Development and Reporting of Prediction Models: Guidance for Authors From Editors of Respiratory, Sleep, and Critical Care Journals.

Prediction models aim to use available data to predict a health state or outcome that has not yet been observed. Prediction is primarily relevant to clinical practice, but is also used in research, and administration. While prediction modeling involves estimating the relationship between patient factors and outcomes, it is distinct from casual inference. Prediction modeling thus requires unique considerations for development, validation, and updating. This document represents an effort from editors at 31 respiratory, sleep, and critical care medicine journals to consolidate contemporary best practices and recommendations related to prediction study design, conduct, and reporting. Herein, we address issues commonly encountered in submissions to our various journals. Key topics include considerations for selecting predictor variables, operationalizing variables, dealing with missing data, the importance of appropriate validation, model performance measures and their interpretation, and good reporting practices. Supplemental discussion covers emerging topics such as model fairness, competing risks, pitfalls of "modifiable risk factors", measurement error, and risk for bias. This guidance is not meant to be overly prescriptive; we acknowledge that every study is different, and no set of rules will fit all cases. Additional best practices can be found in the Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis (TRIPOD) guidelines, to which we refer readers for further details.

The role of adenosine in the maturation of sleep homeostasis in rats.

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.

Suppression of preoptic sleep-regulatory neuronal activity during corticotropin-releasing factor-induced sleep disturbance.

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.

Neuronal activity in the preoptic hypothalamus during sleep deprivation and recovery sleep.

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.

Adenosine A(2A) receptors regulate the activity of sleep regulatory GABAergic neurons in the preoptic hypothalamus.

The median preoptic nucleus (MnPN) and the ventrolateral preoptic area (VLPO) are two hypothalamic regions that have been implicated in sleep regulation, and both nuclei contain sleep-active GABAergic neurons. Adenosine is an endogenous sleep regulatory substance, which promotes sleep via A1 and A2A receptors (A2AR). Infusion of A2AR agonist into the lateral ventricle or into the subarachnoid space underlying the rostral basal forebrain (SS-rBF), has been previously shown to increase sleep. We examined the effects of an A2AR agonist, CGS-21680, administered into the lateral ventricle and the SS-rBF on sleep and c-Fos protein immunoreactivity (Fos-IR) in GABAergic neurons in the MnPN and VLPO. Intracerebroventricular administration of CGS-21680 during the second half of lights-on phase increased sleep and increased the number of MnPN and VLPO GABAergic neurons expressing Fos-IR. Similar effects were found with CGS-21680 microinjection into the SS-rBF. The induction of Fos-IR in preoptic GABAergic neurons was not secondary to drug-induced sleep, since CGS-21680 delivered to the SS-rBF significantly increased Fos-IR in MnPN and VLPO neurons in animals that were not permitted to sleep. Intracerebroventricular infusion of ZM-241385, an A2AR antagonist, during the last 2 h of a 3-h period of sleep deprivation caused suppression of subsequent recovery sleep and reduced Fos-IR in MnPN and VLPO GABAergic neurons. Our findings support a hypothesis that A2AR-mediated activation of MnPN and VLPO GABAergic neurons contributes to adenosinergic regulation of sleep.

Maturation of sleep homeostasis in developing rats: a role for preoptic area neurons.

The present study evaluated the hypothesis that developmental changes in hypothalamic sleep-regulatory neuronal circuits contribute to the maturation of sleep homeostasis in rats during the fourth postnatal week. In a longitudinal study, we quantified electrographic measures of sleep during baseline and in response to sleep deprivation (SD) on postnatal days 21/29 (P21/29) and P22/30 (experiment 1). During 24-h baseline recordings on P21, total sleep time (TST) during the light and dark phases did not differ significantly. On P29, TST during the light phase was significantly higher than during the dark phase. Mean duration of non-rapid-eye-movement (NREM) sleep bouts was significantly longer on P29 vs. P21, indicating improved sleep consolidation. On both P22 and P30, rats exhibited increased NREM sleep amounts and NREM electroencephalogram delta power during recovery sleep (RS) compared with baseline. Increased NREM sleep bout length during RS was observed only on P30. In experiment 2, we quantified activity of GABAergic neurons in median preoptic nucleus (MnPN) and ventrolateral preoptic area (VLPO) during SD and RS in separate groups of P22 and P30 rats using c-Fos and glutamic acid decarboxylase (GAD) immunohistochemistry. In P22 rats, numbers of Fos(+)GAD(+) neurons in VLPO did not differ among experimental conditions. In P30 rats, Fos(+)GAD(+) counts in VLPO were elevated during RS. MnPN neuronal activity was state-dependent in P22 rats, but Fos(+)GAD(+) cell counts were higher in P30 rats. These findings support the hypothesis that functional emergence of preoptic sleep-regulatory neurons contributes to the maturation of sleep homeostasis in the developing rat brain.

