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Hypothalamic control of sleep
Sleep Medicine 8 (2007) 291–301 www.elsevier.com/locate/sleep
Hypothalamic control of sleep Ronald Szymusiak a
a,b,c,*
, Irma Gvilia
a,b,e
, Dennis McGinty
a,d
Research Service, V.A. Greater Los Angeles Healthcare System, 16111 Plummer Street, North Hills, CA 91343, USA b Department of Medicine, University of California at Los Angeles, USA c Department of Neurobiology, University of California at Los Angeles, USA d Department of Psychology, School of Medicine, University of California at Los Angeles, USA e I. Beritashvili Institute of Physiology, Tbilisi, Georgia Received 6 March 2007; accepted 6 March 2007 Available online 30 April 2007
Abstract A sleep-promoting function for the rostral hypothalamus was initially inferred from the presence of chronic insomnia following damage to this brain region. Subsequently, it was determined that a unique feature of the preoptic hypothalamus and adjacent basal forebrain is the presence of neurons that are activated during sleep compared to waking. Preoptic area ‘‘sleep-active’’ neurons have been identified by single and multiple-unit recordings and by the presence of the protein product of the c-Fos gene in the neurons of sleeping animals. Sleep-active neurons are located in several subregions of the preoptic area, occurring with high density in the ventrolateral preoptic area (vlPOA) and the median preoptic nucleus (MnPN). Neurons in the vlPOA contain the inhibitory neuromodulator, galanin, and the inhibitory neurotransmitter, GABA. A majority of MnPN neurons activated during sleep contain GABA. Anatomical tracer studies reveal projections from the vlPOA and MnPN to multiple arousal-regulatory systems in the posterior and lateral hypothalamus and the rostral brainstem. Cumulative evidence indicates that preoptic area neurons function to promote sleep onset and sleep maintenance by inhibitory modulation of multiple arousal systems. Recent studies suggest a role for preoptic area neurons in the homeostatic aspects of the regulation of both rapid eye movement (REM) and non-REM (NREM) sleep and as a potential target for endogenous somnongens, such as cytokines and adenosine. Ó 2007 Elsevier B.V. All rights reserved. Keyword: Hypothalamus
1. Introduction The hypothalamus, extending from the lamina terminalis and the preoptic area (POA) to the posterior hypothalamus and diencephalic/midbrain junction, has long been a focus of brain investigations of sleep-wake regulation. Among the first modern conceptualizations of the central organization of sleep-wake control was that of von Economo [1], who postulated the existence of sleep-promoting structures in the rostral hypothalamus * Corresponding author. Present address: Research Service, V.A. Greater Los Angeles Healthcare System, 16111 Plummer Street, North Hills, CA 91343, USA. Tel.: +1 818 891 7711x7568; fax: +1 818 895 9575. E-mail address: [email protected] (R. Szymusiak).
1389-9457/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.sleep.2007.03.013
that function in opposition to wake-promoting systems in the posterior hypothalamus. This functional-anatomical framework evolved from von Economo’s careful correlations between disturbances in sleep and consciousness in patients with viral encephalitis and subsequent localization of inflammatory brain lesions upon post mortem examination [1]. This basic organizational plan of hypothalamic sleep- and arousal-regulatory neural systems has been repeatedly confirmed and elaborated by contemporary research in sleep neurobiology. The finding that rostral hypothalamic damage causes chronic reductions in sleep has been confirmed many times, with increasingly selective methods of brain tissue destruction [2–7]. Results of lesion studies, demonstrating sleep deficits following rostral hypothalamic damage,
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are complemented by experimental findings that electrical, thermal or chemical stimulation of the POA can be sleep-promoting [8–11]. A unique feature of the POA and immediately adjacent portions of the magnocelluar basal forebrain is the prevalence in these areas of neurons that increase activity during sleep onset and sleep, compared to waking. Neurons with ‘‘sleep-active’’ discharge patterns are rarely recorded in most brain areas, but can be found in comparative abundance in certain regions of the POA and basal forebrain. Sleep-active neurons have been identified by single- and multiple-unit recordings in naturally sleeping animals, and by the presence of the protein product of the c-Fos gene in POA neurons of sleeping animals. We will review recent studies of the physiology and anatomy of putative sleep-regulatory neurons in the POA and describe their anatomical and functional relationships to arousal-regulatory neurons in the posterior hypothalamus and brainstem. This work reveals that sleep-active neurons are located in several subregions of the POA, occurring with particularly high density in the ventrolateral preoptic area (vlPOA) and the median preoptic nucleus (MnPN). Sleep-active neurons can also be found in the medial extension of vlPOA, the dorsolateral preoptic area and the adjacent basal forebrain, where they are intermingled with other cell types. POA sleep-active neurons are inhibitory in nature; vlPOA neurons contain the inhibitory neuromodulator galanin and the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). A majority of MnPN neurons expressing c-Fos-immunoreactivity (IR) during sleep are GABAergic. Anatomical tracer studies reveal projections from the vlPOA and MnPN to multiple arousal-regulatory systems in the posterior hypothalamus and brainstem. Cumulative evidence from single-unit recordings, c-Fos protein immunohistochemistry and anatomical studies support a role for POA neurons in promoting non-rapid eye movement (NREM) sleep onset and sleep maintenance by inhibitory modulation of multiple arousal systems. Recent findings suggest an additional involvement of these neurons in the homeostatic regulation of sleep and in modulating REM sleep-generating neural circuits in the brainstem. 2. Sleep-related neuronal activity in the preoptic area and basal forebrain
actively promoted sleep could be identified. Early reports confirmed the presence of neurons in the POA that exhibited elevated discharge during sleep compared to waking [12,13]. We conducted a systematic examination of neuronal discharge during natural wakefulness and sleep in the lateral POA and adjacent basal forebrain in adult cats [14,15]. Microelectrodes were directed at brain regions where electrical stimulation had been reported to be most effective in promoting electroencephalographic (EEG) synchrony and sleep [8]. We confirmed the presence of neurons with strongly sleep-related discharge [14]. Discharge rate of 20 recorded sleep-active neurons averaged 0.7 ± 0.1 spikes/s during alert, active wakefulness and 9.4 ± 1.0 spikes/s during stable NREM sleep. Activity of these neurons increased prior to sleep onset, as defined by EEG changes, during waking to NREM sleep transitions. Discharge rates were higher during episodes of drowsiness or quiet waking that evolved into bouts of stable NREM sleep, than during episodes of quiet waking that transitioned to alert wakefulness [14,15]. Discharge of sleep-active neurons during active REM sleep (REM sleep-containing high density of phasic events, including ponto-geniulo-occipital waves, rapid eye movements and phasic muscle twitches) was similar to discharge levels during active waking, but discharge rates in quiet REM sleep were intermediate between waking and NREM sleep values [14]. Using tests for antidromic activation from stimulating electrodes placed in subcortical white matter and in the rostral midbrain reticular formation, we demonstrated that some neurons with sleep-related activity were longaxoned projection neurons [15]. Throughout the POA and basal forebrain regions examined, sleep-active neurons constituted only 20%– 25% of the recorded cells. Neurons that displayed maximal activation during waking and REM sleep, with diminished discharge during NREM sleep, were the most frequently encountered cell type. In several regions, sleep-active neurons were intermingled with wake-active cell types and comprised only about 10% of sampled neurons. The highest density of sleep-active neurons was found in ventral-lateral portions of the POA. This latter site was identified by Sterman and Clemente [8] as among the most potent for electrical stimulation-induced enhancement of sleep in the cat. 3. Thermosensitivity of hypothalamic sleep-active neurons
The findings that lesions involving the POA and surrounding areas can cause insomnia [2–7] and that stimulation of these areas can promote sleep [8–11] led to the search for POA neurons that were activated selectively during sleep. This search coincided with the emerging perspective that the regulation of sleep by the brain was an active process, and that neuronal systems that
Cellular mechanisms involved in the control of body temperature and the control of sleep intersect in the rostral hypothalamus. The medial and lateral portions of the POA are critically involved in body temperature regulation. POA lesions can cause profound disturbances in thermoregulation and local warming or
