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In the mammalian brain, adult neurogenesis has been extensively studied in the hippocampal sub-granular zone and the sub-ventricular zone of the anterolateral ventricles. However, growing evidence suggests that new cells are not only “born” constitutively in the adult hypothalamus, but many of these cells also differentiate into neurons and glia and serve specific functions. The preoptic-hypothalamic area plays a central role in the regulation of many critical functions, including sleep–wakefulness and circadian rhythms. While a role for adult hippocampal neurogenesis in regulating hippocampus-dependent functions, including cognition, has been extensively studied, adult hypothalamic neurogenic process and its contributions to various hypothalamic functions, including sleep–wake regulation are just beginning to unravel. This review is aimed at providing the current understanding of the hypothalamic adult neurogenic processes and the extent to which it affects hypothalamic functions, including sleep–wake regulation. We propose that hypothalamic neurogenic processes are vital for maintaining the proper functioning of the hypothalamic sleep–wake and circadian systems in the face of regulatory challenges. Sleep–wake disturbance is a frequent and challenging problem of aging and age-related neurodegenerative diseases. Aging is also associated with a decline in the neurogenic process. We discuss a hypothesis that a decrease in the hypothalamic neurogenic process underlies the aging of its sleep–wake and circadian systems and associated sleep–wake disturbance. We further discuss whether neuro-regenerative approaches, including pharmacological and non-pharmacological stimulation of endogenous neural stem and progenitor cells in hypothalamic neurogenic niches, can be used for mitigating sleep–wake and other hypothalamic dysfunctions in aging.

Aging is a well-recognized risk factor for sleep disruption. The adverse effects of aging on sleep include sleep disruption by frequent awakenings, a decline in both non-rapid eye movement (nonREM) and REM sleep amounts, and a weaker homeostatic response to sleep loss. Evidence also suggests that sleep in females is more sensitive to changes in the ovarian steroidal milieu. The Fischer-344 rats are commonly used experimental subjects in various behavioral and physiological studies, including sleep and aging. Most sleep studies in Fischer-344 rats have used only male subjects to avoid interactions between the estrus and sleep-waking cycles. The changes in sleep-wake organization of female Fischer-344 rats especially with advancing age is not well-characterized. We determined spontaneous sleep-waking across estrus stages in young (3–4 months) cycling females. We also compared spontaneous and homeostatic sleep response profiles of young male, and female and old (24–25 months) male and female Fischer-344 rats. The results suggest that: i) sleep-wake architectures across stages of estrus cycle in young females were largely comparable except for a significant suppression of REM sleep at proestrus night and an increase in REM sleep the following day; ii) that despite hormonal differences, sleep-wake architecture in male and female rats of corresponding ages were comparable except for the suppression of REM sleep at proestrus night and higher nonREM delta power in recovery sleep; and iii) that aging significantly affected sleep-wake amounts, sleep-wake stability, and homeostatic response to sleep loss in both male and female rats and that the adverse effects of aging were largely comparable in both sexes.

Introduction We have previously shown that pharmacological elevation of corticotropin releasing factor (CRF) signaling in the brain results in exacerbation of sleep disturbances evoked by the exposure of rats to an acute stressor, the dirty cage of a male rat. In the present study we (1) assessed wake-sleep behavior of mice after the exposure to the dirty cage stress paradigm, and (2) examined the effect of chemogenetic silencing of CRF neurons in the hypothalamic paraventricular nucleus (PVN) on sleep occurring following the exposure to this stressor. Methods First, a group of mice (n=12) was implanted with EEG/EMG electrodes. In two weeks, post-surgery, six mice were transferred to dirty cages of male rats and recorded for 24 hours. Control mice were transferred to clean cages. In the second study, a group of CRF-ires-cre mice (n=8) received bilateral injections of AAV-hSyn-DIO-hM4Di-mCherry targeting the PVN. The other group of CRF-ires-cre mice (n=8) was injected AAV-hSyn-DIO-mCherry (control vector). All mice were implanted with EEG/EMG electrodes. Dirty cage experiments were started following a 4-week postsurgical period to allow gene recombination and expression. Mice were subjected to intraperitoneal (IP) administration of clozapine-n-oxide (CNO; 3 mg/kg) at ZT1, placed into dirty cages, and recorded for post-stress sleep. Results: Results In mice expressing hM4Di inhibitory DREADDs (designer receptors activated by designer drugs) versus mice injected with control AAV, IP CNO (3 mg/kg) resulted in a significant decrease of post-stress sleep onset latency, decrease of time spent in wakefulness (first hour, 74±5.3 vs. 89±11.0, second hour, 37.2±10.3% vs. 81.3±9.3%; third hour, 40.1±3.3% vs. 47.1±14.3%; fourth hour, 44.4±6.0 vs. 55.5±9.9), and increase in non-rapid eye movement (NREM) sleep time (26.0±5.4% vs. 11.0±11.1%; 62.8%±9.8 vs. 18.7 ± 9.6%; 59.9±3.2% vs. 52.9±14.5%; 55.6±6.2 vs. 44.5±10.0). The hM4Di expressing mice exhibited longer episodes of NREM sleep, compared to mice injected with control AAV (first hour, 133.3±80.1sec vs. 21±1.7sec; second hour, 43256±83.4sec vs. 73.5±44.1sec; third hour, 459.2±139.8sec vs. 139±80.6sec; fourth hour, 233.1±82.6sec vs. 190±72.3sec). Conclusion Chemogenetic silencing of CRF neurons in the PVN attenuates acute stress-induced sleep disturbance in mice. Support (if any) Supported by Department of Veterans Affairs Merit Review Grant # BX00155605 and SRNSF (Georgia) grant FR-18-12533

Growing evidence supports a role for the medullary parafacial zone in non-rapid eye movement (non-REM) sleep regulation. Cell-body specific lesions of the parafacial zone or disruption of its GABAergic/glycinergic transmission causes suppression of non-REM sleep, whereas, targeted activation of parafacial GABAergic/glycinergic neurons reduce sleep latency and increase non-REM sleep amount, bout duration, and cortical electroencephalogram (EEG) slow-wave activity. Parafacial GABAergic/glycinergic neurons also express sleep-associated c-fos immunoreactivity. Currently, it is not clear if parafacial neurons are non-REM sleep-active and/or REM sleep-active or play a role in the initiation or maintenance of non-REM sleep. We recorded extracellular discharge activity of parafacial neurons across the spontaneous sleep-waking cycle using microwire technique in freely behaving rats. Waking-, non-REM sleep-, and REM sleep-active neuronal groups were segregated by the ratios of their discharge rate changes during non-REM and REM sleep versus waking and non-REM sleep versus REM sleep. Parafacial neurons exhibited heterogeneity in sleep-waking discharge patterns, but 34 of 86 (40%) recorded neurons exhibited increased discharge rate during non-REM sleep compared to waking. These neurons also exhibited increased discharge prior to non-REM sleep onset, similar to median preoptic nucleus (MnPO) and ventrolateral preoptic area (VLPO) sleep-active neurons. However, unlike MnPO and VLPO sleep-active neurons, parafacial neurons were weakly-moderately sleep-active and exhibited a stable rather than decreasing discharge across sustained non-REM sleep episode. We show for the first time that the medullary parafacial zone contains non-REM sleep-active neurons. These neurons are likely functionally important brainstem compliments to the preoptic-hypothalamic sleep-promoting neuronal networks that underlie sleep onset and maintenance.

