). Sensory stimuli activate NE and 5HT systems and may inhibit sleep through these pathways.

arousing input mediated by wake-promoting neurons would inhibit VLPO neurons, which would then remove inhibition from wake-promoting neurons, facilitating asousal. In this way, the system can switch decisively between sleep and waking [82]. Since hypnogenic systems inhibit arousal systems, and vice versa, this mechanism would also tend to help sustain either sleep and wake states, once they had been initiated. The arousal systems have diffuse projections reaching virtually every part of the brain and spinal cord. By inhibiting the arousal systems, the localized POA hypnogenic system can regulate global brain functions, ranging from EEG regulation to autonomic activity and motor activity. A critical component of the POA hypnogenic system originates in WSNs. The POA hypnogenic neurons receive inputs from the circadian clock and from neurochemical sleep factors such as adenosine that may mediate homeostatic and physiological modulation of sleep.

Sleep-active neurons other than WSNs are recognized in Figure 8. Not all POA sleep-active neurons are WSNs [54]. The role of non-WSN sleep-active neurons is uncertain, but their presence suggests that there may be more than one component of the POA hypnogenic system. This view is also consistent the interpretations of other investigators who have emphasized findings that sleep may be modulated by several different sleep factors [25] or pre-sleep conditioning procedures [83]. Although POA thermal stimuli are potent regulators of sleep, the temperature-sensitive POA hypnogenic system may be only one component of a more complex system.

SOME CONTRADICTORY DATA Several studies have presented data that were interpreted to challenge the hypothesis that the hypnogenic process, in general, or the POA hypnogenic mechanism, in particular, are coupled to thermoregulatory processes. There has been great interest in the hypnogenic effects of cytokines such as IL-1 and TNF, although these agents also produce fevers. Krueger and Takahashi [84] reviewed several studies showing that it is possible to selectively block either the hypnogenic or pryrogenic effects of these agents, suggesting that these are mediated by separate pathways. We agree with this interpretation, but understand it to mean only that the hypnogenic thermoregulatory system is separate from that involved in fevers, and that certain cytokines stimulate both of these systems, as well as other systems. There is no doubt that many thermoregulatory processes are not coupled to sleep. Clearly, processes such as vasodilation, shivering, and sweating serve to regulate temperature and metabolism within either waking or NREM sleep. It is likely that the POA contains both state-independent and sleepcoupled thermoregulatory mechanisms. ICV administration of neurotransmitters, peptides or other molecules may modulate sleep and induce fever or hypothermia that are not coupled to sleep, and also modulate additional processes such as hormonal or immune functions. In some cases, only stateindependent thermoregulatory processes may be stimulated. Experimental manipulations that induce

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changes in thermoregulation without a change in sleep do not contradict our hypothesis. Induction of fever and sleep may occur at the same time. These represent additive effects of non-sleep and sleep-coupled thermoregulatory processes. However, if sleep begins, even during a fever, body temperature falls [85]. Lesions of VLPO and adjacent POA areas that caused a reduction in NREM sleep did not change the amplitude of circadian temperature rhythm or the 24-h mean temperature in four rats [10]. This was interpreted to show that sleep and thermoregulatory deficits were dissociated. In fact, we reported previously that POA lesions that suppressed sleep produced only transient changes in baseline brain temperature in normal laboratory ambient temperatures [6,86]. However, when the same animals were tested at elevated ambient temperatures, they exhibited elevated heat loss (panting) thresholds and hyperthermia. Impaired heat loss would be expected after loss of WSNs. Thus, animals with lesions may exhibit heat loss deficits that are not revealed unless they are subjected to a thermal challenge. This result suggests that the hypothesis that VLPO lesions produce sleep deficits without thermoregulatory deficits remains to be appropriately tested. Lu et al. [10] also reported that ventromedial POA lesions were followed by greatly increased amplitude of the circadian temperature rhythm, but with a lesser reduction in sleep than with VLPO lesions. This could be explained by the hypothesis that the neural substrate of stateindependent thermoregulation may be partly localized in the ventromedial POA. Studies in rodents found an inverse relationship between body temperature and sleep percentage [87], in general agreement with our hypothesis, but it was also reported that delta activity was not related to temperature or to change (reduction) in temperature within the same NREM episode [88]. Reduction in temperature within a NREM episode could be related to Thypo−Tset, since this is equivalent to thermolytic drive. However, it should also be kept in mind that the time courses of heat loss and generation of slow wave EEG activity are different. When subjects are studied in a mildly warm standard laboratory ambient temperature, heat exchange is limited, and a substantial change in core body temperature cannot be achieved in a few minutes. EEG changes can occur quickly. Our model predicts that a measure of thermolytic drive such as the core

