James M. Krueger, Ph.D - US grants

Our objective is to establish the relationship between Interleukin-1 (IL-1) (also called endogenous pyrogen) and the regulation of sleep-wake cycles. The fundamental proposition that underlies this concern is that there is an endogenous factor(s) that modulates sleep-wake cycles. We hypothesize that IL-1 is one of these substances. An obvious prerequisite to this hypothesis is to show that IL-1 induces sleep. Preliminary results from our laboratory show that IL-1 induces dose-dependent increases in rabbit slow-wave sleep (SWS) and that these sleep responses are independent from febrile responses that are also induced by IL-1. In these studies we used rabbits whic do not have pronounced circadian sleep-wake cycles and restricted our analysis to SWS. We now propose to determine the effects of IL-1 on the architecture of sleep-wake cycles using cats. Our studies in cats will also employ antipyretics to determine if, as in rabbits, febrile responses can be separated from sleep responses. These studies will be carried out using cats with chronically implanted EEG, and EMG electrodes, brain thermistors and ventricular guide tubes. Following intraventricular infusion of various doses of IL-1, responses will be monitored for 32 hours. The effects of IL-1 on brain temperature, SWS and REM sleep as well as the relationship of these responses to each other and wakefulness will be determined. We also propose to microinject IL-1 into brain to determine the effective site(s) responsible for sleep responses. In this case rabbits will be provided with chronically implanted EEG electrodes, brain thermistors, and brain guide tubes. Following injections of 1-2 ul of solutions of IL-1, rabbits will be recorded from for 6 or more hours. During this period duration of SWS, brain temperature and quantitative changes in EEG slow waves will be determined. Anticipated results are of broad significance since IL-1 may provide a new endogenous agent that can be used to examine the mechanisms that underlie sleep.

Robust increases in nonrapid-eye movement sleep (NREMS) occur in response to sleep deprivation, infection, and mild increases in ambient temperature (Tamb). We hypothesize that interleukin-1Beta(IL-1Beta) mediates NREMS responses to sleep deprivation, infection, and Tamb. The IL-1 family includes 3 structurally related proteins IL-1alpha, IL-1Beta, and the IL-1 receptor antagonist (IL-1ra), and the type I and II-IL-1 receptors. Much data implicates IL-1Beta in NREMS regulation, e.g., exogenous IL-1Beta enhances NREMS in several species. Preliminary data also support the hypothesis, e.g., NREMS rebound after sleep deprivation is attenuated if animals are pretreated with anti-IL-1Beta or the IL-1ra. Four specific aims (SAs) are proposed to further test the hypothesis. Four blockers of the family of IL-1 molecules, each having a unique action, will be used in combination with sleep deprivation (SA #1), injection of somnogenic microbial products (SA#2), and mild increased in Tamb (SA#3). The blockers are anti-IL-1alpha, anti-IL-1Beta, the IL-1ra, and a soluble IL-1 type I receptor. In these experiments, it will be determined whether central or systemic changes in the IL-1 family are relevant to NRMES responses in rabbits. In separate concurrent experiments (SA#4) we will determine levels of IL-1alpha, IL-1Beta and the IL-1ra in blood and specific brain regions after sleep deprivation, injection of microbial products, and mild increases in Tamb. It is predicted that regardless of the method used to increase sleep propensity, CNS IL-1Beta will increase. If increased IL-1Beta is blocked, NREMDS response will also be blocked. Further, we predict that the IL-1ra will also increase in brain with a delay relative to IL-1Beta. Expected data will have bearing on whether the IL-1 family serves a physiological role in sleep regulation. Sleep remains an enigma; we know neither its functions nor the exact cellular and molecular mechanisms responsible for sleep. Knowledge of the molecular causes of sleep is likely to be a necessary step toward our understanding of sleep function; expected results will greatly aid this endeavor.

Our objective is to establish the relationship between Interleukin-1 (IL-1) (also called endogenous pyrogen) and the regulation of sleep-wake cycles. The fundamental proposition that underlies this concern is that there is an endogenous factor(s) that modulates sleep-wake cycles. We hypothesize that IL-1 is one of these substances. An obvious prerequisite to this hypothesis is to show that IL-1 induces sleep. Preliminary results from our laboratory show that IL-1 induces dose-dependent increases in rabbit slow-wave sleep (SWS) and that these sleep responses are independent from febrile responses that are also induced by IL-1. In these studies we used rabbits whic do not have pronounced circadian sleep-wake cycles and restricted our analysis to SWS. We now propose to determine the effects of IL-1 on the architecture of sleep-wake cycles using cats. Our studies in cats will also employ antipyretics to determine if, as in rabbits, febrile responses can be separated from sleep responses. These studies will be carried out using cats with chronically implanted EEG, and EMG electrodes, brain thermistors and ventricular guide tubes. Following intraventricular infusion of various doses of IL-1, responses will be monitored for 32 hours. The effects of IL-1 on brain temperature, SWS and REM sleep as well as the relationship of these responses to each other and wakefulness will be determined. We also propose to microinject IL-1 into brain to determine the effective site(s) responsible for sleep responses. In this case rabbits will be provided with chronically implanted EEG electrodes, brain thermistors, and brain guide tubes. Following injections of 1-2 ul of solutions of IL-1, rabbits will be recorded from for 6 or more hours. During this period duration of SWS, brain temperature and quantitative changes in EEG slow waves will be determined. Anticipated results are of broad significance since IL-1 may provide a new endogenous agent that can be used to examine the mechanisms that underlie sleep.