Aging is associated with sleep-wake disruption, dampening of circadian amplitudes, and a reduced homeostatic sleep response. Aging is also associated with a decline in hypothalamic cell proliferation. We hypothesized that the aging-related decline in cell-proliferation contributes to the dysfunction of preoptic-hypothalamic sleep-wake and circadian systems and consequent sleep-wake disruption. We determined if cytosine-β-D-arabinofuranoside (AraC), an antimitotic agent known to suppress hypothalamic cell proliferation and neurogenesis, causes sleep-wake instability in young mice. The sleep-wake profiles were compared during baseline, during 4 weeks of artificial cerebrospinal fluid (aCSF) + 5-bromo-2'-deoxyuridine (BrdU) or AraC+BrdU infusion into the lateral ventricle, and 8 weeks after treatments. The sleep-wake architecture after AraC treatment was further compared with sleep-wake profiles in aged mice. Compared to aCSF+BrdU, 4 weeks of AraC+BrdU infusion significantly decreased (-96%) the number of BrdU+ cells around the third ventricular wall and adjacent preoptic-hypothalamic area and produced a) sleep disruption during the light phase with decreases in non-rapid eye movement (nonREM) (-9%) and REM sleep (-21%) amounts, and increased numbers of shorter (2 min; 142 versus 98 episodes/12 h) and decreased numbers of longer (5 min; 19 versus 26 episodes/12 h) nonREM sleep episodes; and b) wake disruption during the dark phase, with increased numbers of shorter (138 versus 91 episodes/12 h) and decreased numbers of longer active waking (17 versus 24 episodes/12 h) episodes. AraC-treated mice also exhibited lower delta activity within nonREM recovery sleep. The sleep-wake architecture of AraC-treated mice was similar to that observed in aged mice. These findings are consistent with a hypothesis that a decrease in hypothalamic cell proliferation/neurogenesis is detrimental to sleep-wake and circadian systems and may underlie sleep-wake disturbance in aging.

Corticotropin releasing factor (CRF) is implicated in sleep and arousal regulation. Exogenous CRF causes sleep suppression that is associated with activation of at least two important arousal systems: pontine noradrenergic and hypothalamic orexin/hypocretin neurons. It is not known whether CRF also impacts sleep-promoting neuronal systems. We hypothesized that CRF-mediated changes in wake and sleep involve decreased activity of hypothalamic sleep-regulatory neurons localized in the preoptic area. To test this hypothesis, we examined the effects of intracerebroventricular administration of CRF on sleep-wake measures and c-Fos expression in GABAergic neurons in the median preoptic nucleus (MnPN) and ventrolateral preoptic area (VLPO) in different experimental conditions. Administration of CRF (0.1 nmol) during baseline rest phase led to delayed sleep onset and decreases in total amount and mean duration of non-rapid eye movement (NREM) sleep. Administration of CRF during acute sleep deprivation (SD) resulted in suppression of recovery sleep and decreased c-Fos expression in MnPN/VLPO GABAergic neurons. Compared with vehicle controls, intracerebroventricular CRF potentiated disturbances of both NREM and REM sleep in rats exposed to a species-specific psychological stressor, the dirty cage of a male conspecific. The number of MnPN/VLPO GABAergic neurons expressing c-Fos was reduced in the CRF-treated group of dirty cage-exposed rats. These findings confirm the involvement of CRF in wake-sleep cycle regulation and suggest that increased CRF signaling in the brain 1) negatively affects homeostatic responses to sleep loss, 2) exacerbates stress-induced disturbances of sleep, and 3) suppresses the activity of sleep-regulatory neurons of the MnPN and VLPO.

Alcohol causes adenosine buildup, which inhibits wake-active neurons via adenosine A1 receptors thus disinhibiting sleep active neurons and also stimulates sleep-active neurons via A2A receptors, causing sleep. This editorial highlights the study entitled, "Lesions of the basal forebrain cholinergic neurons attenuates sleepiness and adenosine after alcohol consumption" by Sharma and colleagues. They report that the wake-promoting basal forebrain (BF) cholinergic neurons play a crucial role in mediating acute alcohol-induced sleep via adenosinergic signaling.

The preoptic hypothalamus is implicated in sleep regulation. Neurons in the median preoptic nucleus (MnPO) and the ventrolateral preoptic area (VLPO) have been identified as potential sleep regulatory elements. However, the extent to which MnPO and VLPO neurons are activated in response to changing homeostatic sleep regulatory demands is unresolved. To address this question, we continuously recorded the extracellular activity of neurons in the rat MnPO, VLPO and dorsal lateral preoptic area (LPO) during baseline sleep and waking, during 2 h of sleep deprivation (SD) and during 2 h of recovery sleep (RS). Sleep-active neurons in the MnPO ( n = 11) and VLPO ( n = 13) were activated in response to SD, such that waking discharge rates increased by 95.8 ± 29.5% and 59.4 ± 17.3%, respectively, above waking baseline values. During RS, non-rapid eye movement (REM) sleep discharge rates of MnPO neurons initially increased to 65.6 ± 15.2% above baseline values, then declined to baseline levels in association with decreases in EEG delta power. Increase in non-REM sleep discharge rates in VLPO neurons during RS averaged 40.5 ± 7.6% above baseline. REM-active neurons ( n = 16) in the LPO also exhibited increased waking discharge during SD and an increase in non-REM discharge during RS. Infusion of A2A adenosine receptor antagonist into the VLPO attenuated SD-induced increases in neuronal discharge. Populations of LPO wake/REM-active and state-indifferent neurons and dorsal LPO sleep-active neurons were unresponsive to SD. These findings support the hypothesis that sleep-active neurons in the MnPO and VLPO, and REM-active neurons in the LPO, are components of neuronal circuits that mediate homeostatic responses to sustained wakefulness.