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minus skin temperature difference could be associated with delta activity. It has been reported that hand and foot skin vasodilation anticipates sleep onset [28] and increases discretely at sleep onset [89]. These studies have been interpreted to support a hypothesis that vasodilation, itself, is the critical event in promoting sleep. In support of this model, it was shown that, during the daytime, both administration of melatonin and assumption of a resting posture induced (1) hand and foot vasodilation, (2) lowered body temperature, and (3) a correlated increase in sleepiness [90]. Other events such as ‘‘lights off’’, relaxation, and withdrawal of arousing stimuli also contribute to vasodilation. In this model, the mechanisms by which vasodilation would orchestrate the complex processes of sleep are not spelled out. These data are also compatible with our model which would consider the vasodilation as an effector process that is induced by the activation of the POA hypnogenic thermoregulatory system. Since skin warming can increase the activity of POA WSNs [91], or facilitate thermoeffector responses in brainstem or spinal cord [92], skin warming may facilitate sleep and induce vasodilation through hypothalamic mechanisms. Experiments that distinguish these models need to be carried out.

FUNCTIONAL BASIS OF THE CENTRAL ROLE OF THERMOSENSITIVE NEURONS IN SLEEP REGULATION The central role of POA/BF thermosensitive neurons can be understood in terms of an energy conserving function of sleep. Sleep conserves energy through reduced metabolic rate (MR). Processes that contribute to lowered MR include the effects of lowered body temperature on cellular metabolism (the so-called Q10 effect), suppression of motor activity in both REM and NREM, and increased insulation derived from nesting and posture. Early in consolidated sleep periods, a heat loss process associated with vasodilation produces lowered body temperature [93]. The reduction in cerebral metabolic rate during NREM sleep may be viewed as supporting energy conservation. Support for the energy conservation function of sleep comes from well-controlled studies of longterm (2–5 weeks) sleep deprivation in rats [94]. In

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sleep-deprived rats many functions were spared, but rats lost weight and fat reserves and failed to maintain body temperature in a distinctly warm environment (29°C) in spite of doubling of food intake and metabolic rate (MR). Most food intake in homeotherms (80–90%) is used to maintain body temperature [95]. The weight loss and hypothermia in these rats show that during sleep-deprivation excessive heat is lost which is not compensated even by a doubling of heat production. Thus, loss of ability to retain heat and conserve energy, appears to be a fundamental effect of sleep loss in the rat. Small animals sleep up to 18 h daily and the largest animals sleep 2–3 h. In an analysis of 80 mammalian species, the constitutional variables exhibiting the best correlations with sleep time were body mass and metabolic rate [96]. Body mass and metabolic rate are well-correlated (negatively) but, in one analysis, the increased sleep in small animals exhibited partial correlation with body mass rather than metabolic rate. A possible explanation is that energy conservation is more critical in small animals with greater surface/mass ratios, and greater potential mass specific heat loss through the skin. An alternative model that sleep provides a compensatory response for a higher MR is also plausible. Berger and Phillips [95] summarize additional evidence for an energy-conservation model of sleep. An energy-conserving function of sleep may apply particularly to small animals. In the human, a relatively large animal, the energy savings during sleep, compared to a resting baseline, is equivalent to a slice of bread [96]. A behavior with the profound functional significance of sleep in humans would seem to be based on more then a slice of bread. It is possible, perhaps even likely, that in large mammals additional functional mechanisms are superimposed on energy conservation. For example, in the adenosine hypothesis, a reduction in cerebral energy reserves is hypothesized to regulate sleep [97]. We previously hypothesized that body and brain cooling associated with sleep served functions in addition to energy conservation. We hypothesized that mammalian evolution led to the selection of high brain and body temperatures to maximize psychophysiological performance, aerobic capacity, and the substrates of alertness, all of which are positively correlated with body temperature [98]. Aerobic capacity permits animals to run for extended periods. However, high awake mammalian temperatures are relatively close to those that can

cause tissue damage. There is anecdotal evidence that sleep deprivation can increase susceptibility to heat stroke. We hypothesized that the effects of relatively high temperatures are cumulative, like those of sleep deprivation on daytime sleepiness, and that cooling during sleep provides compensation. We hypothesized that sleep homeostasis could be based on awake heat loads, and developed a quantitative model based on this concept [99]. Stage 3–4 sleep in humans [43] and deep sleep in rats [44] is increased after awake body heating. One direct test of this hypothesis was supportive. Rats exposed to 12 h of ambient cooling, sufficient to lower body temperature, combined with sleep deprivation exhibited a much smaller rebound in delta EEG activity than did animals subjected to 12 h of sleep deprivation alone, although a small delayed rebound was observed [100]. This study suggests that body cooling reduces the homeostatic compensation for sleep deprivation. However, other evidence related to this concept is inconsistent. For example, the increase in delta activity after sleep deprivation was not correlated with ‘‘awake’’ temperature in the sleep deprived animals [101]. Further exploration of this hypothesis is needed. Research Agenda 1. Further identification of the phenotypes of POA hypnogenic neurons. 2. Critical assessment of the sleep set point hypothesis. 3. Description of mechanisms by which the circadian clock and putative sleep factors regulate the activity of the POA hypnogenic neurons. 4. Differentiation of functional outputs of the temperature-sensitive and non-temperaturesensitive POA hypnogenic processes. 5. Further analysis of the mechanistic basis of peripheral vasodilation in sleep regulation.