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cooling of the rostral hypothalamus can evoke wholebody thermoregulatory responses [4]. The POA contains the highest density of intrinsically thermosensitive neurons of any brain region. Neurons that exhibit increased activity in response to local warming (warm-sensitive neurons) and neurons that exhibit increased activity in response to local cooling (cold-sensitive neurons) are localized in the medial and lateral POA [16]. Mild local warming of the POA evokes many of the thermoregulatory responses that normally accompany sleep onset, including reductions in shivering and non-shivering thermogenesis, peripheral vasodilation, increased heat loss and a fall in body temperature [17]. In addition, POA warming can evoke the behavioral and electrographic signs of sleep, and promote EEG slow-wave activity during sleep [9,18]. Collectively, these findings suggest the hypothesis that preoptic area sleep-regulatory neurons are thermosensitive. We evaluated this hypothesis by quantifying the discharge of POA thermosensitive neurons during waking and sleep in rats and cats. Local POA temperature was manipulated by chronically implanted, water-perfused thermodes. Bundles of microwires were positioned within the thermal field of the thermode, to permit determination of neuronal responses to increases and decreases in local temperature. Following characterization of neuronal thermosensitivity, cellular activity was quantified through 2–3 spontaneous sleep-waking cycles. We found that the majority of warm-sensitive neurons exhibit sleep-related discharge and that most cold-sensitive neurons have a waking-active discharge profile [19,20]. Warm-sensitive neurons with sleep-related activity exhibited approximately a 50% increase in discharge rate during NREM sleep compared to waking. In addition, the thermosensitivity of warm-sensitive neurons was increased during NREM sleep, that is, in response to the same increase in local temperature, warm-sensitive neurons responded with larger increases in discharge rate during NREM sleep compared to waking [21]. Since activation of warm-sensitive neurons is associated with increased heat loss and a fall in body temperature, these findings suggest a cellular mechanism underlying the fall in body temperature that normally accompanies sleep onset; that is, the increase in thermosensitivity and sleep-related activation of POA warm-sensitive neurons. The findings that local POA warming promotes sleep onset and increases EEG slow-wave activity during sleep indicates that warm-sensitive neurons have a functional role in regulating sleep onset and sleep depth. 4. c-Fos expression in preoptic area neurons during sleep Significant progress in characterizing the neuroanatomy and the neurochemistry of hypothalamic sleepregulatory neurons has been achieved by using immunostaining methods that allow mapping of acti-
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vated neurons at a larger scale than is possible with single-cell electrophysiology. Expression of c-Fos, an immediate-early gene, has been found to be correlated with increased activity in a variety of neurons [22]. Studies employing immunohistochemical detection of the protein product of the c-Fos gene have localized putative sleepregulatory neurons to the ventral lateral preoptic area (vlPOA) and the median preoptic nucleus (MnPN) of the hypothalamus (Figs. 1 and 3). Sherin et al. first examined expression of Fos in the brains of rats that were allowed spontaneous sleep-waking behavior either during the light/rest or the dark/active periods [23]. The number of c-Fos-immunoreactive neurons (IRNs) in the vlPOA of animals killed during the light cycle was significantly higher than in animals killed during the dark cycle. FosIRN counts in these animals were positively correlated with the amount of preceding sleep. To determine the role of circadian factors, the normal sleep-waking behavior and circadian phase were dissociated by depriving animals of sleep for 9 or 12 h periods during the light cycle. After sleep deprivation, some animals were killed immediately, whereas others were killed after a recovery sleep for 45, 90 or 180 min before the sacrifice. Following sleep deprivation, significant numbers of Fos-IRNs in the vlPOA were observed only in animals that were permitted a recovery sleep prior to sacrifice and the average number of Fos-IRNs in the vlPOA of these animals was positively correlated with the time spent asleep during the 1-h period prior to sacrifice. Elevated expression of c-Fos in the light period versus the dark period, positive correlation between the average number of Fos-IRNs and the amount of preceding sleep, and significant increases in Fos-IRNs during recovery sleep following sleep deprivation supported the hypothesis that the vlPOA was a critical sleep-promoting site. Rats that were sacrificed at the termination of sleep deprivation and not permitted recovery sleep did not exhibit increased numbers of Fos-IRNs in the vlPOA, suggesting that c-Fos activation in this nucleus is dependent upon the occurrence of sleep and is not related to sleepiness or sleep propensity. We examined neuronal discharge patterns in the rat vlPOA during sleep and wakefulness [24]. The goal was to determine if a high density of neurons with sleep-related discharge could be localized to the same region where neurons exhibiting sleep-related Fos-IR were so prevalent, and to characterize the activity of vlPOA neurons during NREM versus REM sleep, since studies of Fos-IR cannot temporally resolve such differences. We found that 50% of all neurons recorded in the ventral-most aspects of the lateral POA displayed elevated discharge during sleep compared to waking. This was a higher concentration of sleep-active neurons than we had previously found in other rostral hypothalamic sites. Most vlPOA neurons were activated during both NREM and REM sleep compared to waking; for
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Fig. 1. Examples of c-Fos protein immunoreactivity in the ventrolateral preoptic area (vlPOA) in one rat that was predominately asleep (% total sleep time >75%; A and B) and one rat that was predominately awake (%total sleep time <10%; C and D) during the 2-h period prior to sacrifice. Panels on the left (A and C) are photomicrographs of preoptic area tissue single immunostained for c-Fos protein (black precipitate). Panels on the right B and D) are camera lucida drawings of the same brain sections from which the photomicrographs were taken, with individual Fos-IR neurons shown as black rectangles. The grid shown in B and D is the counting grid used to quantify numbers of Fos-IR neurons in the core of the vlPOA. Note the density of Fos-IR neurons in the vlPOA of the sleeping animal compared to the absence of Fos-IR in the awake animal. Abbreviations: ac, anterior commissure; oc, optic chiasm.
the group of vlPOA neurons with sleep-related discharge, mean discharge rates during NREM and REM sleep did not differ significantly. Most vlPOA neurons exhibited increases in activity during the immediate transition period between waking and NREM sleep, suggesting that activation of vlPOA neurons occurred late in the sequence of neuronal events leading to the onset of sleep; vlPOA neurons also displayed progressively increased activation from light to deep NREM sleep (Fig. 2). In response to 12–16 h of sleep deprivation, discharge of vlPOA neurons increased during recovery sleep, but discharge rates during forced waking were similar to waking rates recorded in non-sleepdeprived rats. This latter finding was consistent with the observation that Fos-IR was enhanced in the vlPOA of sleep-deprived rats only if rats were permitted recovery sleep prior to sacrifice. Gong et al. confirmed the existence of sleep-active neurons in the vlPOA and identified a second group of such neurons in the MnPN [25]. Expression of c-Fos was examined under conditions of spontaneous sleep during the light/rest period and short-term (2 h) sleep restriction, achieved with gentle handling, during the
same period. More neurons exhibiting Fos-IR were present in the MnPN and the vlPOA in rats that were predominately asleep during the 2 h prior to sacrifice, compared to rats that were predominately awake (Fig. 3). The number of Fos-IRNs in both the MnPN and the vlPOA was positively correlated with total sleep time recorded during the 2 h prior to sacrifice. To confirm and extend findings with Fos-IR, we examined the discharge of MnPN neurons across the sleep-waking cycle in unanesthetized, unrestrained rats [26]. In a sample of 89 MnPN neurons, 76% exhibited higher discharge rate during NREM and/or REM sleep compared to waking. Fifty-eight percent of the population displayed similarly elevated discharge rates during both NREM and REM sleep compared to waking. Most of these cells showed a gradual increase in firing rate in anticipation of sleep onset. Peak levels of activity were observed early in the development of NREM sleep episodes that followed sustained episodes of waking. In contrast to vlPOA neurons that displayed increased activity from the early to late portions of individual NREM sleep episodes, discharge of MnPN neurons declined across sustained NREM sleep episodes in the
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Spikes/sec
awake
nonRem sleep
295 REM
30 20 10 0
EMG
EEG 100 sec Fig. 2. Example of extracellularly recorded activity of a vlPOA neuron in an adult rat during waking, during an extended NREM sleep episode and during REM sleep. Top panel is a rate histogram, displaying discharge rate of the neuron in (spikes/s) 1 s bins. Middle panel is dorsal neck electromyogram and the bottom panel is neocortical electroencephalogram.