Sleep homeostasis in rats undergoes significant maturational changes during postweaning development, but the underlying mechanisms of this process are unknown. In the present study we tested the hypothesis that the maturation of sleep is related to the functional emergence of adenosine (AD) signaling in the brain. We assessed postweaning changes in 1) wake-related elevation of extracellular AD in the basal forebrain (BF) and adjacent lateral preoptic area (LPO), and 2) the responsiveness of median preoptic nucleus (MnPO) sleep-active cells to increasing homeostatic sleep drive. We tested the ability of exogenous AD to augment homeostatic responses to sleep deprivation (SD) in newly weaned rats. In groups of postnatal day (P)22 and P30 rats, we collected dialysate from the BF/LPO during baseline (BSL) wake-sleep, SD, and recovery sleep (RS). HPLC analysis of microdialysis samples revealed that SD in P30 rats results in significant increases in AD levels compared with BSL. P22 rats do not exhibit changes in AD levels in response to SD. We recorded neuronal activity in the MnPO during BSL, SD, and RS at P22/P30. MnPO neurons exhibited adult-like increases in waking neuronal discharge across SD on both P22 and P30, but discharge rates during enforced wake were higher on P30 vs. P22. Central administration of AD (1 nmol) during SD on P22 resulted in increased sleep time and EEG slow-wave activity during RS compared with saline control. Collectively, these findings support the hypothesis that functional reorganization of an adenosinergic mechanism of sleep regulation contributes to the maturation of sleep homeostasis. NEW & NOTEWORTHY Brain mechanisms that regulate the maturation of sleep are understudied. The present study generated first evidence about a potential mechanistic role for adenosine in the maturation of sleep homeostasis. Specifically, we demonstrate that early postweaning development in rats, when homeostatic response to sleep loss become adult like, is characterized by maturational changes in wake-related production/release of adenosine in the brain. Pharmacologically increased adenosine signaling in developing brain facilitates homeostatic responses to sleep deprivation.

Sleep fragmentation (SF) is prevalent in human sleep-related disorders. In rats, sustained SF has a potent suppressive effect on adult hippocampal dentate gyrus (DG) neurogenesis. Adult-generated DG neurons progressively mature over several weeks, and participate in certain hippocampal-dependent cognitive functions. We predicted that suppression of neurogenesis by sustained SF would affect hippocampal-dependent cognitive functions in the time window when new neurons would reach functional maturity. Sprague-Dawley rats were surgically-prepared with electroencephalogram (EEG) and electromyogram (EMG) electrodes for sleep state detection. We induced sleep-dependent SF for 12 days, and compared SF animals to yoked sleep fragmentation controls (SFC), treadmill controls (TC) and cage controls (CC). Rats were injected with bromodeoxyuridine on treatment days 4 and 5. Rats were returned to home cages for 14 days. Cognitive performance was assessed in a Barnes maze with 5 days at a constant escape position followed by 2 days at a rotated position. After Barnes maze testing rats were perfused and DG sections were immunolabeled for BrdU and neuronal nuclear antigen (NeuN), a marker of mature neurons.SF reduced BrdU-labeled cell counts by 32% compared to SFC and TC groups. SF reduced sleep epoch duration, but amounts of rapid eye movement (REM) sleep did not differ between SF and SFC rats, and non-rapid eye movement (NREM) was reduced only transiently. In the Barnes maze, SF rats exhibited a progressive decrease in escape time, but were slower than controls. SF animals used different search strategies. The use of a random, non-spatial search strategy was significantly elevated in SF compared to the SFC, TC and CC groups. The use of random search strategies was negatively correlated with NREM sleep bout length during SF. Sustained sleep fragmentation reduced DG neurogenesis and induced use of a non-spatial search strategy, which could be seen 2 weeks after terminating the SF treatment. The reduction in neurogenesis induced by sleep fragmentation is likely to underlie the delayed changes in cognitive function.

The perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the regulation of behavioral arousal. The PF-LHA predominantly contains neurons that are active during behavioral and cortical activation and quiescent during non-rapid eye movement (nonREM) sleep, that is, are nonREM-off neurons. Some in vitro and in vivo studies indicate that PF-LHA neurons, including hypocretin-expressing neurons, are under GABAergic control. However, a role of GABA in suppressing the discharge of PF-LHA neurons during spontaneous nonREM sleep has not been confirmed. We recorded the sleep-wake discharge profiles of PF-LHA neurons and simultaneously assessed the contributions of local GABA(A) receptor activation and blockade on their wake- and nonREM sleep-related discharge activities by delivering GABA(A) receptor agonist, muscimol (500 nm, 5 microM, and 10 microM) and its antagonist, bicuculline (5 microM, 10 microM, and 20 microM), adjacent to the recorded neurons via reverse microdialysis. Muscimol dose-dependently decreased the discharge of PF-LHA neurons including nonREM-off neurons. Muscimol-induced suppression of discharge during nonREM sleep was significantly weaker than the suppression produced during waking. In the presence of bicuculline, PF-LHA neurons, including nonREM-off neurons, exhibited elevated discharge, which was dose-dependent and was significantly higher during nonREM sleep, compared to waking. These results suggest that GABA(A) receptor mediated increased GABAergic tone contributes to the suppression of PF-LHA neurons, including nonREM-off neurons, during spontaneous nonREM sleep.

The adult hippocampal dentate gyrus (DG) exhibits cell proliferation and neurogenesis throughout life. We examined the effects of daily administration of eszopiclone (Esz), a commonly used hypnotic drug and gamma-aminobutyric acid (GABA) agonist, compared with vehicle, on DG cell proliferation and neurogenesis, and on sleep-wake patterns. Esz was administered during the usual sleep period of rats, to mimic typical use in humans. Esz treatment for 7 days did not affect the rate of cell proliferation, as measured by 5-bromo-2'-deoxyuridine (BrdU) immunostaining. However, twice-daily Esz administration for 2 weeks increased survival of newborn cells by 46%. Most surviving cells exhibited a neuronal phenotype, identified as BrdU-neuronal nuclei (NeuN) double-labeling. NeuN is a marker of neurons. Non-rapid eye movement sleep was increased on day 1, but not on days 7 or 14 of Esz administration. Delta electroencephalogram activity was increased on days 1 and 7 of treatment, but not on day 14. There is evidence that enhancement of DG neurogenesis is a critical component of the effects of antidepressant treatments of major depressive disorder (MDD). Adult-born DG cells are responsive to GABAergic stimulation, which promotes cell maturation. The present study suggests that Esz, presumably acting as a GABA agonist, has pro-neurogenic effects in the adult DG. This result is consistent with evidence that Esz enhances the antidepressant treatment response of patients with MDD with insomnia.

The perifornical-lateral hypothalamic area (PF-LHA) plays a central role in the regulation of behavioral arousal. The PF-LHA contains several neuronal types including wake-active hypocretin (HCRT) neurons that have been implicated in the promotion and/or maintenance of behavioral arousal. Adenosine is an endogenous sleep factor and recent evidence suggests that activation and blockade of adenosine A(1) receptors within the PF-LHA promote and suppress sleep, respectively. Although, an in vitro study indicates that adenosine inhibits HCRT neurons via A(1) receptor, the in vivo effects of A(1) receptor mediated adenosinergic transmission on PF-LHA neurons including HCRT neurons are not known. First, we determined the effects of N(6)-cyclopentyladenosine (CPA), an adenosine A(1) receptor agonist, on the sleep-wake discharge activity of the PF-LHA neurons recorded via microwires placed adjacent to the microdialysis probe used for its delivery. Second, we determined the effects of CPA and that of an A(1) receptor antagonist, 1,3-dipropyl-8-phenylxanthine (CPDX) into the PF-LHA on cFos-protein immunoreactivity (Fos-IR) in HCRT and non-HCRT neurons around the microdialysis probe used for their delivery. The effect of CPA on Fos-IR was studied in rats that were kept awake during lights-off phase, whereas the effect of CPDX was examined in undisturbed rats during lights-on phase. CPA significantly suppressed the sleep-wake discharge activity of PF-LHA neurons. Doses of CPA (50 muM) and CPDX (50 muM) that suppressed and induced arousal, respectively, in our earlier study [Alam MN, Kumar S, Rai S, Methippara M, Szymusiak R, McGinty D (2009) Brain Res 1304:96-104], significantly suppressed and increased Fos-IR in HCRT and non-HCRT neurons. These findings suggest that wake-promoting PF-LHA system is subject to increased endogenous adenosinergic inhibition and that adenosine acting via A(1) receptors, in part, inhibits HCRT neurons to promote sleep.

