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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., SCOPUS: ar.j, info:eu-repo/semantics/published

Sleep homeostasis is a fundamental property of vigilance state regulation that is highly conserved across species. Neuronal systems and circuits that underlie sleep homeostasis are not well understood. In Drosophila, a neuronal circuit involving neurons in the ellipsoid body and in the dorsal Fan-shaped body is a candidate for both tracing sleep need during waking and translating it to increased sleep drive and expression. Sleep homeostasis in rats and mice involves multiple neuromodulators acting on multiple wake- and sleep-promoting neuronal systems. A functional central homeostat emerges from A1 receptor mediated actions of adenosine on wake-promoting neurons in the basal forebrain and hypothalamus, and A2A adenosine receptor-mediated actions on sleep-promoting neurons in the preoptic hypothalamus and nucleus accumbens.

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

The 21st century has brought with it a welcome call for increased rigor in observational research methods (1, 2). It is not that observational research methods are inherently flawed – they are not (3, 4). Observational studies can contribute valuable evidence supporting causal associations when designed and conducted using rigorous methods. The “flaws” are a result of reliance on outdated methodology, inadequate attention to threats to validity (such as confounding), opaque reporting of results, lack of replication, and a failure to interpret findings within the context of the limitations of observational research methodology. Aware of this situation and influenced by our experience as journal editors, we convened an ad hoc group of 47 editors of 35 respiratory, sleep, and critical care journals to offer guidance to authors, peer reviewers, and researchers on the design and reporting of observational causal inference studies. This guidance takes the form of a call for investigators to consider making major changes to their approach to such studies. This document represents our current best understanding of approaches to causal inference, an active area of research. We anticipate that best practice in this, as in any scientific endeavor, will continue to evolve, requiring this document to be updated every 5 to 10 years. We believe these changes will increase the rigor, validity, and value of the work we publish in our journals.

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.

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.

Brain activation during wakefulness is sustained by multiple arousal systems. Dysfunction of one or more arousal systems is a feature of neurological disorders associated with hypersomnolence and/or sleep-wake cycle disturbance. Narcolepsy, Alzheimer’s disease (AD), Parkinson’s disease (PD), and traumatic brain injury appear to involve hypocretin (HCT) and possibly histamine insufficiency as a mechanism related to excessive daytime sleepiness. Loss of cholinergic neurons in AD and of dopamine neurons in PD contributes to sleep-wake disturbance. GABAergic neurons in the preoptic hypothalamus and rostral medulla promote sleep through inhibition of arousal systems. Pathology of preoptic sleep regulatory circuits is correlated with sleep disturbance in AD. An unidentified endogenous somnogen that potentiates the actions of gamma-aminobutyric acid (GABA) is present in the cerebrospinal fluid (CSF) of patients with primary hypersomnia. Descending pathways from the dorsal lateral pons to the ventral medulla and spinal cord are responsible for the inhibition of spinal motoneurons during rapid eye movement (REM) sleep and are implicated in human REM sleep behavior disorder.

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.

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 is accompanied by a reorganization of thermoregulatory control that includes reduced metabolism and activation of heat loss responses to achieve a lower core body temperature (CBT). Compared to waking, regulatory responses to thermal challenge are reduced during sleep, particularly REM sleep. Vulnerability to thermal stress during sleep may explain why many species prefer a thermally neutral and thermally stable environment in which to sleep. Brain mechanisms that regulate sleep and thermoregulation are anatomically functionally related. Within the preoptic and anterior hypothalamus, populations of sleep-regulatory neurons are warm-sensitive, exhibiting increases in activity in responses to increases in CBT and skin temperature (Tsk). Activation of warm-sensing neurons in the POAH promotes sleep. In humans, activation of heat loss mechanisms at the time of sleep onset impacts sleep amount and sleep quality. An elevated distal-to-proximal Tsk gradient at sleep onset reflects increased peripheral blood flow and heat loss and is associated with short sleep latencies and increased sleep efficiency and sleep depth. Increases in peripheral blood flow and Tsk can exert feed-forward excitatory effects on brain mechanisms that promote sleep, and function to reinforce circadian and homeostatic aspects of sleep regulation.

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.