Fig. 3. Examples of the distribution of Fos-IR in the rostral and caudal portions of the median preoptic nucleus (MnPN) of an asleep and an awake rat. Shown for each MnPN site is a photomicrograph of preoptic tissue single immunostained for c-Fos protein (black precipitate) and a corresponding camera lucida drawing, showing the counting grids used for quantification of cell counts. Note the presence of Fos-IR neurons in the rostral and caudal MnPN of the sleeping, but not the awake animal. Modified from Ref. [25], with permission.
absence of intervening waking (Fig. 4). Comparisons between sleep-related activity in MnPN versus vlPOA neurons suggest a more important role for MnPN neurons in initiating transitions from waking to stable NREM sleep, while vlPOA neurons may predominately function to maintain sleep stability and continuity. Subsequent investigation of Fos-IR in MnPN and vlPOA neurons in animals undergoing short-term sleep restriction (see below) supports this. 5. Neurochemistry of MnPN and vlPOA sleep-regulatory neurons A partial understanding of the functional organization of POA sleep regulatory neurons comes from the
findings on the neurochemical nature of sleep-active neurons in this area. Combining Fos-immunostaining with in situ hybridization for galanin — an inhibitory neuromodulator, Gaus et al. showed that about 80% of sleep-active cells in the vlPOA of rats that had been sleeping an average of 84% of the hour prior to death expressed the neuropeptide galanin; conversely, 52% of galanin-expressing cells were sleep-active [27]. In a previous study from this group [28], galanin in vlPOA neurons was found to be highly co-localized with GABA. Gong et al. further examined the neurotransmitter phenotype of MnPN and vlPOA sleep-active neurons [29]. To evaluate the hypothesis that MnPN and vlPOA sleep-active neurons are GABAergic, the authors combined immunostaining for c-Fos protein with
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Fig. 4. Example of discharge of a rat MnPN neuron recorded during episodes of spontaneous waking and sleep. Top panel is a hypnogram, showing the temporal distribution of waking, NREM and REM sleep. Unit, raw recording of MnPN neuronal activity; Rate, rate histogram (spikes/s) in 1 s bins; EEG, neocortical electroencephalogram; EMG, dorsal neck electromyogram. Modified from Ref. [26], with permission.
immunostaining for glutamic acid decarboxylase (GAD), a marker of GABAergic cells. The number of Fos single-, GAD single- and Fos + GAD double-IRNs was quantified throughout the MnPN and the vlPOA in rats exhibiting varying amounts of spontaneous sleep during a 2-h recording period beginning two hours after lights-on [29]. The numbers of total Fos-IRNs and Fos + GAD-IRNs in both the MnPN and the vlPOA were positively correlated with the amount of preceding sleep; a majority of MnPN and vlPOA neurons that were Fos-positive following sustained spontaneous sleep also stained for GAD (Fig. 5). The same study examined patterns of Fos + GAD-IR in the MnPN and the vlPOA after 24-h sleep deprivation. Fos + GAD-IR cell counts in the MnPN were significantly elevated in rats that were permitted 2-h recovery period following 24-h sleep deprivation compared to both sleep deprivation control and spontaneously sleeping rats. Although the three groups of rats did not exhibit significantly different sleep amounts, there was a group effect on the sleep EEG. EEG delta power in NREM sleep was significantly higher in the recovery versus the control sleep deprivation and spontaneously sleeping groups. The number of GABAergic neurons expressing Fos-IR in the MnPN and the vlPOA of sleep-deprived versus relevant control rats was slightly, but significantly, elevated even in the absence of the opportunity for recovery sleep [29]. These findings demonstrated that sleep deprivation is associated with increased activation of GABAergic neurons in the MnPN and the vlPOA, suggesting involvement of these neurons in homeostatic regulation of sleep.
6. Descending modulation of hypothalamic and brainstem arousal systems by sleep-regulatory neurons in the preoptic area Anatomical evidence suggests that preoptic area neurons function to promote sleep by descending inhibitory modulation of multiple arousal systems located in the posterior hypothalamus and brainstem. The most striking anatomical relationship is between neurons in the vlPOA and histaminergic neurons in the tuberomammillary nucleus (TMN), as originally described by Sherin et al. [23,28]. The vlPOA provides a dense projection to the histaminergic cell body regions of the TMN and is a major source of afferents to this nucleus. Discharge of TMN neurons across the sleep-waking cycle is the reciprocal of that observed in most vlPOA neurons, that is, elevated discharge during waking and reduced activity during NREM and REM sleep [30,31]. Electrical stimulation of the vlPOA area in a horizontal rat brain slice preparation containing the rostral and caudal portions of the hypothalamus, evokes GABA-mediated inhibitory postsynaptic potentials in histaminergic neurons in the TMN [32]. Collectively, anatomical and physiological evidence suggests that GABA- and galanin-containing vlPOA neurons function to inhibit the activity of TMN cells during NREM and REM sleep. The vlPOA and adjacent medial and dorsal regions are also a source of afferents to the midline and lateral dorsal raphe nucleus (DRN) and to the locus coeruleus (LC) [28,33]. The MnPN projects to these brainstem monoam-
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Fig. 5. (A and B). Examples of Fos single and Fos + GAD double immunolabeled neurons in the median preoptic nucleus from a rat that was predominately asleep (A) and a rat that was predominately awake (B) during the 2 h prior to sacrifice. Solid arrows indicated double-labeled cells, with the black precipitate of Fos-IR localized to the cell nucleus and orange-brown GAD staining present throughout the soma and dendrites. Open arrows indicate GAD single immunoreactive neurons. Calibration = 20lm. (C) Regression line scatter plots of the number of Fos + GAD-IR neurons versus total sleep time in the MnPN and vlPOA of 11 rats permitted 2 h of spontaneous sleep and waking prior to sacrifice. In each of the three POA subregions examined, there were significant positive correlations between total sleep time and Fos + GAD-IR cell counts. Modified from Ref. [29], with permission.
inergic nuclei as well [34]. Discharge of presumed serotonergic neurons in the DRN and of presumed noradrenergic neurons in the LC also exhibit the ‘‘REM-off’’ discharge pattern that is observed in TMN neurons and is the reciprocal pattern to that observed in most VLPO and MnPN sleep-active neurons [35]. Additional evidence of functional descending inhibitory projections from the POA to the DRN comes from the finding that local warming of the POA, a manipulation that activates sleep-active neurons (see above), causes suppression of waking discharge in REM-off, presumed serotonergic neurons in the DRN [36]. The hypocretin (orexin) neuronal system in the posterior hypothalamus has been implicated in several physiological functions, including regulation of behavioral and electrographic arousal. The cell bodies of hypocretin neurons are confined to the perifornical region of the lateral hypothalamus (PFLH) and the adjacent
dorsomedial hypothalamus. Hypocretin neurons have widespread projections throughout the brain and spinal cord [37–39]. Abnormalities of the hypocretin peptides and/or receptors lead to arousal deficits in experimental animals [40,41] and loss of hypocretin neurons has been implicated in the pathophysiology of human narcolepsy/ cataplexy syndrome [42,43]. Anterograde and retrograde tracer studies have documented projections from the vlPOA and MnPN to the hypocretin neuronal field in the PFLH [44]. A subset of projection neurons from the MnPN to the PFLH immunostain for GAD [45]. Projection neurons from both the MnPN and the vlPOA to the PFLH express c-Fos protein immunoreactivity during sleep [46]. Discharge of hypocretin neurons across the sleep-waking cycle is similar to that described for the monoamines, with maximal activity during waking and minimal firing during NREM and REM sleep [47–49]. Local warming of the preoptic area evokes
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suppression of waking-related neuronal activity in the PFLH [50,51] and inhibition of preoptic area neurons by local perfusion of muscimol induces Fos-IR in hypocretin neurons [52]. Suppression of hypocretin neuronal activity during sleep appears to be a consequence of increased endogenous GABA-mediated inhibition. Local microdialysis perfusion of the GABA-A receptor antagonist bicuculline into the PFLH of sleeping rats results in intense expression of Fos-IR in hypocretin neurons ipsilateral to the dialysis probe [53].
Prostaglandin D2 (PGD2) is another important endogenous sleep factor that has a site of action at least partially localized to the preoptic area [64]. Infusion of PGD2 into the subarachnoid space just rostral to the optic chiasm promotes sleep and increases the number of Fos-IRNs in the vlPOA [65].