The lamina terminalis (LT) consists of the organum vasculosum of the LT (OVLT), the median preoptic nucleus (MnPO) and the subfornical organ (SFO). All subdivisions of the LT project to the ventrolateral periaqueductal gray (vlPAG). The LT and the vlPAG are implicated in several homeostatic and behavioral functions, including body fluid homeostasis, thermoregulation and the regulation of sleep and waking. By combining visualization of c-Fos protein and retrograde neuroanatomical tracer we have examined the functional correlates of LT-vlPAG projection neurons. Rats were injected with retrograde tracer into the vlPAG and, following a 1-week recovery period, they were subjected to either hypertonic saline administration (0.5 M NaCl, 1 mL/100 g i.p.), 24-h water deprivation, isoproterenol administration (increases circulating angiotensin II; 50 microg/kg s.c.), heat exposure (39 degrees C for 60 min) or permitted 180 min spontaneous sleep. Retrogradely labeled neurons from the vlPAG and double-labelled neurons were then identified and quantified throughout the LT. OVLT-vlPAG projection neurons were most responsive to hypertonic saline and water deprivation. SFO-vlPAG projection neurons were most active following isoproterenol administration, and MnPO-vlPAG projection neurons displayed significantly more Fos immunostaining following water deprivation, heat exposure and sleep. These results support the existence of functional subdivisions of LT-vlPAG-projecting neurons, and indicate three patterns of activity that correspond to thermal and sleep wake regulation, osmotic or hormonal stimuli.

The perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the regulation of arousal. The PF-LHA contains wake-active neurons that are quiescent during non-REM sleep and in the case of neurons expressing the peptide hypocretin (HCRT), quiescent during both non-REM and REM sleep. Adenosine is an endogenous sleep factor and recent evidence suggests that adenosine via A(1) receptors may act on PF-LHA neurons to promote sleep. We examined the effects of bilateral activation as well as blockade of A(1) receptors in the PF-LHA on sleep-wakefulness in freely behaving rats. The sleep-wake profiles of male Wistar rats were recorded during reverse microdialysis perfusion of artificial cerebrospinal fluid (aCSF) and two doses of adenosine A(1) receptor antagonist, 1,3-dipropyl-8-phenylxanthine (CPDX; 5 microM and 50 microM) or A(1) receptor agonist, N(6)-cyclopentyladenosine (CPA; 5 microM and 50 microM) into the PF-LHA for 2 h followed by 4 h of aCSF perfusion. CPDX perfused into the PF-LHA during lights-on phase produced arousal (F=7.035, p

The median preoptic nucleus (MnPN) of the hypothalamus contains sleep-active neurons including sleep-active GABAergic neurons and is involved in the regulation of nonREM/REM sleep. The hypocretinergic (HCRT) neurons of the perifornical-lateral hypothalamic area (PF-LHA) and serotonergic (5-HT) neurons of the dorsal raphe nucleus (DRN) are mostly active during waking and have been implicated in the regulation of arousal. MnPN GABAergic neurons project to the PF-LHA and DRN. It is hypothesized that MnPN promotes sleep by inhibiting multiple arousal systems including HCRT and other wake-active neurons within the PF-LHA and 5-HT neurons in the DRN. We examined the effects of inactivation of MnPN neurons by locally microinjecting 0.2 µl of 1 mM or 10 mM solutions of a GABA A receptor agonist, muscimol, into the MnPN on Fos expression (Fos-IR) in the PF-LHA neurons including HCRT neurons and 5-HT neurons in the DRN in anesthetized rats. Compared to artificial cerebrospinal fluid control, microinjection of muscimol into the MnPN resulted in significantly higher percentages of HCRT and non-HCRT neurons in the PF-LHA and 5-HT neurons in the DRN that exhibited Fos-IR. The percentage of melanin-concentrating hormone (MCH)+/Fos+ neurons in the PF-LHA did not change after muscimol treatments. These results support a hypothesis that the activation of MnPN neurons contributes to the suppression of wake-promoting systems including HCRT and other unidentified neurons in the PF-LHA and 5-HT neurons in the DRN. These results also suggest that MCH neurons may not be under MnPN inhibitory control. These findings are consistent with a hypothesized role of MnPN in sleep regulation.

The hypnogenic function of the rostral hypothalamic region, particularly the preoptic area (POA) was established previously on the basis of lesion, neuronal unit recording, and neurochemical and thermal stimulation studies. Recent studies have mapped the locations of putative sleep-promoting neurons in the POA using c-Fos immunostaining techniques and confirmed these findings with electrophysiological methods. Segregated groups of sleep-active neurons have been localized in the ventrolateral POA (vlPOA) and median preoptic nucleus (MnPN). MnPN and vlPOA sleep-active neurons express the inhibitory transmitter, GABA. In vlPOA neurons, GABA is co-localized with a second inhibitory transmitter, galanin. Descending projections from these sites terminate in putative arousal-promoting cell groups, including histaminergic, serotonergic, orexinergic, noradrenergic, and cholinergic neurons. These findings suggest the hypothesis that non-REM sleep occurs as a consequence of GABAergic and galaninergic inhibition of arousal-promoting neurons resulting from activation of vlPOA and MnPN sleep-promoting neurons. In support of this hypothesis, it was shown that putative sleep-promoting and arousal-promoting neurons exhibit reciprocal changes in discharge across the sleep-wake cycle and that GABA release in wake-promoting sites increases during nonREM sleep. In addition, some POA sleep-active neurons are warm-sensitive. Local POA warming inhibits discharge of multiple arousal-promoting neuronal groups. POA warming, unit recording, and lesion studies also show that POA regulates the amount of delta EEG activity within nonREM sleep, and index of the depth of sleep. Finally, there is evidence that arousal systems inhibit vlPOA and MnPN neurons and the POA hypnogenic mechanism. Mutually-inhibitory interactions between sleep-promoting and arousal-promoting systems are hypothesized to form a functional sleep-wake switch.