CLINICAL APPLICATIONS OF THE THERMOREGULATORY HYPOTHESIS We have placed the POA thermoregulatory system at the center of NREM sleep control. This necessarily leads to predictions that disorders closely

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linked to sleep would be modulated by thermoregulatory processes. We can offer two types of data that are congruent with such predictions. The first example is concerned with obstructive sleep apnea (OSA), in which loss of upper airway dilator tone during sleep permits collapse of the upper airway. In some cases OSA is associated with reduced upper airway dimensions, but this does not explain all characteristics of the disease. Many patients who go on to develop OSA begin to gain weight and snore in early adulthood, and daytime sleepiness, a cardinal symptom of OSA, is not completely reversed by adequate CPAP treatment in many patients. Males have larger airways than females, but more OSA. Based on these considerations, we hypothesized that some OSA patients have an exaggerated energy-conserving hypnogenic drive, which generates weight gain, daytime sleepiness, and reduced upper airway dilator activity during sleep. To test an element of this hypothesis, in chronic cat preparations, we examined the effects of activation of the POA hypnogenic system by local warming on the inspiratory activity of a laryngeal airway dilator muscle, the posterior cricoaretinoid (PCA). POA warming induced an inhibition of PCA activity, particularly during NREM sleep [102]. The lead time of onset of the PCA inspiratory burst relative to the diaphragmatic activity was also reduced by POA warming. This study shows that activation of WSNs during sleep can regulate the activity of airway dilator muscles, and supports the hypothesis of a role for the POA in OSA. An association between sleep mechanisms and the affective disorder, depression, has been under study for 30 years. Sleep disturbance is a cardinal symptom of depression. Surprisingly, in a majority of patients, one night of sleep deprivation relieves depression as measured by standard rating scales, and the depression recurs after the next sustained sleep period [103]. Depression is usually worse following morning awakening. These facts have led to the idea that sleep is depressogenic. Wehr [103] has hypothesized that the depressogenic feature of sleep is that it induces the physiological drive of ‘‘heat’’, meaning that, during sleep, brain temperature is above Tset, so that heat loss mechanisms would be activated. In support of this model, Wehr has emphasized the following findings. (1) Most patients with depression have elevated nocturnal body temperatures while ill; nocturnal temperatures

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normalize during remission. (2) Antidepressant medications, including tricyclics, MAO inhibitors, and SSRIs, lower body temperature. (3) Depressives have blunted nocturnal TSH secretion, and TSH secretion is inhibited by heat exposure. Nocturnal TSH secretion is partially normalized by sleep deprivation in depressed patients. (4) Patients with winter seasonal affective disorder (SAD) exhibit a fall in nocturnal temperature from winter, when they are depressed, to summer, when they are euthymic. Daytime bright light exposure in winter, which relieves SAD, lowers winter nocturnal body temperature. (5) Preliminary results suggest that the relief of depression induced by a night of sleep deprivation is attenuated if sleep deprivation is carried out in a high ambient temperature. These findings suggest a hypothesis that the thermoregulatory-coupled hypnogenic mechanism is not sufficiently effective in depressed patients. In terms of our model, hypnogenic WSNs could be less activated during sleep in depressed patients, leading to nocturnal temperature elevations during sleep and sleep disturbance. The emergence of depressive symptoms may be due to the cumulative effect of elevated temperature, or to changes in the regulation of the set point. Alternatively, the abnormality could be downstream from WSNs, such that some element of the thermolytic cascade associated with sleep is compromised in these patients.