7. Pharmacology of sleep-regulatory neurons in the preoptic area
As reviewed above, aspects of the functional and anatomical organization of sleep-regulatory neurons in the POA are understood in some detail. Neurons that exhibit sleep-related c-Fos protein immunoreactivity are localized in the vlPOA and MnPN. Electrophysiological recordings in these areas demonstrate that most sleepactive neurons exhibit elevated discharge during both NREM and REM sleep compared to waking. Additional sleep-active neurons are located more diffusely in the medial extension of the vlPOA, in the dorsolateral preoptic area and in adjacent portions of the magnocellular basal forebrain. Projections from the preoptic area to several brain regions implicated in the control of arousal have been documented. Therefore, it can be hypothesized that activation of MnPN and vlPOA neurons at the transition from waking to sleep results in GABA- and/or galanin-mediated inhibition of neurons in these arousal systems. This hypothesis is supported by findings that patterns of neuronal activity across the sleep-waking cycle in the MnPN and vlPOA are, for the most part, reciprocal to those observed in the TMN, PFLH, DRN and LC. Destruction of sleep-active neurons in one or more subregions of the POA should partially release arousal systems from sleep-related inhibitory modulation and result in chronic insomnia. The presence of persistent sleep deficits following destruction of rostral hypothalamic tissue is a robust and consistent finding in the sleep neurobiological literature. That this insomnia occurs as a result of disinhibition of arousal systems is supported by the finding that infusion of GABAergic agonist into the posterior lateral hypothalamus reverses the insomnia that follows destruction of POA neurons in cats [66]. Many POA neurons are responsive to changes in brain and/or peripheral temperature, and a significant subpopulation of sleep-active neurons are warm-sensitive. As a result, local warming of the POA in experimental animals can suppress wakingrelated neuronal activity in posterior hypothalamic and rostral brainstem arousal systems and promote sleep onset and EEG synchrony. The close functional and anatomical relationships between sleep-regulatory and thermoregulatory neurons in the preoptic area account for the ability of whole-body warming or
Critical to a complete understanding of the hypothalamic regulation of sleep and for the development of novel therapies to treat sleep disorders associated with insomnia and/or disturbed sleep is knowledge about which endogenous neurotransmitters/neuromodulators regulate the excitability of preoptic area neurons. Currently, there are significant gaps in our knowledge, but some recent progress has been made. Anatomical studies demonstrate that the vlPOA receives synaptic input from the same monoaminergic systems to which it projects [54]. Identified GABAergic neurons in the vlPOA recorded in vitro are inhibited by noradrenalin and serotonin [55]. This suggests that activation of monoaminergic systems can suppress activity in vlPOA neurons and function to promote sustained episodes of waking [56]. The inhibitory neuromodulator adenosine has been implicated in sleep regulation, and elevations in extracellular adenosine appear to be an important mechanism underlying increased sleep propensity, increased sleep amount and increased EEG slow-wave activity that occurs as a consequence of sustained waking [57]. Adenosine appears to promote sleep by inhibition of wakepromoting neurons, including cholinergic neurons in the magnocellular basal forebrain [57,58]. However, recent findings indicate that adenosine may also promote sleep by way of excitatory effects on POA sleepactive neurons through both direct and indirect actions. Bath application of adenosine produced an A1 receptormediated suppression of spontaneous inhibitory postsynaptic potentials (IPSPs) in rat vlPOA neurons recorded in vitro [59]. Administration of an adenosine A2A receptor agonist exerted direct excitatory effects on a subset of rat vlPOA neurons recorded in vitro [60]. The functional importance of this A2A effect is demonstrated by our recent finding that perfusion of A2A agonist into the lateral POA in rats has a sleep-promoting effect [61]. The cytokine, interleukin 1b (IL1-b), is also somnogenic and has been implicated in homeostatic sleep control [62]. The sleep-promoting effect of intracererbroventricularly-administered IL1-b in rats is accompanied by increased activation of c-Fos in MnPN neurons [63].
8. Summary and perspective: a role for preoptic area neurons in the homeostatic regulation of NREM and REM sleep
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increases in skin temperature to promote sleep in man [67,68]. It also provides a mechanistic explanation of why sleep onset is reliably accompanied by increased heat loss, diminished metabolism and a fall in body temperature [69]. Important details of the physiology of POA sleep-regulatory neurons remain to be determined. The extent to which the circadian modulation of sleep-wake propensity does or does not involve changes in the activity or excitability of POA sleep-active neurons is unknown. Direct and indirect projections from the suprachiasmatic nucleus (SCN) to the MnPN and vlPOA have been documented [54,70]. However, the demonstration and characterization of functional synaptic inputs from the SCN and related nuclei onto MnPN and/or VLPO sleep-active neurons are lacking. The ability of endogenous neuromodulators implicated in homeostatic sleep regulation, such as adenosine IL-1b and PGD2, to activate MnPN and vlPOA neurons suggests possible mechanisms by which sleep deprivation or sleep restriction can evoke compensatory increases in sleep propensity and sleep amount. The relationship of POA neuronal activity to homeostatic sleep need has not been extensively investigated. Published evidence from c-Fos [23] and unit recording studies [24] indicates that vlPOA neurons are not activated in response to sleep deprivation, unless animals are permitted recovery sleep. Responses of MnPN neurons to sleep deprivation are more consistent with a potential role in homeostatic aspects of sleep control [26,29]. We have recently evaluated patterns of Fos-IR in MnPN and vlPOA neurons following acute total sleep deprivation and selective REM sleep restriction, in an attempt to clarify relationships of POA neuronal activation to homeostatic sleep pressure versus the actual occurrence of sleep. In one set of experiments, attempts were made to subject groups of rats to experimental conditions that differentially manipulated the level of sleep pressure and the actual amount of sleep [71]. One group of rats was permitted 2 h of spontaneous sleep beginning 1 h after lights-on (moderate sleep pressure, high sleep amount), 2 h of spontaneous sleep beginning 1 h after lights-off (low sleep pressure, low sleep amount) and 2 h of total sleep deprivation beginning 1 h after lights-out (high sleep pressure, no sleep). Consistent with previous findings, Fos-IR cell counts in the vlPOA were higher during spontaneous sleep in the light than during sleep deprivation in the light. Levels of sleep pressure did have some effect on vlPOA neurons, as cell counts were lower during spontaneous sleep in the dark compared to sleep deprivation in the light. In the MnPN, highest FosIR cell counts were observed in the sleep-deprived condition, indicating maximal activation of these cells in response to sleep pressure as opposed to the occurrence of sleep.
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A second set of experiments was designed to expose rats to conditions that differentially manipulated levels of REM sleep homeostatic pressure and actual REM sleep amount. Expression of c-Fos in MnPN and vlPOA neurons was examined under conditions of spontaneous sleep with differing amounts of REM sleep, REM sleep restriction and REM sleep recovery following REM sleep restriction [72]. Across all conditions, the number of Fos-IRNs in the MnPN was highest in REM sleeprestricted rats displaying the highest levels of REM sleep homeostatic pressure/drive, that is, those rats exhibiting the most frequent attempts to enter REM sleep. In vlPOA, the number of Fos-IRNs also increased with increasing REM pressure during REM restriction. These finding provides the first evidence that activation of subsets of MnPN and vlPOA neurons is more strongly related to REM sleep pressure than to REM sleep amount, since accumulated REM sleep time in REM sleep-restricted rats was significantly lower than in all other groups. Collectively, these experiments indicate that MnPN neurons are strongly responsive to homeostatic need for NREM and REM sleep, independent of sleep amount. These findings suggest a role for these neurons in promoting sleep onset subsequent to episodes of sustained waking, and in modulating the activity of brainstem REM sleep-generating mechanisms to evoke REM rebound in response to total sleep and/or selective REM sleep restriction. By comparison, vlPOA neurons are only moderately activated in response to increased homeostatic sleep pressure following total sleep deprivation, but do become strongly activated during recovery sleep. This suggests that these neurons are involved in consolidating sleep and promoting sleep maintenance in response to sustained waking. A subset of vlPOA neurons does become activated in response to elevated REM sleep pressure occurring as a consequence of selective REM restriction. Therefore, both MnPN and vlPOA neurons may function to modulate activity among REM sleep-generating circuits in the brainstem during normal and deprived sleep by inhibition of brainstem serotonergic and noradrenergic neurons. The role of adenosine, IL-b and other endogenous somnogenic substances in mediating changes in the activity of MnPN and vlPOA neurons in response to homeostatic sleep need remains to be clarified.
Acknowledgements Supported by the Medical Research Service of the Departments of Veterans Affairs, and National Institutes of Health Grants MH63323 and HL60296. The authors thank Md. Noor Alam, Hui Gong, Natalia Suntsova, Melvi Methhipara and Aaron Uschakov for their critical contributions.