THE SUBGRANULAR CELL LAYER IN THE DENTATE GYRUS (DG) OF THE ADULT HIPPOCAMPUS CONTAINS PROGENITOR CELLS, WHICH HAVE THE POTENTIAL TO proliferate and differentiate into neurons. These progenitors mature locally into granule cells of the DG, sending axonal projections to area CA3 and dendrites into the molecular layer.1.2 Adult neurogenesis has been demonstrated in birds and several mammals, including humans. The processes of cell proliferation, migration, maturation, and survival are all subject to modulation by experiential events.3 Stress is an important negative regulator of cell proliferation.4,5 Previously we reported that 96 hours of total sleep deprivation (TSD) affects neurogenesis in the rat DG by reducing cell proliferation in the subgranular cell layer, and the percentage of new cells later expressing a neuronal marker.6,7 The inhibitory effects of extended sleep deprivation have been confirmed.8 However, mammalian sleep is physiologically heterogeneous. The two primary stages of mammalian sleep, non-rapid eye movement (NREM) and rapid eye movement (REM) sleep, have very different, even opposite, electrophysiologic and metabolic properties, compared with waking, and could have different effects on neurogenesis. Revealing the relative impact of REM and NREM sleep on neurogenesis is a necessary step in understanding the mechanisms underlying suppression of neurogenesis in response to sleep loss. The aim of the present study was to assess the effect of REM sleep deprivation (REMD) on neurogenesis in the DG of the adult rat. To achieve REM deprivation, rats lived on a treadmill that was briefly activated when REM was detected by fast Fourier transform analysis of the electroencephalogram (EEG) and electromyogram (EMG) activity. Yoked control (YC) animals lived on the same treadmill and were subjected to the same treadmill movements.

The median preoptic nucleus (MnPO) has been implicated in the regulation of hydromineral balance and cardiovascular regulation. The MnPO also contains neurons that are active during sleep and in response to increasing homeostatic pressure for sleep. The potential role of these neurons in the regulation of arousal prompted an analysis of the efferent projections from the MnPO. Anterograde and retrograde neuroanatomical tracers were utilized to characterize the neural connectivity from the MnPO to several functionally important sleep- and arousal-regulatory neuronal systems in the rat brain. Anterograde terminal labeling from the MnPO was confirmed within the core and extended ventrolateral preoptic nucleus. Within the lateral hypothalamus, labeled axons were observed in close apposition to proximal and distal dendrites of hypocretin/orexin immunoreactive (IR) cells. Projections from the MnPO to the locus coeruleus were observed within and surrounding the tyrosine hydroxylase–IR cell cluster. Labeled axons from the MnPO were mostly observed within the lateral division of the dorsal raphe nucleus and heavily within the ventrolateral periaqueductal gray. Few anterogradely labeled appositions were present juxtaposed to choline acetyltransferase–IR somata within the magnocellular preoptic area. The use of retrogradely transported neuroanatomical tracers placed within the prospective efferent terminal fields supported and confirmed findings from the anterograde tracer experiments. These anatomical findings support the hypothesis that MnPO neurons function to promote sleep by inhibition of orexinergic and monoaminergic arousal systems and disinhibition of sleep regulatory neurons in the ventrolateral preoptic area.

The adult hippocampal dentate gyrus (DG) is a site of continuing neurogenesis. This process is influenced by a variety of physiological and experiential stimuli including total sleep deprivation (TSD). In humans, sleep fragmentation (SF) is a more common sleep condition than TSD. SF is associated with several prevalent diseases. We assessed a hypothesis that SF would suppress adult neurogenesis in the DG of the adult rat. An intermittent treadmill system was used; the treadmill was on for 3 s and off for 30 s (SF). For sleep fragmentation control (SFC), the treadmill was on for 15 min and off for 150 min. SF was conducted for three durations: 1, 4 and 7 days. To label proliferating cells, the thymidine analog, 5-bromo-2-deoxyuridine (BrdU), was injected 2 h prior to the end of each experiment. Expression of the intrinsic proliferative marker, Ki67, was also studied. SF rats exhibited an increased number of non-rapid eye movement (NREM) sleep bouts with no change in the percent of time spent in this stage. The numbers of both BrdU-positive cells and Ki67-positive cells were reduced by approximately 70% (P

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.

The hypocretin (HCRT) system of the perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the facilitation of behavioral arousal. HCRT neurons receive serotonergic afferents from the dorsal raphe nucleus. Although in-vitro pharmacological studies suggest that serotonin (5-HT) inhibits HCRT neurons, the in-vivo effects of 5-HT on HCRT neurons in the PF-LHA and associated behavioral changes have not been described. We examined the effects of 5-HT delivered locally into the PF-LHA using reverse microdialysis on its neuronal activity and the consequent sleep-wake changes in rats. First, we quantified Fos expression (Fos-IR) in HCRT and other PF-LHA neurons following unilateral 5-HT perfusion in awake rats. Second, we determined the transient effects of 5-HT perfusion on the extracellular activity of the PF-LHA neurons recorded via microwires placed adjacent to the microdialysis probe. Third, we examined the effects of 5-HT perfusion into the PF-LHA on the sleep-wake profiles of the rats during the lights-off period. Unilateral perfusion of 5-HT into the PF-LHA in awake rats dose-dependently decreased the number of HCRT neurons exhibiting Fos-IR. 5-HT also inhibited the discharge activity of four of five responsive wake-related, putative HCRT neurons. However, unilateral perfusion of 5-HT into the PF-LHA did not produce significant behavioral changes during the 2-h recording period. These results confirm the in-vitro findings that 5-HT exerts inhibitory influences on HCRT neurons but further suggest that the inactivation of a limited number of HCRT neurons by unilateral 5-HT microdialysis may not be sufficient to induce behavioral changes.

Previous work shows that sleep deprivation impairs hippocampal-dependent learning and long-term potentiation (LTP). Brain-derived neurotrophic factor (BDNF), cAMP response-element-binding (CREB) and calcium–calmodulin-dependent protein kinase II (CAMKII) are critical modulators of hippocampal-dependent learning and LTP. In the present study we compared the effects of short- (8 h) and intermediate-term (48 h) sleep deprivation (SD) on the expression of BDNF and its downstream targets, Synapsin I, CREB and CAMKII in the neocortex and the hippocampus. Rats were sleep deprived using an intermittent treadmill system which equated total movement in the SD and control treadmill animals (CT), but permitted sustained periods of rest in CT animals. Animals were divided into SD (treadmill schedule: 3 s on/12 s off) and two treadmill control groups, CT1 (15 min on/60 min off) and CT2 (30 min on/120 min off – permitting more sustained sleep). Real-time Taqman RT-PCR was used to measure changes in mRNA; BDNF protein levels were determined using ELISA. In the hippocampus, 8 h treatments reduced BDNF, Synapsin I, CREB and CAMKII gene expression in both SD and control groups. Following 48 h of experimental procedures, the expression of all these four molecular markers of plasticity was reduced in SD and CT1 groups compared to the CT2 and cage control groups. In the hippocampus, BDNF protein levels after 8 h and 48 h treatments paralleled the changes in mRNA. In neocortex, neither 8 h nor 48 h SD or control treatments had significant effects on BDNF, Synapsin I and CAMKII mRNA levels. Stepwise regression analysis suggested that loss of REM sleep underlies the effects of SD on hippocampal BDNF, Synapsin I and CREB mRNA levels, whereas loss of NREM sleep underlies the effects on CAMKII mRNA.