SUMMARY: THE POA HYPNOGENIC SYSTEM Much evidence supports a hypothesis that a POA thermosensitive neuronal system regulates sleep. The activation of POA WSNs (or deactivation of CSNs) is necessary and sufficient for control of NREM sleep. Activation of WSNs also occurs during spontaneous NREM sleep. Activation of WSNs is expected to induce thermolytic processes and body cooling. The occurrence of vasodilation at sleep onset and the inverse relationship between sleep propensity and body temperature support the hypothesis that hypnogenic mechanisms are coupled to thermolytic mechanisms. The output of the POA temperature-sensitive hypnogenic system inhibits established arousal systems, providing a mechanistic basis for the sleep-onset process. Species differences in 24-h sleep amounts and deficits produced by prolonged sleep deprivation in rats have

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been related to regulation of heat loss or metabolic rate. These processes are controlled by POA thermosensitive neurons. The circadian rhythm in sleep propensity in humans is coupled to the body temperature rhythm. Thus, a hypothesis that sleep is regulated by POA thermosensitive neurons is consistent with critical biological features of sleep. This model is congruent with the energy-conservation theory of sleep function, but other effects of brain or body cooling during sleep may also have functional consequences. Another line of work has identified POA sleepactive neurons by mapping expression of the neuronal activation-induced protooncogene, c-fos. This work has led to the discovery of segregated sleepactive neuronal populations in the VLPO and MnPN. Sleep-related c-fos expression was also found in additional POA sites, but this was not segregated from wake-related c-fos expression. Sleep-active neurons in VLPO and MnPN express the inhibitory neurotransmitter, GABA, and send projections to the sites of established arousal systems in the basal forebrain, posterior hypothalamus and midbrain. Thus, activation of sleep-active neurons is expected to inhibit arousal systems. In support of this model, discharge of putative histaminergic and serotonergic arousal-related neurons exhibits reciprocal changes with sleep-active neurons across the sleep–wake cycle. Neurotransmitters associated with arousal systems induce inhibition of VLPO neurons. The mutually inhibitory interactions of hypnogenic and arousal systems suggests a ‘‘sleep–wake switch’’ model which provides a mechanistic basis for decisive switching between sleep and wake states. Some critical issues are unresolved. In humans, the energy savings during sleep are too small to explain a behavior that dominates our everyday lives, and additional functional mechanisms may be important. Dissociations between sleep and the usual body temperature decreases have been reported, and there is evidence for a non-temperature sensitive POA hypnogenic system. Preliminary evidence suggests that sleep-active neurons identified by c-fos and WSNs are co-localized, but the exact relationship of these systems requires further study. Further, the POA hypnogenic system may be regulated primarily by non-thermal neuromodulatory inputs. For example, the sleep-promoting factors, adenosine and PGD2 were shown to excite sleepactive neurons. The neuronal targets for many other sleep-promoting substances have not been studied.

Many questions remain concerning the regulation of both thermosensitive and non-thermosensitive sleep-promoting neurons and the coupling of functional and mechanistic controls of sleep.

ACKNOWLEDGEMENTS This work was supported by the research service of the Veterans Administration, and by the National Institutes of Health (MH 47480, HL 60296). We thank Md. Noor Alam, Hui Gong, Tim Hays, and Teresa Steininger for their critical contributions.

REFERENCES 1. von Economo C. Sleep as a problem of localization. J Nerv Ment Dis 1930; 71: 249–259. 2. Swanson LW. The hypothalamus. Bjorklund A, Hokfelt T, Swanson L (eds). Handbook of Chemical Neuroanatomy. Vol. 5: Integrated Systems of the CNS, Part I. Amsterdam: Elsevier 1987; 1–124. 3. Szymusiak R. Magnocellular nuclei of the basal forebrain: substrates of sleep and arousal regulation. Sleep 1995; 18: 478–500. 4. Sherin JE, Shiromani PJ, McCarley RW, Saper CB. Activation of ventrolateral preoptic neurons during sleep. Science Washington 1996; 271: 216–219. 5. Asala SA, Okano Y, Honda K, Inoue S. Effects of medial preoptic lesions on sleep and wakefulness in unrestrained rats. Neurosci Let 1990; 114: 300–304. 6. Szymusiak R, Danowski J, McGinty D. Exposure to heat restores sleep in cats with preoptic/anterior hypothalamic cell loss. Brain Res 1991; 541: 134–138. 7. Nauta WJH. Hypothalamic regulation of sleep in rats. An experimental study. J Neurophysiol 1946; 9: 285–316. 8. McGinty DJ, Sterman MB. Sleep suppression after basal forebrain lesions in the cat. Science Washington 1968; 160: 1253–1255. 9. Sallanon M, Sakai K, Denoyer M, Jouvet M. Long lasting insomnia induced by preoptic neuron lesions and its transient reversal by muscimol injection into the posterior hypothalamus. J Neurosci 1989; 32: 669–683. 10. Lu J, Greco M, Shiromani P, Saper C. Effect of lesions of the ventrolateral preoptic nucleus on NREM and REM sleep. J Neuroscience 2000; 20: 3830–3842. 11. Schmidt M, Valatx J-L, Sakai K, Fort P, Jouvet M. Role of the lateral preoptic area in sleep-related erectal mechanisms and sleep generation in the rat. J Neuroscience 2000; 20: 6640–6647. 12. John J, Kumar V. Effect of NMDA lesion of the medial preoptic neurons on sleep and other functions. Sleep 1998; 21: 587–598. 13. Szymusiak R, McGinty D. Sleep suppression following kainic acid-induced lesions of the basal forebrain. Exp Neurol 1986; 94: 598–614.