Growth hormone-releasing hormone activates sleep regulatory neurons of the rat preoptic hypothalamus.

We examined whether growth hormone-releasing hormone (GHRH) may promote non-rapid eye movement (NREM) sleep via activation of GABAergic neurons in the preoptic area. Male Sprague-Dawley rats were implanted with EEG, EMG electrodes and a unilateral intracerebroventricular cannula. Groups of rats received injections (3 microl icv) with gonadotropin-releasing hormone (GHRH) (0.1 nmol/100 g body wt) or equal volume of physiological saline at the onset of the dark period and were permitted spontaneous sleep for 90 min. Separate groups of rats were sleep deprived by gentle handling for 90 min, beginning at the time of GHRH or saline injection, at the onset of the dark period. Other groups of rats received intracerebroventricular octreotide (somatostatin analog OCT) injections, intracerebroventricular injection of one of two doses of competitive GHRH antagonist, or intracerebroventricular saline injection at light onset and were then permitted 90 min spontaneous sleep-waking. Rats were killed immediately after the 90-min sleep/wake monitoring period. Brain tissue was processed for immunohistochemistry for c-Fos protein and glutamic acid decarboxylase (GAD). Single c-Fos and dual Fos-GAD cell counts were determined in the median preoptic nucleus (MnPN), and in the core and the extended parts of the ventrolateral preoptic nucleus (cVLPO and exVLPO). Intracerebroventricular GHRH elicited a significant increase in NREM sleep amount. Double-labeled Fos+GAD cell counts were significantly elevated after GHRH injection in the MnPN and VLPO in both undisturbed and sleep-deprived groups. OCT and GHRH antagonist significantly decreased NREM sleep amount compared with control rats. OCT injection increased single c-Fos-labeled cell counts in the MnPN, but not in the VLPO. Double-labeled cell counts were significantly reduced after OCT and the high dose of GHRH antagonist injection in all areas examined. These findings identify GABAergic neurons in the MnPN and VLPO as potential targets of the sleep-regulatory actions of GHRH.

Hypothalamic versus neocortical control of sleep.

PURPOSE OF REVIEW: Regions of the neocortex most strongly activated during waking exhibit increased sleep intensity during subsequent sleep. The novel concept that aspects of sleep homeostasis are determined locally in the cortex contrasts with the established views that global changes in neocortical activity during sleep are achieved through inhibition of ascending arousal systems that originate in the brainstem and hypothalamus. RECENT FINDINGS: Experiments in animals and humans document asymmetries in neocortical electroencephalogram (EEG) slow-wave activity (SWA), a marker of homeostatic sleep need, as a result of functional activity during waking. In addition to local, use-dependent augmentation of EEG SWA and evoked potentials, expression of plasticity-related genes and of sleep-regulatory cytokines and neuromodulators have been shown to be elevated in a use-dependent manner in neocortex. The functional consequences of local sleep are hypothesized to involve regulation of synaptic plasticity, synaptic homeostasis and energy balance. SUMMARY: The evidence for use-dependent modulation of neocortical activity during sleep is compelling and provides novel insights into sleep function. However, local changes in neocortex are generally expressed on a background of global sleep. It remains to be determined if events initiated in the cortex have global sleep-promoting effects and how neocortical and hypothalamic mechanisms of sleep control interact.

Functional correlates of activity in neurons projecting from the lamina terminalis to the ventrolateral periaqueductal gray.

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.

Chronic Suppression of Hypothalamic Cell Proliferation and Neurogenesis Induces Aging-Like Changes in Sleep-Wake Organization in Young Mice.

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-ß-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.

The median preoptic nucleus reciprocally modulates activity of arousal-related and sleep-related neurons in the perifornical lateral hypothalamus.