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Hypothalamic control of sleep Ronald Szymusiak a
a,b,c,*
, Irma Gvilia
a,b,e
, Dennis McGinty
a,d
Research Service, V.A. Greater Los Angeles Healthcare System, 16111 Plummer Street, North Hills, CA 91343, USA b Department of Medicine, University of California at Los Angeles, USA c Department of Neurobiology, University of California at Los Angeles, USA d Department of Psychology, School of Medicine, University of California at Los Angeles, USA e I. Beritashvili Institute of Physiology, Tbilisi, Georgia Received 6 March 2007; accepted 6 March 2007 Available online 30 April 2007
Abstract A sleep-promoting function for the rostral hypothalamus was initially inferred from the presence of chronic insomnia following damage to this brain region. Subsequently, it was determined that a unique feature of the preoptic hypothalamus and adjacent basal forebrain is the presence of neurons that are activated during sleep compared to waking. Preoptic area ‘‘sleep-active’’ neurons have been identified by single and multiple-unit recordings and by the presence of the protein product of the c-Fos gene in the neurons of sleeping animals. Sleep-active neurons are located in several subregions of the preoptic area, occurring with high density in the ventrolateral preoptic area (vlPOA) and the median preoptic nucleus (MnPN). Neurons in the vlPOA contain the inhibitory neuromodulator, galanin, and the inhibitory neurotransmitter, GABA. A majority of MnPN neurons activated during sleep contain GABA. Anatomical tracer studies reveal projections from the vlPOA and MnPN to multiple arousal-regulatory systems in the posterior and lateral hypothalamus and the rostral brainstem. Cumulative evidence indicates that preoptic area neurons function to promote sleep onset and sleep maintenance by inhibitory modulation of multiple arousal systems. Recent studies suggest a role for preoptic area neurons in the homeostatic aspects of the regulation of both rapid eye movement (REM) and non-REM (NREM) sleep and as a potential target for endogenous somnongens, such as cytokines and adenosine. Ó 2007 Elsevier B.V. All rights reserved. Keyword: Hypothalamus
1. Introduction The hypothalamus, extending from the lamina terminalis and the preoptic area (POA) to the posterior hypothalamus and diencephalic/midbrain junction, has long been a focus of brain investigations of sleep-wake regulation. Among the first modern conceptualizations of the central organization of sleep-wake control was that of von Economo [1], who postulated the existence of sleep-promoting structures in the rostral hypothalamus * Corresponding author. Present address: Research Service, V.A. Greater Los Angeles Healthcare System, 16111 Plummer Street, North Hills, CA 91343, USA. Tel.: +1 818 891 7711x7568; fax: +1 818 895 9575. E-mail address: [email protected] (R. Szymusiak).
1389-9457/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.sleep.2007.03.013
that function in opposition to wake-promoting systems in the posterior hypothalamus. This functional-anatomical framework evolved from von Economo’s careful correlations between disturbances in sleep and consciousness in patients with viral encephalitis and subsequent localization of inflammatory brain lesions upon post mortem examination [1]. This basic organizational plan of hypothalamic sleep- and arousal-regulatory neural systems has been repeatedly confirmed and elaborated by contemporary research in sleep neurobiology. The finding that rostral hypothalamic damage causes chronic reductions in sleep has been confirmed many times, with increasingly selective methods of brain tissue destruction [2–7]. Results of lesion studies, demonstrating sleep deficits following rostral hypothalamic damage,
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are complemented by experimental findings that electrical, thermal or chemical stimulation of the POA can be sleep-promoting [8–11]. A unique feature of the POA and immediately adjacent portions of the magnocelluar basal forebrain is the prevalence in these areas of neurons that increase activity during sleep onset and sleep, compared to waking. Neurons with ‘‘sleep-active’’ discharge patterns are rarely recorded in most brain areas, but can be found in comparative abundance in certain regions of the POA and basal forebrain. Sleep-active neurons have been identified by single- and multiple-unit recordings in naturally sleeping animals, and by the presence of the protein product of the c-Fos gene in POA neurons of sleeping animals. We will review recent studies of the physiology and anatomy of putative sleep-regulatory neurons in the POA and describe their anatomical and functional relationships to arousal-regulatory neurons in the posterior hypothalamus and brainstem. This work reveals that sleep-active neurons are located in several subregions of the POA, occurring with particularly high density in the ventrolateral preoptic area (vlPOA) and the median preoptic nucleus (MnPN). Sleep-active neurons can also be found in the medial extension of vlPOA, the dorsolateral preoptic area and the adjacent basal forebrain, where they are intermingled with other cell types. POA sleep-active neurons are inhibitory in nature; vlPOA neurons contain the inhibitory neuromodulator galanin and the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). A majority of MnPN neurons expressing c-Fos-immunoreactivity (IR) during sleep are GABAergic. Anatomical tracer studies reveal projections from the vlPOA and MnPN to multiple arousal-regulatory systems in the posterior hypothalamus and brainstem. Cumulative evidence from single-unit recordings, c-Fos protein immunohistochemistry and anatomical studies support a role for POA neurons in promoting non-rapid eye movement (NREM) sleep onset and sleep maintenance by inhibitory modulation of multiple arousal systems. Recent findings suggest an additional involvement of these neurons in the homeostatic regulation of sleep and in modulating REM sleep-generating neural circuits in the brainstem. 2. Sleep-related neuronal activity in the preoptic area and basal forebrain
actively promoted sleep could be identified. Early reports confirmed the presence of neurons in the POA that exhibited elevated discharge during sleep compared to waking [12,13]. We conducted a systematic examination of neuronal discharge during natural wakefulness and sleep in the lateral POA and adjacent basal forebrain in adult cats [14,15]. Microelectrodes were directed at brain regions where electrical stimulation had been reported to be most effective in promoting electroencephalographic (EEG) synchrony and sleep [8]. We confirmed the presence of neurons with strongly sleep-related discharge [14]. Discharge rate of 20 recorded sleep-active neurons averaged 0.7 ± 0.1 spikes/s during alert, active wakefulness and 9.4 ± 1.0 spikes/s during stable NREM sleep. Activity of these neurons increased prior to sleep onset, as defined by EEG changes, during waking to NREM sleep transitions. Discharge rates were higher during episodes of drowsiness or quiet waking that evolved into bouts of stable NREM sleep, than during episodes of quiet waking that transitioned to alert wakefulness [14,15]. Discharge of sleep-active neurons during active REM sleep (REM sleep-containing high density of phasic events, including ponto-geniulo-occipital waves, rapid eye movements and phasic muscle twitches) was similar to discharge levels during active waking, but discharge rates in quiet REM sleep were intermediate between waking and NREM sleep values [14]. Using tests for antidromic activation from stimulating electrodes placed in subcortical white matter and in the rostral midbrain reticular formation, we demonstrated that some neurons with sleep-related activity were longaxoned projection neurons [15]. Throughout the POA and basal forebrain regions examined, sleep-active neurons constituted only 20%– 25% of the recorded cells. Neurons that displayed maximal activation during waking and REM sleep, with diminished discharge during NREM sleep, were the most frequently encountered cell type. In several regions, sleep-active neurons were intermingled with wake-active cell types and comprised only about 10% of sampled neurons. The highest density of sleep-active neurons was found in ventral-lateral portions of the POA. This latter site was identified by Sterman and Clemente [8] as among the most potent for electrical stimulation-induced enhancement of sleep in the cat. 3. Thermosensitivity of hypothalamic sleep-active neurons
The findings that lesions involving the POA and surrounding areas can cause insomnia [2–7] and that stimulation of these areas can promote sleep [8–11] led to the search for POA neurons that were activated selectively during sleep. This search coincided with the emerging perspective that the regulation of sleep by the brain was an active process, and that neuronal systems that
Cellular mechanisms involved in the control of body temperature and the control of sleep intersect in the rostral hypothalamus. The medial and lateral portions of the POA are critically involved in body temperature regulation. POA lesions can cause profound disturbances in thermoregulation and local warming or