D. MCGINTY AND R. SZYMUSIAK

340

14. John J, Kumar V, Gopinath G. Recovery of sleep after fetal preoptic transplantation in the medial preoptic area-lesioned rats. Sleep 1998; 21: 601– 606. 15. Sterman MB, Clemente CD. Forebrain inhibitory mechanisms: sleep patterns induced by basal forebrain stimulation in the behaving cat. Exp Neurol 1962; 6: 103–117. 16. Zhang J, Obal F Jr, Zheng T, Fang J, Taishi P, Krueger J. Intrapreoptic microinjection of GHRH or it antagonist alters sleep in rats. J Neurosci 1999; 19: 2187–2194. 17. Mendelson WB, Martin JV, Perlis M, Wagner R. Enhancement of sleep by microinjection of triazolam into the medial preoptic area. Neuropsychopharm 1989; 2: 61–66. 18. Ueno R, Ishikawa Y, Nakayama T, Hayaishi O. Prostaglandin D2 induces sleep when microinjected into the preoptic area of conscious rats. Biochem Biophys Res Com 1982; 109: 576–582. 19. Radulovacki M, Virus RM, Djuricic-Nedelson M, Green R. Adenosine analogs and sleep in rats. J Pharmacol Exp Ther 1984; 228: 268–274. 20. Porkka-Heiskanen T, Strecker RE, Thakkar M, Bjorkum AA, Greene RW, McCarley RW. Adenosine: A mediator of the sleep-inducing effects of prolonged wakefulness. Science Washington 1997; 276: 1265– 1268. 21. Portas CM, Thakkar M, Rainnie DG, Greene RW, McCarley RW. Role of adenosine in behavioral state modulation: a microdialysis study in the freely moving cat. Neuroscience 1997; 79: 225–235. 22. Matsumura H, Nakajima T, Osaka T, Satoh S, Kawase K, Kubo E, Kantha SS, Kasahara K, Hayaishi O. Prostaglandin D2-sensitive, sleep-promoting zone defined in the ventral surface of the rostral basal forebrain. Proc Natl Acad Sci USA 1994; 91: 11998– 12002. 23. Scammel T, Gerashchenko D, Urade Y, Onoe H, Saper C, Hayaishi O. Activation of ventrolateral preoptic neurons by the somnogen prostaglandin D2. Proc Natl Acad Sci USA 1998; 95: 7754–7759. 24. Morairty S, Thomson D, Szymusiak R, Hays T, McGinty D. The somnogenic effects of prostaglandin D2 infusion in rats with preoptic/anterior hypothalamic lesions. Sleep Res 1996; 25: 18. 25. Krueger J, Obal F Jr, Opp M, Toth L, Johannsen L, Cady A. Somnogenic cytokines and models concerning their effects on sleep. Yale J Biol Med 1990; 63: 157–172. 26. Susic V, Masirevic G, Totic S. The effects of deltasleep-inducing peptide (DSIP) on wakefulness and sleep patterns in the cat. Brain Res 1987; 414: 262–270. 27. de Lecea L, Criado ??, Prospero-Garcia O et al. A cortical neuropeptide with neuronal depressant and sleep-modulating properties. Nature (London) 1996; 381: 242–245. 28. Inoue S, Honda K, Komoda Y. Sleep as neuronal

29.

30.

31.