The perifornical-lateral hypothalamic area (PF/LH) contains neuronal groups playing an important role in control of waking and sleep. Among the brain regions that regulate behavioral states, one of the strongest sources of projections to the PF/LH is the median preoptic nucleus (MnPN) containing a sleep-active neuronal population. To evaluate the role of MnPN afferents in the control of PF/LH neuronal activity, we studied the responses of PF/LH cells to electrical stimulation or local chemical manipulation of the MnPN in freely moving rats. Single-pulse electrical stimulation evoked responses in 79% of recorded PF/LH neurons. No cells were activated antidromically. Direct and indirect transsynaptic effects depended on sleep-wake discharge pattern of PF/LH cells. The majority of arousal-related neurons, that is, cells discharging at maximal rates during active waking (AW) or during AW and rapid eye movement (REM) sleep, exhibited exclusively or initially inhibitory responses to stimulation. Sleep-related neurons, the cells with elevated discharge during non-REM and REM sleep or selectively active in REM sleep, exhibited exclusively or initially excitatory responses. Activation of the MnPN via microdialytic application of L-glutamate or bicuculline resulted in reduced discharge of arousal-related and in excitation of sleep-related PF/LH neurons. Deactivation of the MnPN with muscimol caused opposite effects. The results indicate that the MnPN contains subset(s) of neurons, which exert inhibitory control over arousal-related and excitatory control over sleep-related PF/LH neurons. We hypothesize that MnPN sleep-active neuronal group has both inhibitory and excitatory outputs that participate in the inhibitory control of arousal-promoting PF/LH mechanisms.

Rapid eye movement sleep deprivation contributes to reduction of neurogenesis in the hippocampal dentate gyrus of the adult rat.

STUDY OBJECTIVES: The dentate gyrus (DG) of the adult hippocampus contains progenitor cells, which have potential to differentiate into neurons. Previously we reported that 96 hours of total sleep deprivation reduces neurogenesis in the DG of adult rats. Loss of either non-rapid eye movement (NREM) or rapid eye movement (REM) sleep could have contributed to the effect of total sleep deprivation. The present study assessed the effect of 4 days of REM sleep deprivation (REMD) on neurogenesis. DESIGN: REMD was achieved by brief treadmill movement initiated by automatic online detection of REM sleep. A yoked-control (YC) rat was placed in the same treadmill and experienced the identical movement regardless the stage of the sleep-wake cycle. The thymidine analog 5- bromo- 2'- deoxy-uridine and the intrinsic proliferation marker, Ki-67, were both used to label proliferating cells. SETTING: Basic neurophysiology laboratory. PARTICIPANTS: Male Sprague-Dawley male rats (300-320 g). RESULTS: REM sleep was reduced by 85% in REMD rats and by 43% in YC, compared with cage control animals and by 79% in REMD rats compared with YC. NREM sleep and slow wave activity within NREM did not differ in REMD and YC groups. Cell proliferation was reduced by 63 % in REMD compared with YC rats, and by 82% and 51%, respectively, in REMD and YC rats compared with cage controls. Across all animals, cell proliferation exhibited a positive correlation with the percentage of REM sleep (r = 0.84, P < 0.001). Reduced cell proliferation in REMD rats was confirmed with the intrinsic proliferation marker, Ki-67. REMD also reduced the percentage of proliferating cells that later expressed a mature neuronal marker. CONCLUSIONS: The present findings support a hypothesis that REM sleep-associated processes facilitate proliferation of granule cells in the adult hippocampal DG.

Hypothalamic regulation of sleep and arousal.

Normal waking is associated with neuronal activity in several chemically defined ascending arousal systems. These include monoaminergic neurons in the brainstem and posterior hypothalamus, cholinergic neurons in the brainstem and basal forebrain, and hypocretin (orexin) neurons in the lateral hypothalamus. Collectively, these systems impart tonic activation to their neuronal targets in the diencephalon and neocortex that is reflected in the low-voltage fast-frequency electroencephalogram patterns of wakefulness. Neuronal discharge in these arousal systems declines rapidly at sleep onset. Transitions from waking to sleep, therefore, involve coordinated inhibition of multiple arousal systems. An important source of sleep-related inhibition of arousal arises from neurons located in the preoptic hypothalamus. These preoptic neurons are strongly activated during sleep, exhibiting sleep/waking state-dependent discharge patterns that are the reciprocal of that observed in the arousal systems. The majority of preoptic sleep regulatory neurons synthesize the inhibitory neurotransmitter GABA. Anatomical and functional evidence supports the hypothesis that GABAergic neurons in the median preoptic nucleus (MnPN) and ventrolateral preoptic area (VLPO) exert inhibitory control over the monoaminergic systems and the hypocretin system during sleep. Recent findings indicate that MnPN and VLPO neurons integrate homeostatic aspects of sleep regulation and are important targets for endogenous sleep factors, such as adenosine and growth hormone releasing hormone.