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cooling of the rostral hypothalamus can evoke wholebody thermoregulatory responses [4]. The POA contains the highest density of intrinsically thermosensitive neurons of any brain region. Neurons that exhibit increased activity in response to local warming (warm-sensitive neurons) and neurons that exhibit increased activity in response to local cooling (cold-sensitive neurons) are localized in the medial and lateral POA [16]. Mild local warming of the POA evokes many of the thermoregulatory responses that normally accompany sleep onset, including reductions in shivering and non-shivering thermogenesis, peripheral vasodilation, increased heat loss and a fall in body temperature [17]. In addition, POA warming can evoke the behavioral and electrographic signs of sleep, and promote EEG slow-wave activity during sleep [9,18]. Collectively, these findings suggest the hypothesis that preoptic area sleep-regulatory neurons are thermosensitive. We evaluated this hypothesis by quantifying the discharge of POA thermosensitive neurons during waking and sleep in rats and cats. Local POA temperature was manipulated by chronically implanted, water-perfused thermodes. Bundles of microwires were positioned within the thermal field of the thermode, to permit determination of neuronal responses to increases and decreases in local temperature. Following characterization of neuronal thermosensitivity, cellular activity was quantified through 2–3 spontaneous sleep-waking cycles. We found that the majority of warm-sensitive neurons exhibit sleep-related discharge and that most cold-sensitive neurons have a waking-active discharge profile [19,20]. Warm-sensitive neurons with sleep-related activity exhibited approximately a 50% increase in discharge rate during NREM sleep compared to waking. In addition, the thermosensitivity of warm-sensitive neurons was increased during NREM sleep, that is, in response to the same increase in local temperature, warm-sensitive neurons responded with larger increases in discharge rate during NREM sleep compared to waking [21]. Since activation of warm-sensitive neurons is associated with increased heat loss and a fall in body temperature, these findings suggest a cellular mechanism underlying the fall in body temperature that normally accompanies sleep onset; that is, the increase in thermosensitivity and sleep-related activation of POA warm-sensitive neurons. The findings that local POA warming promotes sleep onset and increases EEG slow-wave activity during sleep indicates that warm-sensitive neurons have a functional role in regulating sleep onset and sleep depth. 4. c-Fos expression in preoptic area neurons during sleep Significant progress in characterizing the neuroanatomy and the neurochemistry of hypothalamic sleepregulatory neurons has been achieved by using immunostaining methods that allow mapping of acti-
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vated neurons at a larger scale than is possible with single-cell electrophysiology. Expression of c-Fos, an immediate-early gene, has been found to be correlated with increased activity in a variety of neurons [22]. Studies employing immunohistochemical detection of the protein product of the c-Fos gene have localized putative sleepregulatory neurons to the ventral lateral preoptic area (vlPOA) and the median preoptic nucleus (MnPN) of the hypothalamus (Figs. 1 and 3). Sherin et al. first examined expression of Fos in the brains of rats that were allowed spontaneous sleep-waking behavior either during the light/rest or the dark/active periods [23]. The number of c-Fos-immunoreactive neurons (IRNs) in the vlPOA of animals killed during the light cycle was significantly higher than in animals killed during the dark cycle. FosIRN counts in these animals were positively correlated with the amount of preceding sleep. To determine the role of circadian factors, the normal sleep-waking behavior and circadian phase were dissociated by depriving animals of sleep for 9 or 12 h periods during the light cycle. After sleep deprivation, some animals were killed immediately, whereas others were killed after a recovery sleep for 45, 90 or 180 min before the sacrifice. Following sleep deprivation, significant numbers of Fos-IRNs in the vlPOA were observed only in animals that were permitted a recovery sleep prior to sacrifice and the average number of Fos-IRNs in the vlPOA of these animals was positively correlated with the time spent asleep during the 1-h period prior to sacrifice. Elevated expression of c-Fos in the light period versus the dark period, positive correlation between the average number of Fos-IRNs and the amount of preceding sleep, and significant increases in Fos-IRNs during recovery sleep following sleep deprivation supported the hypothesis that the vlPOA was a critical sleep-promoting site. Rats that were sacrificed at the termination of sleep deprivation and not permitted recovery sleep did not exhibit increased numbers of Fos-IRNs in the vlPOA, suggesting that c-Fos activation in this nucleus is dependent upon the occurrence of sleep and is not related to sleepiness or sleep propensity. We examined neuronal discharge patterns in the rat vlPOA during sleep and wakefulness [24]. The goal was to determine if a high density of neurons with sleep-related discharge could be localized to the same region where neurons exhibiting sleep-related Fos-IR were so prevalent, and to characterize the activity of vlPOA neurons during NREM versus REM sleep, since studies of Fos-IR cannot temporally resolve such differences. We found that 50% of all neurons recorded in the ventral-most aspects of the lateral POA displayed elevated discharge during sleep compared to waking. This was a higher concentration of sleep-active neurons than we had previously found in other rostral hypothalamic sites. Most vlPOA neurons were activated during both NREM and REM sleep compared to waking; for
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Fig. 1. Examples of c-Fos protein immunoreactivity in the ventrolateral preoptic area (vlPOA) in one rat that was predominately asleep (% total sleep time >75%; A and B) and one rat that was predominately awake (%total sleep time <10%; C and D) during the 2-h period prior to sacrifice. Panels on the left (A and C) are photomicrographs of preoptic area tissue single immunostained for c-Fos protein (black precipitate). Panels on the right B and D) are camera lucida drawings of the same brain sections from which the photomicrographs were taken, with individual Fos-IR neurons shown as black rectangles. The grid shown in B and D is the counting grid used to quantify numbers of Fos-IR neurons in the core of the vlPOA. Note the density of Fos-IR neurons in the vlPOA of the sleeping animal compared to the absence of Fos-IR in the awake animal. Abbreviations: ac, anterior commissure; oc, optic chiasm.
the group of vlPOA neurons with sleep-related discharge, mean discharge rates during NREM and REM sleep did not differ significantly. Most vlPOA neurons exhibited increases in activity during the immediate transition period between waking and NREM sleep, suggesting that activation of vlPOA neurons occurred late in the sequence of neuronal events leading to the onset of sleep; vlPOA neurons also displayed progressively increased activation from light to deep NREM sleep (Fig. 2). In response to 12–16 h of sleep deprivation, discharge of vlPOA neurons increased during recovery sleep, but discharge rates during forced waking were similar to waking rates recorded in non-sleepdeprived rats. This latter finding was consistent with the observation that Fos-IR was enhanced in the vlPOA of sleep-deprived rats only if rats were permitted recovery sleep prior to sacrifice. Gong et al. confirmed the existence of sleep-active neurons in the vlPOA and identified a second group of such neurons in the MnPN [25]. Expression of c-Fos was examined under conditions of spontaneous sleep during the light/rest period and short-term (2 h) sleep restriction, achieved with gentle handling, during the
same period. More neurons exhibiting Fos-IR were present in the MnPN and the vlPOA in rats that were predominately asleep during the 2 h prior to sacrifice, compared to rats that were predominately awake (Fig. 3). The number of Fos-IRNs in both the MnPN and the vlPOA was positively correlated with total sleep time recorded during the 2 h prior to sacrifice. To confirm and extend findings with Fos-IR, we examined the discharge of MnPN neurons across the sleep-waking cycle in unanesthetized, unrestrained rats [26]. In a sample of 89 MnPN neurons, 76% exhibited higher discharge rate during NREM and/or REM sleep compared to waking. Fifty-eight percent of the population displayed similarly elevated discharge rates during both NREM and REM sleep compared to waking. Most of these cells showed a gradual increase in firing rate in anticipation of sleep onset. Peak levels of activity were observed early in the development of NREM sleep episodes that followed sustained episodes of waking. In contrast to vlPOA neurons that displayed increased activity from the early to late portions of individual NREM sleep episodes, discharge of MnPN neurons declined across sustained NREM sleep episodes in the
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Spikes/sec
awake
nonRem sleep
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30 20 10 0
EMG
EEG 100 sec Fig. 2. Example of extracellularly recorded activity of a vlPOA neuron in an adult rat during waking, during an extended NREM sleep episode and during REM sleep. Top panel is a rate histogram, displaying discharge rate of the neuron in (spikes/s) 1 s bins. Middle panel is dorsal neck electromyogram and the bottom panel is neocortical electroencephalogram.