The lamina terminalis consists of the organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus (MnPO) and subfornical organ. The MnPO and ventrolateral preoptic area (vlPOA) are known to contain high densities of neurons that are sleep active. The prevalence of sleep-active neurons in the OVLT and subfornical organ is unknown. The vlPOA and subdivisions of the lamina terminalis project to hypothalamic regions involved in the control of behavioral, electrographic or autonomic arousal, including the lateral hypothalamic area (LHA) and paraventricular nucleus (PVN). The extent to which projection neurons are active during sleep is unknown. We quantified c-Fos protein immunoreactivity (IR) in the lamina terminalis and vlPOA in sleeping and awake rats that received injections of retrograde tracer into either the LHA or PVN. Fos IR was also examined in lamina terminalis neurons following tracer injections into the vlPOA. Significantly more projection neurons from the MnPO, OVLT and vlPOA to the LHA were Fos-immunoreactive in sleeping vs. awake animals. Waking Fos IR was more prevalent in lamina terminalis neurons projecting to the PVN although a subset of MnPO projection neurons in sleeping rats was Fos-immunoreactive. Almost 50% of vlPOA-PVN projection neurons expressed Fos IR during sleep, compared with 3% during waking. Significantly more neurons in the OVLT and MnPO projecting to the vlPOA were Fos-immunoreactive in sleeping vs. awake rats. Inhibition of LHA and PVN neurons arising from OVLT, MnPO and vlPOA neurons may contribute to suppression of behavioral, electroencephalographic and sympathetic nervous system activation during sleep.

The median preoptic nucleus (MnPN) and the ventral lateral preoptic area (vlPOA) of the hypothalamus express sleep-related Fos immunoreactivity, and a subset of Fos-immunoreactive neurons (IRNs) in these nuclei contain glutamic acid decarboxylase (GAD), a marker of GABAergic cells. We recently showed that the numbers of Fos-positive (Fos+) and Fos+ GAD-IRNs in both the MnPN and the vlPOA are positively correlated with the total amount of preceding sleep. The present study was designed to clarify whether or not activation of sleep-related neurons in the rat MnPN and vlPOA is associated with rapid eye movement (REM) sleep regulation. Expression of c-fosin MnPN and vlPOA neurons was examined under conditions of spontaneous sleep, REM sleep restriction, and REM sleep recovery after REM sleep restriction. Across all conditions, the number of Fos-IRNs was highest in REM-sleep-restricted rats displaying the highest levels of REM sleep homeostatic pressure/drive, i.e., those rats exhibiting the most frequent attempts to enter REM sleep. This 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.

Sleep is hypothesized to play a critical role in facilitating brain growth and plasticity. Neurogenesis in the adult hippocampus is a recently established model of brain plasticity. We determined how adult neurogenesis was affected by sleep deprivation (SD) using an intermittent treadmill deprivation system, which permits comparisons with movement-matched controls. Immunohistochemistry for bromodeoxyuridine (BrdU) and neuron-specific nuclear antigen (NeuN) was used to quantify the proliferative and neurogenic processes. Following 96 h SD, proliferation of new cells was reduced by 50%. Three weeks following the termination of SD, the proportion of BrdU-labeled cells exhibiting a mature neuronal phenotype was reduced by 35%. Combining the suppression of proliferation and failure in development of mature neuronal phenotype, we estimate that 96 h SD reduced new dentate gyrus neurons generated during the deprivation period by 60%. Suppression of proliferation was not associated with elevation of the stress hormone corticosterone. Current evidence suggests that the detrimental effects of SD on neurogenesis requires accumulated sleep debt and is not quickly reversible. We speculate that the suppression of neurogenesis by SD is associated with suppression of protein synthesis.

Evidence suggests that adenosine (AD) is an endogenous sleep factor. The hypnogenic action of AD is mediated through its inhibitory A1and excitatory A2Areceptors. Although AD is thought to be predominantly active in the wake-active region of the basal forebrain (BF), a hypnogenic action of AD has been demonstrated in several other brain areas, including the preoptic area. We hypothesized that in lateral preoptic area (LPOA), a region with an abundance of sleep-active neurons, AD acting via A1receptors would induce waking by inhibition of sleep-active neurons and that AD acting via A2Areceptors would promote sleep by stimulating the sleep-active neurons. To this end, we studied the effects on sleep of an AD transport inhibitor, nitrobenzyl-thio-inosine (NBTI) and A1and A2Areceptor agonists/antagonists by microdialyzing them into the LPOA. The results showed that, in the sleep-promoting area of LPOA: 1) A1receptor stimulation or inhibition of AD transport by NBTI induced waking and 2) A2Areceptor stimulation induced sleep. We also confirmed that NBTI administration in the wake promoting area of the BF increased sleep. The effects of AD could be mediated either directly or indirectly via interaction with other neurotransmitter systems. These observations support a hypothesis that AD mediated effects on sleep-wake cycles are site and receptor dependent.

The perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the regulation of behavioural arousal. The PF-LHA contains several cell types including neurones expressing the peptides, hypocretin (HCRT; also called orexin) and melanin-concentrating hormone (MCH). Evidence suggests that most of the PF-LHA neurones, including HCRT neurones, are active during waking and quiescent during non-rapid eye movement (non-NREM) sleep. The PF-LHA contains local GABAergic interneurones and also receives GABAergic inputs from sleep-promoting regions in the preoptic area of the hypothalamus. We hypothesized that increased GABA-mediated inhibition within PF-LHA contributes to the suppression of neuronal activity during non-REM sleep. EEG and EMG activity of rats were monitored for 2 h during microdialytic delivery of artificial cerebrospinal fluid (aCSF) or bicuculline, a GABAA receptor antagonist, into the PF-LHA in spontaneously sleeping rats during the lights-on period. At the end of aCSF or bicuculline perfusion, rats were killed and c-Fos immunoreactivity (Fos-IR) in HCRT, MCH and other PF-LHA neurones was quantified. In response to bicuculline perfusion into the PF-LHA, rats exhibited a dose-dependent decrease in non-REM and REM sleep time and an increase in time awake. The number of HCRT, MCH and non-HCRT/non-MCH neurones exhibiting Fos-IR adjacent to the microdialysis probe also increased dose-dependently in response to bicuculline. However, significantly fewer MCH neurones exhibited Fos-IR in response to bicuculline as compared to HCRT and other PF-LHA neurones. These results support the hypothesis that PF-LHA neurones, including HCRT neurones, are subject to increased endogenous GABAergic inhibition during sleep. In contrast, MCH neurones appear to be subject to weaker GABAergic control during sleep.