The ventrolateral division of the periaqueductal gray (vlPAG) and the adjacent deep mesencephalic reticular nucleus have been implicated in the control of sleep. The preoptic hypothalamus, which contains populations of sleep-active neurons, is an important source of afferents to the vlPAG. The perifornical lateral hypothalamus (LH) contains populations of wake-active neurons and also projects strongly to the vlPAG. We examined nonREM and REM sleep-dependent expression of c-Fos protein in preoptic-vlPAG and LH-vlPAG projection neurons identified by retrograde labeling with Fluoro-gold (FG). Separate groups of rats (n=5) were subjected to 3 hours total sleep deprivation (TSD) followed by 1 hour recovery sleep (RS), or to 3 hours of selective REM sleep deprivation (RSD) followed by RS. A third group of rats (n=5) was subjected to TSD without opportunity for RS (awake group). In the median preoptic nucleus (MnPN), the percentage of FG+ neurons that were also Fos+ was higher in TSD-RS animals compared to both RSD-RS rats and awake rats. There were significant correlations between time spent in deep nonREM sleep during the 1-hour prior to sacrifice across groups and the percentage of double-labeled cells in MnPN and ventrolateral preoptic area (VLPO). There were no significant correlations between percentage of double labeled neurons and time spent in REM sleep for any of the preoptic nuclei examined. In the LH, percentage of double-labeled neurons was highest in awake rats, intermediate in TSD-RS rats and lowest in the RSD-RS group. These results suggest that neurons projecting from MnPN and VLPO to the vlPAG are activated during nonREM sleep and support the hypothesis that preoptic neurons provide inhibitory input to vlPAG during sleep. Suppression of excitatory input to the vlPAG from the LH during sleep may have a permissive effect on REM sleep generation.

We examined the effects of eszopiclone (ESZ), a GABA-A receptor agonist in current clinical use as a hypnotic medication, on the activity of subcortical wake- and sleep-active neuronal populations in the rat brain. Sleep-wake states were quantified after i.p. injections of ESZ (3 and 10 mg/kg) or vehicle administered early in the dark phase, when rats are spontaneously awake. Rats were euthanized 2 h post-injection and brain tissue was processed for c-Fos protein immunoreactivity (IR) and for neurotransmitter markers. ESZ at 3 and 10 mg/kg increased time spent in non-rapid-eye-movement (nonREM) sleep, but had no significant effect on Fos-IR in GABAergic neurons in the preoptic hypothalamus that normally express c-Fos during sleep. Among wake-active cell types examined, Fos-IR in hypocretin (HCRT) neurons in the perifornical lateral hypothalamus (LH) was reduced following 3 and 10 mg/kg ESZ. At 10 mg/kg, ESZ suppressed Fos-IR in cholinergic and noncholinergic neurons in the basal forebrain and in serotonergic and nonserotonegic neurons in the dorsal raphe. Having determined that HCRT neurons were responsive to the low dose of systemic ESZ, we unilaterally perfused ESZ directly into the LH of awake rats, using reverse microdialysis. Perfusion of ESZ at 50 μM into the LH for 2 h suppressed waking-related Fos-IR in HCRT neurons, but not in nonHCRT neurons ipsilateral to the dialysis probe. Bilateral LH perfusion of ESZ at 50 μM for 2 h early in the dark phase significantly increased sleep. These findings demonstrate that sleep induction by ESZ does not require activation of GABAergic sleep-regulatory neurons in the preoptic hypothalamus, and identify suppression of HCRT neurons in the LH and suppression of basal forebrain and dorsal raphe neurons as potential mechanisms underlying the sleep-promoting effects of ESZ.

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

Absence epilepsy (AE) in humans and the genetic AE model in WAG/Rij rats are both associated with abnormalities in sleep architecture that suggest insufficiency of the sleep-promoting mechanisms. In this study we compared the functionality of sleep-active neuronal groups within two well-established sleep-promoting sites, the ventrolateral and median preoptic nuclei (VLPO and MnPN, respectively), in WAG/Rij and control rats. Neuronal activity was assessed using c-Fos immunoreactivity and chronic single-unit recording techniques. We found that WAG/Rij rats exhibited a lack of sleep-associated c-Fos activation of GABAergic MnPN and VLPO neurons, a lower percentage of MnPN and VLPO cells increasing discharge during sleep and reduced firing rates of MnPN sleep-active neurons, compared to non-epileptic rats. The role of sleep-promoting mechanisms in pathogenesis of absence seizures was assessed in non-epileptic rats using electrical stimulation and chemical manipulations restricted to the MnPN. We found that fractional activation of the sleep-promoting system in waking was sufficient to elicit absence-like seizures. Given that reciprocally interrelated sleep-promoting and arousal neuronal groups control thalamocortical excitability, we hypothesize that malfunctioning of sleep-promoting system results in impaired ascending control over thalamocortical rhythmogenic mechanisms during wake-sleep transitions thus favoring aberrant thalamocortical oscillations. Our findings suggest a pathological basis for AE-associated sleep abnormalities and a mechanism underlying association of absence seizures with wake-sleep transitions.

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.