Fig. 3. Examples of the distribution of Fos-IR in the rostral and caudal portions of the median preoptic nucleus (MnPN) of an asleep and an awake rat. Shown for each MnPN site is a photomicrograph of preoptic tissue single immunostained for c-Fos protein (black precipitate) and a corresponding camera lucida drawing, showing the counting grids used for quantification of cell counts. Note the presence of Fos-IR neurons in the rostral and caudal MnPN of the sleeping, but not the awake animal. Modified from Ref. [25], with permission.
absence of intervening waking (Fig. 4). Comparisons between sleep-related activity in MnPN versus vlPOA neurons suggest a more important role for MnPN neurons in initiating transitions from waking to stable NREM sleep, while vlPOA neurons may predominately function to maintain sleep stability and continuity. Subsequent investigation of Fos-IR in MnPN and vlPOA neurons in animals undergoing short-term sleep restriction (see below) supports this. 5. Neurochemistry of MnPN and vlPOA sleep-regulatory neurons A partial understanding of the functional organization of POA sleep regulatory neurons comes from the
findings on the neurochemical nature of sleep-active neurons in this area. Combining Fos-immunostaining with in situ hybridization for galanin — an inhibitory neuromodulator, Gaus et al. showed that about 80% of sleep-active cells in the vlPOA of rats that had been sleeping an average of 84% of the hour prior to death expressed the neuropeptide galanin; conversely, 52% of galanin-expressing cells were sleep-active [27]. In a previous study from this group [28], galanin in vlPOA neurons was found to be highly co-localized with GABA. Gong et al. further examined the neurotransmitter phenotype of MnPN and vlPOA sleep-active neurons [29]. To evaluate the hypothesis that MnPN and vlPOA sleep-active neurons are GABAergic, the authors combined immunostaining for c-Fos protein with
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Fig. 4. Example of discharge of a rat MnPN neuron recorded during episodes of spontaneous waking and sleep. Top panel is a hypnogram, showing the temporal distribution of waking, NREM and REM sleep. Unit, raw recording of MnPN neuronal activity; Rate, rate histogram (spikes/s) in 1 s bins; EEG, neocortical electroencephalogram; EMG, dorsal neck electromyogram. Modified from Ref. [26], with permission.
immunostaining for glutamic acid decarboxylase (GAD), a marker of GABAergic cells. The number of Fos single-, GAD single- and Fos + GAD double-IRNs was quantified throughout the MnPN and the vlPOA in rats exhibiting varying amounts of spontaneous sleep during a 2-h recording period beginning two hours after lights-on [29]. The numbers of total Fos-IRNs and Fos + GAD-IRNs in both the MnPN and the vlPOA were positively correlated with the amount of preceding sleep; a majority of MnPN and vlPOA neurons that were Fos-positive following sustained spontaneous sleep also stained for GAD (Fig. 5). The same study examined patterns of Fos + GAD-IR in the MnPN and the vlPOA after 24-h sleep deprivation. Fos + GAD-IR cell counts in the MnPN were significantly elevated in rats that were permitted 2-h recovery period following 24-h sleep deprivation compared to both sleep deprivation control and spontaneously sleeping rats. Although the three groups of rats did not exhibit significantly different sleep amounts, there was a group effect on the sleep EEG. EEG delta power in NREM sleep was significantly higher in the recovery versus the control sleep deprivation and spontaneously sleeping groups. The number of GABAergic neurons expressing Fos-IR in the MnPN and the vlPOA of sleep-deprived versus relevant control rats was slightly, but significantly, elevated even in the absence of the opportunity for recovery sleep [29]. These findings demonstrated that sleep deprivation is associated with increased activation of GABAergic neurons in the MnPN and the vlPOA, suggesting involvement of these neurons in homeostatic regulation of sleep.
6. Descending modulation of hypothalamic and brainstem arousal systems by sleep-regulatory neurons in the preoptic area Anatomical evidence suggests that preoptic area neurons function to promote sleep by descending inhibitory modulation of multiple arousal systems located in the posterior hypothalamus and brainstem. The most striking anatomical relationship is between neurons in the vlPOA and histaminergic neurons in the tuberomammillary nucleus (TMN), as originally described by Sherin et al. [23,28]. The vlPOA provides a dense projection to the histaminergic cell body regions of the TMN and is a major source of afferents to this nucleus. Discharge of TMN neurons across the sleep-waking cycle is the reciprocal of that observed in most vlPOA neurons, that is, elevated discharge during waking and reduced activity during NREM and REM sleep [30,31]. Electrical stimulation of the vlPOA area in a horizontal rat brain slice preparation containing the rostral and caudal portions of the hypothalamus, evokes GABA-mediated inhibitory postsynaptic potentials in histaminergic neurons in the TMN [32]. Collectively, anatomical and physiological evidence suggests that GABA- and galanin-containing vlPOA neurons function to inhibit the activity of TMN cells during NREM and REM sleep. The vlPOA and adjacent medial and dorsal regions are also a source of afferents to the midline and lateral dorsal raphe nucleus (DRN) and to the locus coeruleus (LC) [28,33]. The MnPN projects to these brainstem monoam-
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Fig. 5. (A and B). Examples of Fos single and Fos + GAD double immunolabeled neurons in the median preoptic nucleus from a rat that was predominately asleep (A) and a rat that was predominately awake (B) during the 2 h prior to sacrifice. Solid arrows indicated double-labeled cells, with the black precipitate of Fos-IR localized to the cell nucleus and orange-brown GAD staining present throughout the soma and dendrites. Open arrows indicate GAD single immunoreactive neurons. Calibration = 20lm. (C) Regression line scatter plots of the number of Fos + GAD-IR neurons versus total sleep time in the MnPN and vlPOA of 11 rats permitted 2 h of spontaneous sleep and waking prior to sacrifice. In each of the three POA subregions examined, there were significant positive correlations between total sleep time and Fos + GAD-IR cell counts. Modified from Ref. [29], with permission.
inergic nuclei as well [34]. Discharge of presumed serotonergic neurons in the DRN and of presumed noradrenergic neurons in the LC also exhibit the ‘‘REM-off’’ discharge pattern that is observed in TMN neurons and is the reciprocal pattern to that observed in most VLPO and MnPN sleep-active neurons [35]. Additional evidence of functional descending inhibitory projections from the POA to the DRN comes from the finding that local warming of the POA, a manipulation that activates sleep-active neurons (see above), causes suppression of waking discharge in REM-off, presumed serotonergic neurons in the DRN [36]. The hypocretin (orexin) neuronal system in the posterior hypothalamus has been implicated in several physiological functions, including regulation of behavioral and electrographic arousal. The cell bodies of hypocretin neurons are confined to the perifornical region of the lateral hypothalamus (PFLH) and the adjacent
dorsomedial hypothalamus. Hypocretin neurons have widespread projections throughout the brain and spinal cord [37–39]. Abnormalities of the hypocretin peptides and/or receptors lead to arousal deficits in experimental animals [40,41] and loss of hypocretin neurons has been implicated in the pathophysiology of human narcolepsy/ cataplexy syndrome [42,43]. Anterograde and retrograde tracer studies have documented projections from the vlPOA and MnPN to the hypocretin neuronal field in the PFLH [44]. A subset of projection neurons from the MnPN to the PFLH immunostain for GAD [45]. Projection neurons from both the MnPN and the vlPOA to the PFLH express c-Fos protein immunoreactivity during sleep [46]. Discharge of hypocretin neurons across the sleep-waking cycle is similar to that described for the monoamines, with maximal activity during waking and minimal firing during NREM and REM sleep [47–49]. Local warming of the preoptic area evokes
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suppression of waking-related neuronal activity in the PFLH [50,51] and inhibition of preoptic area neurons by local perfusion of muscimol induces Fos-IR in hypocretin neurons [52]. Suppression of hypocretin neuronal activity during sleep appears to be a consequence of increased endogenous GABA-mediated inhibition. Local microdialysis perfusion of the GABA-A receptor antagonist bicuculline into the PFLH of sleeping rats results in intense expression of Fos-IR in hypocretin neurons ipsilateral to the dialysis probe [53].
Prostaglandin D2 (PGD2) is another important endogenous sleep factor that has a site of action at least partially localized to the preoptic area [64]. Infusion of PGD2 into the subarachnoid space just rostral to the optic chiasm promotes sleep and increases the number of Fos-IRNs in the vlPOA [65].