Interleukin-1b (IL-1) is a pro-inflammatory cytokine that has been implicated in the regulation of nonrapid eye movement (nonREM) sleep. IL-1, IL-1 receptors and the IL-1 receptor antagonist (ra) are present normally in discrete brain regions, including the preoptic area (POA) of the hypothalamus and the adjoining magnocellular basal forebrain (BF). The POA ⁄ BF have been implicated in the regulation of sleep‐wakefulness. We hypothesized that IL-1 promotes nonREM sleep, in part by altering the state-dependent discharge activity of POA ⁄ BF neurons. We recorded the sleep‐wake discharge profiles of 83 neurons in the lateral POA ⁄ BF and assessed the effects of IL-1, IL-1ra, and IL-ra + IL-1 delivered through a microdialysis probe on state-dependent neuronal discharge activity. IL-1 decreased the discharge rate of POA ⁄ BF neurons as a group (n = 55) but wake-related and sleep-related neurons responded differently. IL-1 significantly decreased the discharge rate of wake-related neurons. Of 24 wake-related neurons studied, 19 (79%) neurons exhibited a greater than 20% change in their discharge in the presence of IL-1 during waking. IL-1 suppressed the discharge activity of 18 of 19 responsive neurons. Of 13 sleep-related neurons studied, IL-1 increased the discharge activity of five and suppressed the discharge activity of four neurons. IL-1ra increased the discharge activity of four of nine neurons and significantly attenuated IL-1-induced effects on neuronal activity of POA ⁄ BF neurons (n = 19). These results suggest that the sleep-promoting effects of IL-1 may be mediated, in part, via the suppression of wake-related neurons and the activation of a subpopulation of sleeprelated neurons in the POA ⁄ BF.

Upper airway dilator activity during sleep appears to be diminished under conditions of enhanced sleep propensity, such as after sleep deprivation, leading to worsening of obstructive sleep apnea (OSA). Non-rapid eye movement (NREM) sleep propensity originates in sleep-active neurons of the preoptic area (POA) of the hypothalamus and is facilitated by activation of POA warm-sensitive neurons (WSNs). We hypothesized that activation of WSNs by local POA warming would inhibit activity of the posterior cricoarytenoid (PCA) muscle, an airway dilator, during NREM sleep. In chronically prepared unrestrained cats, the PCA exhibited inspiratory bursts in approximate synchrony with inspiratory diaphragmatic activity during waking, NREM, and REM. Integrated inspiratory PCA activity (IA), peak activity (PA), and the lead time (LT) of the onset of inspiratory activity in PCA relative to diaphragm were significantly reduced in NREM sleep and further reduced during REM sleep compared with waking. Mild bilateral local POA warming (0.5–1.2°C) significantly reduced IA, PA, and LT during NREM sleep compared with a prewarming NREM baseline. In some animals, effects of POA warming on PCA activity were found during waking or REM. Because POA WSN activity is increased during spontaneous NREM sleep and regulates sleep propensity, we hypothesize that this activation contributes to reduction of airway dilator activity in patients with OSA.

Neurones in the median preoptic nucleus (MnPN) and the ventrolateral preoptic area (vlPOA) express immunoreactivity for c-Fos protein following sustained sleep, and display elevated discharge rates during both non-REM and REM sleep compared to waking. We evaluated the hypothesis that MnPN and vlPOA sleep-active neurones are GABAergic by combining staining for c-Fos protein with staining for glutamic acid decarboxylase (GAD). In a group of six rats exhibiting spontaneous total sleep times averaging 82.2 ± 5.1% of the 2 h immediately prior to death, >75% of MnPN neurones that were Fos-immunoreactive (IR) were also GAD-IR. Similar results were obtained in the vlPOA. In a group of 11 rats exhibiting spontaneous sleep times ranging from 20 to 92%, the number of Fos + GAD-IR neurones in MnPN and vlPOA was positively correlated with total sleep time. Compared to control animals, Fos + GAD-IR cell counts in the MnPN were significantly elevated in rats that were sleep deprived for 24 h and permitted 2 h of recovery sleep. These findings demonstrate that a majority of MnPN and vlPOA neurones that express Fos-IR during sustained spontaneous sleep are GABAergic. They also demonstrate that sleep deprivation is associated with increased activation of GABAergic neurones in the MnPN and vlPOA.

Activation of the preoptic area (POA) warm sensitive neurons is known to promote non-REM (NREM) sleep and inhibit neuronal discharge in arousal-related brain structures. The perifornical area of the lateral hypothalamus (PF/LH) was recently recognized to be an additional important arousal promoting region. We studied the behavior of PF/LH neurons in rats during the normal sleep-wake cycle and in response to local POA warming. Most PF/LH neurons were wake-active, and exhibited low discharge throughout NREM. Seventy four percent of these wake-active neurons exhibited moderate or strong activation in REM sleep compared to NREM sleep. A substantial group (26%) exhibited very low discharge in REM as well as NREM sleep. Fifty two percent of units in the PF/LH area were responsive to POA warming; 90% of responsive neurons exhibited a significant reduction (-26.47+/-2.16% for 1 degrees C of POA warming) in their discharge rate. The inhibitory effect of POA warming on PF/LH neurons was not associated with EEG slowing. This study supports the hypothesis that sleep induction by POA warm sensitive neurons is mediated through the inhibition of multiple arousal-related structures.

Several lines of evidence show that the preoptic area (POA) of the hypothalamus is critically implicated in the regulation of sleep. Functionally heterogeneous cell groups with sleep-related discharge patterns are located both in the medial and lateral POA. Recently a cluster of neurons showing sleep-related c-Fos immunoreactivity was found in the median preoptic nucleus (MnPN). To determine the specificity of the state-related behaviour of MnPN neurons we have undertaken the first study of their discharge patterns across the sleep-waking cycle. Nearly 76 % of recorded cells exhibited elevated discharge rates during sleep. Sleep-related units showed several distinct types of activity changes across sleep stages. Two populations included cells displaying selective activation during either non-rapid eye movement (NREM) sleep (10 %) or REM sleep (8 %). Neurons belonging to the predominant population (58 %) exhibited activation during both phases of sleep compared to wakefulness. Most of these cells showed a gradual increase in their firing rates prior to sleep onset, elevated discharge during NREM sleep and a further increase during REM sleep. This specific sleep-waking discharge profile is opposite to that demonstrated by wake-promoting monoaminergic cell groups and was previously found in cells localized in the ventrolateral preoptic area (vlPOA). We hypothesize that these vlPOA and MnPN neuronal populations act as parts of a GABAergic/galaninergic sleep-promoting ('anti-waking') network which exercises inhibitory control over waking-promoting systems. MnPN neurons that progressively increase activity during sustained waking and decrease activity during sustained sleep states may be involved in homeostatic regulation of sleep.