Inactivation of median preoptic nucleus causes c-Fos expression in hypocretin- and serotonin-containing neurons in anesthetized rat.

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 microl 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.

Body temperature and sleep.

Sleep in mammals is accompanied by a decrease in core body temperature (CBT). The circadian clock in the hypothalamic suprachiasmatic nucleus regulates daily rhythms in both CBT and arousal states, and these rhythms are normally coupled. Reductions in metabolic heat production resulting from behavioral quiescence and reduced muscle tone along with changes in autonomic nervous system activity and thermoeffector activity contribute to the sleep-related fall in CBT. Reductions in sympathetic tone to the peripheral vasculature resulting in heat loss through the skin are reflected in a sleep-related increase in distal skin temperature that is a prominent feature of sleep onset in humans. Within a sleep episode, patterns of autonomic nervous system and thermoeffector activity and the ability to defend against heat and cold exposure differ during nonrapid eye movement (NREM) and rapid eye movement sleep. Anatomic and functional integration of the control of arousal states and thermoregulation occur in the preoptic/anterior hypothalamus. Subsets or warm-sensing neurons in the preoptic/anterior hypothalamus implicated in CBT regulation are spontaneously activated during sleep onset and NREM sleep compared to waking and may underlie sleep-related changes in autonomic nervous system and thermoeffector activity.

Characteristics of sleep-active neurons in the medullary parafacial zone in rats.

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.

Preoptic area neurons and the homeostatic regulation of rapid eye movement 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-fos in 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.

Homeostatic regulation of sleep: a role for preoptic area neurons.

The median preoptic nucleus (MnPN) and the ventrolateral preoptic area (vlPOA) contain putative sleep-regulatory neurons that exhibit elevated discharge rates during sleep compared with waking. Expression of c-Fos protein immunoreactivity (IR) in GABAergic neurons in the MnPN and the vlPOA is high in spontaneously sleeping rats and in rats undergoing recovery sleep after sleep deprivation. However, it is unclear whether c-Fos-IR in these neurons is evoked by increases in sleep pressure or by increases in sleep amount. We examined c-Fos-IR in MnPN and vlPOA neurons under experimental conditions that dissociated homeostatic sleep pressure, sleep amount, and time of day. Groups of rats with strong diurnal rhythms in sleep-wake organization were killed after (1) spontaneous sleep in the light, (2) spontaneous sleep in the dark, (3) sleep deprivation (SLD) in the light and (4) recovery sleep after SLD in the light. Numbers of GABAergic neurons expressing c-Fos-IR in the MnPN were significantly higher after SLD in the light compared with spontaneous sleep and recovery sleep in the light. In contrast, Fos-IR in vlPOA GABAergic neurons was most prevalent after spontaneous sleep and recovery sleep in the light. No light-dark differences in Fos-IR were observed in the MnPN after SLD in groups of rats with weak or absent diurnal sleep-waking rhythms. Our findings define potential roles for MnPN and vlPOA GABAergic neurons in homeostatic aspects of sleep regulation.

Hypothalamic control of sleep.

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.

Cell proliferation in the dentate gyrus of the adult rat fluctuates with the light-dark cycle.

This study measured cell proliferation in the hippocampal dentate gyrus in the adult rat at different times within a 12:12h light-dark cycle. The experiments were conducted in animals living in either a complex environment or in standard lab cages. A single dose of the thymidine analog 5-Bromo-2'-deoxyuridine (BrdU) was injected 2h before animals were sacrificed either 4, 11, 16, or 23h after the beginning of the light phase of the light-dark cycle (designated ZT0). In both studies, we found a significant increase in the number of BrdU-positive cells in the subgranular cell layer (SGZ) following BrdU administration at ZT9 and sacrifice at ZT11, compared to other circadian times examined. BrdU administration at ZT9 was timed to primarily identify proliferating cells that were in the S phase of the cell cycle during the light phase. Our results suggest that cell proliferation is enhanced either by sleep or by other variables coupled to the light phase of the circadian cycle.