32. 33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

detoxification and restitution. Behav Brain Res 1995; 69: 91–95. Chastrette N, Cespuglio R, Jouvet M. Proopiomelanocortin (POMC)-derived peptides and sleep in the rat. Part 1 – Hypnogenic properties of ACTH derivatives. Neuropeptides 1990; 15: 61–74. Danguir J, Nicolaidis S. Chronic intracerebroventricular infusion of insulin causes increase in slow wave sleep in rats. Brain Res 1984; 306: 97–103. Cravatt B, Prospero-Garcia O, Siuzdak G, Gilula N, Henriksen S, Boger D, Lerner R. Chemical characterization of a family of brain lipids that induce sleep. Science 1995; 268: 1509. Boulant JA. Hypothalamic mechanisms in thermoregulation. Fed Proc 1981; 40: 2843–2850. Roberts WW, Robinson TCL. Relaxation and sleep induced by warming of the preoptic region and anterior hypothalamus in cats. Exp Neurol 1969; 25: 282–294. Von Euler C, Soderberg U. The influence of hypothalamic thermoreceptive structures on the electroencephalogram and gamma motor activity. Electroenceph Clin Neurophysiol 1957; 9: 391–408. Benedek G, Obal F Jr, Lelkes Z, Obal F. Thermal and chemical stimulations of the hypothalamic heat detectors: the effects on the EEG. Acta Physiol Acad Sci Hung 1982; 60: 27–35. Sakaguchi S, Glotzbach SF, Heller HC. Influence of hypothalamic and ambient temperatures on sleep in kangaroo rats. Am J Physiol 1979; 237: R80–R88. Boulant JA, Dean JB. Temperature receptors in the central nervous system. Ann Rev Physiol 1986; 48: 639–654. McGinty D, Szymusiak R, Thomson D. Preoptic/ anterior hypothalamic warming increases EEG delta frequency activity within non-rapid eye movmement sleep. Brain Res 1994; 667: 273–277. McGinty D, Thomson D, Szymusiak R, Morairty S. Suppression of sleep by local preoptic/anterior hypothalamic cooling. Sleep Res 1996; 25: 18. Valatx JL, Roussel B, Cure M. Sommeil et temperature cerebrale du rat au cours de l’exposition chronique in ambiance chaude. Brain Res 1973; 55: 107–122. Schmidek WR, Hoshino K, Schmidek M, Timo-Iouria C. Influence of environmental temperature on the sleep/wakefulness cycle in the rat. Physiol Behav 1936; 8: 363–371. Roussel B, Turrillo P, Kitahama K. Effect of ambient temperature on the sleep–waking cycle in two strains of mice. Brain Res 1984; 294: 67–73. Horne JA, Reid AJ. Night-time sleep EEG changes following body heating in a warm bath. Electroencephalogr Clin Neurophysiol 1985; 60: 154–157. Morairty S, Szymusiak R, Thomson D, McGinty D. Selective increases in nonrapid eye movement sleep following whole body heating in rats. Brain Research 1993; 617: 10–16. Obal F Jr, Alfoldi P, Rubicsek G. Promotion of sleep

HYPOTHALAMIC SLEEP-PROMOTING MECHANISMS

341

by heat in young rats. Pflugers Arch – Eur J Physiol 1995; 430: 729–738. Dijk DJ, Czeisler CA. Contribution of the circadian pacemaker and the sleep homeostat to sleep propensity, sleep structure, electroencephalic slow waves, and sleep spindle activity in humans. J Neurosci 1995; 15: 3526–3538. Baker F, Selsick H, Driver H, Taylor S, Mitchell D. Different nocturnal body tempertures and sleep with forced air warming in men and in women taking hormonal contraceptives. J Sleep Res 1998; 7: 175– 181. Heller HC, Glotzbach S, Grahn D et al. Sleepdependent changes in the thermoregulatory system. In: Lydic R, Biebuyck J (eds). Clinical Physiology of Sleep. Washington DC: American Physiological Society, 1988; 145–158. Krauchi K, Cajochen C, Werth E, Wirz-Justice A. Warm feet promote the rapid onset of sleep. Nature (London) 1999; 401: 36–37. Alfoldi P, Rubicsek G, Cserni G, Obal JF. Brain and core temperatures and peripheral vasomotion during sleep and wakefulness at various ambient temperatures in the rat. Pflugers Arch 1990; 417: 336–341. Campbell S, Broughton R. Rapid decline in body temperature before sleep: fluffing the physiological pillow. Chronobiol Intern 1994; 11: 126–131. Haskell EH, Palca JW, Walker JM, Berger RJ, Heller HC. The effects of high and low ambient temperatures on human sleep stages. Electroenceph Clin Neurophysiol 1981; 51: 494–501. Benedek G, Obal JF, Jansco-Gabor A, Obal F. Effects of elevated ambient temperatures on the sleepwaking activity of rats with imparied warm reception. Waking Sleeping 1980; 4: 87–94. Alam MN, McGinty D, Szymusiak R. Neuronal discharge of preoptic/anterior hypothalamic thermosensitive neurons: relation to NREM sleep. Am J Physiol 1995; 269: R1240–R1249. Alam MN, McGinty D, Szymusiak R. Thermosensitive neurons of the diagonal band in rats: relation to wakefulness and non-rapid eye movement sleep. Brain Res 1997; 752: 81–89. Boulant JA. Hypothalamic control of thermoregulation: neurophysiological basis. In: Morgane PJ, Panksepp J (eds). Handbook of the Hypothalamus. New York: Dekker, 1980; 1–82. Heller HC, Glotzbach SF. Thermoregulation During Sleep an Hibernation. In: Robertshaw D (ed). Int Rev Physiol, Environ Physiol II. Baltimore: University Park Press, 1977; 147–188. Hays T, Szymusiak R, McGinty D. GABAA receptor modulation of temperature sensitive neurons in the diagonal band of Broca in vitro. Brain Res 1999; 845: 215–223. van den Pol AN, Tsujimoto KL. Neurotransmitters of the Hypothalamic Suprachiasmatic Nucleus: Immunocytochemical Analysis of 25 Neuronal Antigens. Neuroscience 1985; 15: 1049–1086.