7. Pharmacology of sleep-regulatory neurons in the preoptic area
As reviewed above, aspects of the functional and anatomical organization of sleep-regulatory neurons in the POA are understood in some detail. Neurons that exhibit sleep-related c-Fos protein immunoreactivity are localized in the vlPOA and MnPN. Electrophysiological recordings in these areas demonstrate that most sleepactive neurons exhibit elevated discharge during both NREM and REM sleep compared to waking. Additional sleep-active neurons are located more diffusely in the medial extension of the vlPOA, in the dorsolateral preoptic area and in adjacent portions of the magnocellular basal forebrain. Projections from the preoptic area to several brain regions implicated in the control of arousal have been documented. Therefore, it can be hypothesized that activation of MnPN and vlPOA neurons at the transition from waking to sleep results in GABA- and/or galanin-mediated inhibition of neurons in these arousal systems. This hypothesis is supported by findings that patterns of neuronal activity across the sleep-waking cycle in the MnPN and vlPOA are, for the most part, reciprocal to those observed in the TMN, PFLH, DRN and LC. Destruction of sleep-active neurons in one or more subregions of the POA should partially release arousal systems from sleep-related inhibitory modulation and result in chronic insomnia. The presence of persistent sleep deficits following destruction of rostral hypothalamic tissue is a robust and consistent finding in the sleep neurobiological literature. That this insomnia occurs as a result of disinhibition of arousal systems is supported by the finding that infusion of GABAergic agonist into the posterior lateral hypothalamus reverses the insomnia that follows destruction of POA neurons in cats [66]. Many POA neurons are responsive to changes in brain and/or peripheral temperature, and a significant subpopulation of sleep-active neurons are warm-sensitive. As a result, local warming of the POA in experimental animals can suppress wakingrelated neuronal activity in posterior hypothalamic and rostral brainstem arousal systems and promote sleep onset and EEG synchrony. The close functional and anatomical relationships between sleep-regulatory and thermoregulatory neurons in the preoptic area account for the ability of whole-body warming or
Critical to a complete understanding of the hypothalamic regulation of sleep and for the development of novel therapies to treat sleep disorders associated with insomnia and/or disturbed sleep is knowledge about which endogenous neurotransmitters/neuromodulators regulate the excitability of preoptic area neurons. Currently, there are significant gaps in our knowledge, but some recent progress has been made. Anatomical studies demonstrate that the vlPOA receives synaptic input from the same monoaminergic systems to which it projects [54]. Identified GABAergic neurons in the vlPOA recorded in vitro are inhibited by noradrenalin and serotonin [55]. This suggests that activation of monoaminergic systems can suppress activity in vlPOA neurons and function to promote sustained episodes of waking [56]. The inhibitory neuromodulator adenosine has been implicated in sleep regulation, and elevations in extracellular adenosine appear to be an important mechanism underlying increased sleep propensity, increased sleep amount and increased EEG slow-wave activity that occurs as a consequence of sustained waking [57]. Adenosine appears to promote sleep by inhibition of wakepromoting neurons, including cholinergic neurons in the magnocellular basal forebrain [57,58]. However, recent findings indicate that adenosine may also promote sleep by way of excitatory effects on POA sleepactive neurons through both direct and indirect actions. Bath application of adenosine produced an A1 receptormediated suppression of spontaneous inhibitory postsynaptic potentials (IPSPs) in rat vlPOA neurons recorded in vitro [59]. Administration of an adenosine A2A receptor agonist exerted direct excitatory effects on a subset of rat vlPOA neurons recorded in vitro [60]. The functional importance of this A2A effect is demonstrated by our recent finding that perfusion of A2A agonist into the lateral POA in rats has a sleep-promoting effect [61]. The cytokine, interleukin 1b (IL1-b), is also somnogenic and has been implicated in homeostatic sleep control [62]. The sleep-promoting effect of intracererbroventricularly-administered IL1-b in rats is accompanied by increased activation of c-Fos in MnPN neurons [63].
8. Summary and perspective: a role for preoptic area neurons in the homeostatic regulation of NREM and REM sleep
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increases in skin temperature to promote sleep in man [67,68]. It also provides a mechanistic explanation of why sleep onset is reliably accompanied by increased heat loss, diminished metabolism and a fall in body temperature [69]. Important details of the physiology of POA sleep-regulatory neurons remain to be determined. The extent to which the circadian modulation of sleep-wake propensity does or does not involve changes in the activity or excitability of POA sleep-active neurons is unknown. Direct and indirect projections from the suprachiasmatic nucleus (SCN) to the MnPN and vlPOA have been documented [54,70]. However, the demonstration and characterization of functional synaptic inputs from the SCN and related nuclei onto MnPN and/or VLPO sleep-active neurons are lacking. The ability of endogenous neuromodulators implicated in homeostatic sleep regulation, such as adenosine IL-1b and PGD2, to activate MnPN and vlPOA neurons suggests possible mechanisms by which sleep deprivation or sleep restriction can evoke compensatory increases in sleep propensity and sleep amount. The relationship of POA neuronal activity to homeostatic sleep need has not been extensively investigated. Published evidence from c-Fos [23] and unit recording studies [24] indicates that vlPOA neurons are not activated in response to sleep deprivation, unless animals are permitted recovery sleep. Responses of MnPN neurons to sleep deprivation are more consistent with a potential role in homeostatic aspects of sleep control [26,29]. We have recently evaluated patterns of Fos-IR in MnPN and vlPOA neurons following acute total sleep deprivation and selective REM sleep restriction, in an attempt to clarify relationships of POA neuronal activation to homeostatic sleep pressure versus the actual occurrence of sleep. In one set of experiments, attempts were made to subject groups of rats to experimental conditions that differentially manipulated the level of sleep pressure and the actual amount of sleep [71]. One group of rats was permitted 2 h of spontaneous sleep beginning 1 h after lights-on (moderate sleep pressure, high sleep amount), 2 h of spontaneous sleep beginning 1 h after lights-off (low sleep pressure, low sleep amount) and 2 h of total sleep deprivation beginning 1 h after lights-out (high sleep pressure, no sleep). Consistent with previous findings, Fos-IR cell counts in the vlPOA were higher during spontaneous sleep in the light than during sleep deprivation in the light. Levels of sleep pressure did have some effect on vlPOA neurons, as cell counts were lower during spontaneous sleep in the dark compared to sleep deprivation in the light. In the MnPN, highest FosIR cell counts were observed in the sleep-deprived condition, indicating maximal activation of these cells in response to sleep pressure as opposed to the occurrence of sleep.
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A second set of experiments was designed to expose rats to conditions that differentially manipulated levels of REM sleep homeostatic pressure and actual REM sleep amount. Expression of c-Fos in MnPN and vlPOA neurons was examined under conditions of spontaneous sleep with differing amounts of REM sleep, REM sleep restriction and REM sleep recovery following REM sleep restriction [72]. Across all conditions, the number of Fos-IRNs in the MnPN was highest in REM sleeprestricted rats displaying the highest levels of REM sleep homeostatic pressure/drive, that is, those rats exhibiting the most frequent attempts to enter REM sleep. In vlPOA, the number of Fos-IRNs also increased with increasing REM pressure during REM restriction. These finding provides the first evidence that activation of subsets of MnPN and vlPOA neurons is more strongly related to REM sleep pressure than to REM sleep amount, since accumulated REM sleep time in REM sleep-restricted rats was significantly lower than in all other groups. Collectively, these experiments indicate that MnPN neurons are strongly responsive to homeostatic need for NREM and REM sleep, independent of sleep amount. These findings suggest a role for these neurons in promoting sleep onset subsequent to episodes of sustained waking, and in modulating the activity of brainstem REM sleep-generating mechanisms to evoke REM rebound in response to total sleep and/or selective REM sleep restriction. By comparison, vlPOA neurons are only moderately activated in response to increased homeostatic sleep pressure following total sleep deprivation, but do become strongly activated during recovery sleep. This suggests that these neurons are involved in consolidating sleep and promoting sleep maintenance in response to sustained waking. A subset of vlPOA neurons does become activated in response to elevated REM sleep pressure occurring as a consequence of selective REM restriction. Therefore, both MnPN and vlPOA neurons may function to modulate activity among REM sleep-generating circuits in the brainstem during normal and deprived sleep by inhibition of brainstem serotonergic and noradrenergic neurons. The role of adenosine, IL-b and other endogenous somnogenic substances in mediating changes in the activity of MnPN and vlPOA neurons in response to homeostatic sleep need remains to be clarified.
Acknowledgements Supported by the Medical Research Service of the Departments of Veterans Affairs, and National Institutes of Health Grants MH63323 and HL60296. The authors thank Md. Noor Alam, Hui Gong, Natalia Suntsova, Melvi Methhipara and Aaron Uschakov for their critical contributions.
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