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 sleep-active 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, gamma-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 asleep-wake switch. 2001 Harcourt Publishers Ltd

Deficiency of orexin, a newly discovered hypothalamic peptide, is thought to lead to abnormal sleepiness and cataplexy in both human narcolepsy and animal models of the disease. As the POA contains extensive orexin terminals and is established as a sleep/arousal regulatory site, we evaluated a hypothesis that this site is a target for the arousal-inducing effects of orexin. Orexin-A was microinjected into lateral preoptic area (IPOA) and the effects on sleep-wakefulness and brain temperature were studied. Compared to saline vehicle control, orexin-A induced an increase in wakefulness for 70 min and suppressed all sleep stages, especially SWS2 and REM for 80 and 90 min, respectively. Brain temperature was not differentially affected by orexin-A compared to saline control. The orexin-induced arousal and REM suppression are consistent with the orexin-deficiency model of narcolepsy. Our results suggest that the IPOA orexin terminal field or adjacent structures may be a locus of arousal regulation by this peptide and a substrate of sleep-wake regulatory deficits in narcolepsy.

A subset of neurons recorded in the magnocellular basal forebrain (mBF) of cats and rats exhibit elevated discharge rates during waking and REM sleep, and diminished discharge during sleep with cortical EEG synchrony (nonREM sleep). This pattern is observed in mBF neurons in cats with identified ascending projections, and in neurons located in cholinergic regions of the rat mBF. However, the cholinergic versus noncholinergic nature of recorded cells could not be determined with the extracellular recording method employed. During waking, discharge of mBF neurons is strongly movement-related. Peak discharge rates occur during a variety of head and limb movements. Discharge rates during waking immobility are reduced by >50% compared to rates during waking movement. The absence of movement accounts for more of the variance in discharge across the sleep-wake cycle than does the presence of cortical EEG synchronization. Several factors participate in the regulation of mBF neuronal activity across arousal states. Tonic inhibition mediated by adenosine appears to be present during both waking and sleep. In some mBF neurons, increased GABAergic inhibition contributes to nonREM sleep-related reductions in discharge rate. Fluctuations in mBF cell activity during waking behaviors may reflect changing excitatory input from neurons in the pontine and midbrain tegmentum.

Pathways mediating the generation and/or maintenance of sleep reside within the preoptic/anterior hypothalamus (POAH). Reproduction, water balance, thermoregulation, and neuroendocrine functions are also associated with POAH, but it is not fully understood whether sleep is consolidated with these behavioral and physiological functions, or whether sleep-related circuitry is segregated from other POAH regions. Recent studies indicate that sleep mechanisms may be localized to the ventrolateral preoptic area (VLPO) and that this region sends inhibitory projections to waking/arousal-related neurons in the histaminergic tuberomammillary nucleus (TM), the noradrenergic locus coeruleus (LC), and the serotonergic dorsal raphe (DR). The present study is a quantitative investigation of preoptic area efferents to these monoaminergic groups. The results demonstrate that biotinylated dextran injections in the VLPO region reveal a robust innervation of TM that was as much as five times greater than innervation derived from other POAH subregions. The innervation of TM originated almost exclusively from injection sites in the region of galanin neurons. VLPO projections to the LC were moderately dense and were greater than in other POAH regions except for equivalent input from the medial preoptic area. Projections to the dorsal raphe were equivalent to LC innervation and were generally two to three times greater from VLPO than from other POAH regions, except for projections from the lateral preoptic region, which were similar in magnitude. The rostral and caudal levels projected more to the TM, whereas the midrostral region of VLPO strongly innervated the LC core. These findings, with recent studies demonstrating medial and lateral extensions of the sleep-related VLPO neuronal group, indicate that descending arousal state control may be mediated by this specific galaninergic/gamma-aminobutyric acid (GABA)ergic cell group.

1. The cholinergic system of the basal forebrain (BF) is hypothesized to play an important role in behavioural and electrocortical arousal. Adenosine has been proposed as a sleep-promoting substance that induces sleep by inhibiting cholinergic neurons of the BF and brainstem. However, adenosinergic influences on the activity of BF neurons in naturally awake and sleeping animals have not been demonstrated. 2. We recorded the sleep-wake discharge profile of BF neurons and simultaneously assessed adenosinergic influences on wake- and sleep-related activity of these neurons by delivering adenosinergic agents adjacent to the recorded neurons with a microdialysis probe. Discharge rates of BF neurons were recorded through two to three sleep-wake episodes during baseline (artificial cerebrospinal fluid perfusion), and after delivering an adenosine transport inhibitor (s-(p-nitrobenzyl)-6-thioinosine; NBTI), or exogenous adenosine, or a selective adenosine A1 receptor antagonist (8-cyclopentyl-1, 3-dimethylxanthine; CPDX). 3. NBTI and adenosine decreased the discharge rate of BF neurons during both waking and non-rapid eye movement (NREM) sleep. In contrast, CPDX increased the discharge rate of BF neurons during both waking and NREM sleep. These results suggest that in naturally awake and sleeping animals, adenosine exerts tonic inhibitory influences on BF neurons, supporting the hypothesized role of adenosine in sleep regulation. 4. However, in the presence of exogenous adenosine, NBTI or CPDX, BF neurons retained their wake- and sleep-related discharge patterns, i.e. still exhibited changes in discharge rate during transitions between waking and NREM sleep. This suggests that other neurotransmitters/neuromodulators also contribute to the sleep-wake discharge modulation of BF neurons.

We developed a thermoregulatory model of sleep control based on the hypothesis that non-rapid eye-movement sleep participates in homeostatic thermoregulation. This model successfully reproduced several qualitative features of human sleep/wake cycles during entrained as well as the internally desynchronized states. Among the reproduced features, generation mechanisms of the biphasic sleepiness distribution are studied here in the light of the model structure. Harmonic analysis is employed for this purpose. Through linearizations and confining the harmonics of the masking process to the fundamental component, a simplified representation of sleepiness is obtained. The simplified sleepiness is constructed with the fundamental circadian, the second harmonic components, and the constant (DC). The bimodality of the sleepiness is shown to be made by the second harmonic which is added to the fundamental component. The behavior of their amplitudes and phase positions are investigated under the varied sleep/wake durations and phase differences between the oscillators. Since the sleepiness generated by our model is roughly mimicked by the simplified representation under diverse conditions, this simplification can be regarded as adequate. From the behavior of the constituents of respective harmonic components, the fundamental component is shown to originate from the sleep/wake masking process and the circadian oscillators; the second harmonic from the multiplicative interactions between the circadian oscillators and the sleep/wake masking process. These results indicate that the rhythmic processes are principal constituents of the sleepiness, at least in the steady state.

1. 1. Prenatal stress in rats has been shown to produce long-term behavioral, neuroendocrine and neurochemical changes. These changes may model aspects of human depressive illness. 2. 2. In this pilot investigation, adult male offspring exposed to stress in utero and nonstressed controls were studied using 24-hour electroencephalographic sleep recordings. 3. 3. Prenatally stressed animals demonstrated reduced latency to the onset of rapid eye movement (REM) sleep, prolongation of the first REM episode, and diminished slowwave sleep. 4. 4. Although preliminary, the observed changes parallel those seen in studies of human depression. These data further support the face validity of the prenatal stress model as a potential tool for future studies on the pathophysiology of depressive disorder.