60. Meijer J, Reitveld W. Neurophysiology of the suprachiasmatic circadian pacemaker in rodents. Physiol Rev 1989; 69: 671–707. 61. Stephenson LA, Wenger CB, O’Donovan BH, Nadel ER. Circadian rhythm in sweating and cutaneous blood flow. Am J Physiol 1984; 246: R321–R324. 62. Lavie P. Ultradian Rhythms in Physiology and Behavior. In: Schulz H, Lavie P (eds). Ultradian Rhythms in Physiology and Behavior. New York: Springer-Verlag, 1985; 148–164. 63. Czeisler CA, Weitzman ED, Moore-Ede MC, Zimmerman JC. Human sleep: Its duration and organization depend on its circadian phase. Science (Washington) 1980; 210: 1264–1267. 64. Kripke D, Elliot J, Youngstedt S, Smith J. Melatonin: marvel or marker? Ann Med 1998; 30: 81–87. 65. Morgan JI, Curran T. Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. Ann Rev Neurosci 1991; 14: 421–451. 66. Szymusiak R, Alam N, Steininger T, McGinty D. Sleepwaking discharge patterns of ventrolateral preoptic/ anterior hypothalamic neurons in rats. Brain Res 1998; 803: 178–188. 67. Gong H, Szymuisak R, King J, Steininger T, McGinty D. Sleep-related c-Fos expression in the preoptic hypothalamus:effects of ambient warming. Am J Physiol Regulatory Integrative Comp Physiol 2000; 279: R2079–R2088. 68. Cirelli C, Pompeiano M, Arrighi P, Tononi G. Sleepwaking changes after c-fos antisense injections into the medial preoptic area. Neuroreport 1995; 6: 801– 805. 69. Shiromani P, Basheer R, Thakkar J, Wagner D, Greco M, Charness M. Sleep and wakefulness in c-fos and fos B gene knockout mice. Mol Brain Res 2000; 80: 75–87. 70. McGinty D, Gong H, Alam T et al. Warm-sensitive neurons are co-localized the sleep-active neurons in the preoptic hypothalamus. (Abstr). Sleep 2001; 24: A.58. 71. Alam MN, Szymusiak R, Gong H, King J, McGinty D. Adenosinergic modulation of rat basal forebrain neurons during sleep and waking: Neuronal recording with microdialysis. J Physiol (London) 1999; 521: 679–690. 72. Sherin JE, Elmquist JK, Torrealba F, Saper CB. Innervation of histaminergic tuberomammillary neurons by GABAergic and galaninergic neurons in the ventrolateral preoptic nucleus of the rat. J Neurosci 1998; 18: 4705–4721. 73. Steininger T, Gong H, McGinty D, Szymusiak R. Subregional organization of preoptic area/anterior hypothalamic projctions to arousal-related monoaminergic cell groups. J Comp Neurol 2001; 429: 638–653. 74. Jones BE. Basic mechanisms of sleep–wake states. In: Kryger MH, Roth T, Dement WC (eds). Principles and Practice of Sleep Medicine. Philadelphia: WB Saunders Co, 1989; 121–140.

46.

47.

48.

49. 50.

51. 52.

53.

54.

55.

56.

57.

58.

59.

342

75. Gong H, Szymusiak R, King J et al. Co-localization of c-Fos protein and GABA in preoptic area neurons following sleep (Abstr). Sleep 2001; 24: A155. 76. Zardetto-Smith A, Johnson A. Chemical topography of efferent projections from the median preoptic nucleus to pontine monoaminergic cell groups in the rat (letter). Neurosci Lett 1995; 199: 215– 219. 77. Gritti I, Mainville L, Jones BE. Projections of GABAergic and cholinergic basal forebrain and GABAergic peroptic-anterior hypothalamic neurons to the posterior lateral hypothalamus of the rat. J Comp Neurol 1994; 339: 251–268. 78. Krilowicz BL, Szymusiak R, McGinty D. Regulation of posterior lateral hypothalamic arousal related neuronal discharge by preoptic anterior hypothalamic warming. Brain Res 1994; 668: 30–38. 79. Alam MN, Szymusiak R, McGinty D. Local preoptic/ anterior hypothalamic warming alters spontaneous and evoked neuronal activity in the magno-cellular basal forebrain. Brain Res 1995; 696: 221–230. 80. Guzman-Marin R, Alam MdN, Szymuisak R, DruckerColin R, Gong H, McGinty D. Discharge modulation of rat dorsal raphe neurons during sleep and waking: Effects of preoptic/basal forebrain warming. Brain Res 2000; 875: 23–34. 81. Gallopin T, Fort P, Eggermann E et al. Identification of sleep-promoting neurons in vitro. Nature (London) 2000; 404: 992–995. 82. McGinty D, Szymusiak R. The sleep–wake switch: A neuronal alarm clock. Nature Medicine 2000; 6: 510–511. 83. Garcia-Garcia F, Beltran-Parrazal L, Jimenez-Arguiano A et al. Manipulations during forced wakefulness have differential impact on sleep architecture, EEG power spectrum, and Fos induction. Brain Re Bulletin 1998; 47: 317–324. 84. Krueger JM, Takahashi S. Thermoregulation and sleep. Closely linked but separable. Ann NY Acad Sci 1997; 813: 281–286. 85. Walter JS, Meyers P, Krueger JM. Microinjection of interleukin-1 into brain: Separation of sleep and fever responses. Physiol Behav 1989; 45: 169–176. 86. Szymusiak R, Satinoff E. Ambient temperature-dependence of sleep disturbances produced by basal forebrain damage in rats. Brain Res Bull 1984; 12: 295–305. 87. Franken P, Tobler I, Borbely A. Sleep and waking have a major effect on the 24-h rhythm of cortical temperature in the rat. J Biol Rhythms 1992; 7: 341–352. 88. Franken P, Tobler I, Borbely A. Cortical temperature and EEG slow-wave activity in the rat: analysis of vigilance state related changes. Pflugers Arch 1992; 420: 500–507. 89. Krauchi K, Cajochen C, Werth W, Wirz-Justice A. Functional link between distal vasodilation and sleep onset latency? Am J Physiol Regulatory Integrative Comp Physiol 2000; 278: R741–R748.

D. MCGINTY AND R. SZYMUSIAK 90. A relationship between heat loss and sleepiness: effects of postural change and melatonin administration. J Applied Physiol 1997; 83: 134–139. 91. Wit A, Wang S. Temperature-sensitive neurons in the preoptic/anterior hypothalamic region: effects of increasing ambient temperature. Am J Physiol 1968; 215: 1151–1159. 92. Berner N, Heller H. Does the preoptic anterior hypothalamus recieve thermoafferent information? Am J Physiol Regulatory Integrative Comp Physiol 1998; 274: R9–R18. 93. Aschoff J. Circadian control of body temperature. J Therm Biol 1983; 8: 143–147. 94. Rechtschaffen A, Bergmann BM, Everson CA, Kushida CA, Gilliland MA. Sleep deprivation in the rat: X. integration and discussion of the findings. Sleep 1989; 12: 68–87. 95. Berger RJ, Phillips NH. Regulation of energy metabolism and body temperature during sleep and circadian torpor. In: Lydic R, Biebuyck J (eds). Clinical Physiology of Sleep. Washington, DC: American Physiological Society, 1988; 171–189. 96. Zepelin H. Mammalian sleep. In: Kryger MH, Roth T, Dement WC (eds). Principles and Practice of Sleep Medicine. Philadelphia: WB Saunders; 1994; 69–80. 97. Benington JH, Heller CH. Restoration of brain energy metabolism as the function of sleep. Prog Neurobiol 1995; 45: 347–360. 98. McGinty D, Szymusiak R. Keeping cool: a hypothesis about the mechanisms and functions of slow wave sleep. Trends Neurosci 1990; 13: 480–487. 99. Nakao M, McGinty D, Szymusiak R, Yamamoto M. A thermoregulatory model of sleep control. Jap J Physiol 1995; 45: 291–309. 100. Franken P, Tobler I, Borbely A. Effects of 12-h sleep deprivation and of 12-h cold exposure on sleep regulation and cortical temperature in the rat. Physiol Behav 1993; 54: 885–894. 101. Franken P, Dijk DJ, Tobler I, Borbely AA. Sleep deprivation in the rat: effects on EEG power spectra, vigilance states, and cortical temperature. Am J Physiol 1991; 216: R198–R208. 102. McGinty D, Szymusiak R, Thomson D. Preoptic/ anterior hypothalamic (POAH) warming suppresses laryngeal dilator activity during sleep. Sleep 1998; 21 (Suppl): 46. 103. Wehr T. Effects of wakefulness and sleep on depression and mania. In: Montplaisir J, Godbout R (eds). Sleep and Biological Rhythms. New York: Oxford University Press, 1990; 42–86. 104. Hays T, Szymuisak R, McGinty D. Intrinsic nonlinear firing rate responses to temperature found in rat diagonal band neurons in vitro. Sleep Research Online 1999; 2S: 43.