The broad objective of this proposal is to examine the hypothesis that hypothalamic GRF-like peptides are involved in sleep regulation, i.e., GRF (growth hormone releasing factor), VlP (vasoactive intestinal peptide) and PHI (peptide histidine- isoleucine). We hypothesize that GRF stimulation of GH (growth hormone) secretion and promotion of nonREM sleep (NREMS) are parallel, albeit dissociable, functions of hypothalamic neurons which project to both the median eminence and basal forebrain hypnogenic areas. Further, GH and SOM (somatostatin) released by GRF may promote REMS (REM sleep) subsequent to NREMS. VlP and PHI are structurally related to GRF and stimulate secretion of pituitary PRL (prolactin) that can reach the brain by means of a specific transport mechanism. The VIP/PHI-PRL axis is assumed to have a permissive or facilitatory effect on REMS. Some previously proposed sleep factors, e.g., interleukin-l (ILl), may act through these endocrine mechanisms. The hypothesis is based upon preliminary data and previous reports: 1) GH and PRL secretions are coupled to sleep; 2) GRF promotes first NREMS then REMS, while GH and SOM selectively promote REMS; 3) both VIP and PRL increase REMS; and 4) proposed sleep factors affect endocrine regulation. The hypothesis will be tested by studying the effects of GRF, VIP, PHI, and PRL on sleep, thermoregulation and secretion of hormones after intracerebroventricular, intracerebral, systemic injection. Antibodies against GRF and pharmacological lesions of GRF neurons will be used to block endogenous GRF actions to determine the role of endogenous GRF in physiological sleep and in sleep (and GH secretion) elicited by ILl. The contribution of endogenous PRL to physiological sleep and to REMS elicited by VIP and PHI will be studied in animals pretreated with PRL antibodies. Sleep-related variations in GRF secretions will be measured. The experiments will be carried out in rats and rabbits chronically implanted with EEG electrodes, intracerebral cannulas and intracardial catheters. Hormone levels will be measured by means of ELISA or RIA from serial samples. We anticipate that the results will provide evidence that endocrine and sleep regulations involve common regulatory pathways.

Our objective is to establish the relationship between Interleukin-1 (IL-1) (also called endogenous pyrogen) and the regulation of sleep-wake cycles. The fundamental proposition that underlies this concern is that there is an endogenous factor(s) that modulates sleep-wake cycles. We hypothesize that IL-1 is one of these substances. An obvious prerequisite to this hypothesis is to show that IL-1 induces sleep. Preliminary results from our laboratory show that IL-1 induces dose-dependent increases in rabbit slow-wave sleep (SWS) and that these sleep responses are independent from febrile responses that are also induced by IL-1. In these studies we used rabbits whic do not have pronounced circadian sleep-wake cycles and restricted our analysis to SWS. We now propose to determine the effects of IL-1 on the architecture of sleep-wake cycles using cats. Our studies in cats will also employ antipyretics to determine if, as in rabbits, febrile responses can be separated from sleep responses. These studies will be carried out using cats with chronically implanted EEG, and EMG electrodes, brain thermistors and ventricular guide tubes. Following intraventricular infusion of various doses of IL-1, responses will be monitored for 32 hours. The effects of IL-1 on brain temperature, SWS and REM sleep as well as the relationship of these responses to each other and wakefulness will be determined. We also propose to microinject IL-1 into brain to determine the effective site(s) responsible for sleep responses. In this case rabbits will be provided with chronically implanted EEG electrodes, brain thermistors, and brain guide tubes. Following injections of 1-2 ul of solutions of IL-1, rabbits will be recorded from for 6 or more hours. During this period duration of SWS, brain temperature and quantitative changes in EEG slow waves will be determined. Anticipated results are of broad significance since IL-1 may provide a new endogenous agent that can be used to examine the mechanisms that underlie sleep.

The broad objective of this proposal is to investigate the role of interleukin-1 (IL1) in the regulation of sleep/wake cycles. We hypothesize that IL1 plays a key role in the physiological regulation of sleep. Major evidence in support of this hypothesis includes: 1) IL1 and many substances that induce IL1 production enhance non-rapid-eye-movement sleep (NREMS), 2) substances that inhibit IL1 production or activity inhibit NREMS, 3) IL1B-mRNA and IL1 receptors are found in the normal brain, 4) IL1 immunoreactive hypothalamic neurons are in the normal brain, and 5) cerebrospinal fluid levels of IL1 are higher during NREMS than during wakefulness. Despite this recent knowledge, there remains a clear need to determine sleep-related molecular mechanisms of IL1 somnogenic actions. To address these issues, using rabbit and rat sleep assay models, we propose to: Investigate substances that are know to: a) mimic IL1, e.g. IL1 fragments; and b) alter IL1 activity and/or production, e.g. anti IL1 antibodies and anti IL1-receptor antibodies; such studies will help determine what part of the iL1 molecule is responsible for this somnogenic actions, provide experimental tools that later can be used in other sleep studies and reaffirm in two species often used in sleep research that IL1, indeed, enhances NREMS. We will also investigate a positive and a negative feedback loop involved in IL1 regulation. Substances to be investigated that form part of these feedback loops include interferon and the corticotropin-releasing factor-glucocorticoid axis. Using antibodies or surgical procedures, both feedback loops will be disrupted; the effects of opening these loops on sleep and on IL1 dose-response curves will be determined. Expected results will establish a role for IL1 in sleep regulation and provide information concerning the molecular events and their order, involved in IL1-altered sleep. Such information will help provide us with an understanding of one of the major enigmas of neurobiology, the function of sleep. It will also help us understand the adaptive role sleep may play in infectious disease and possibly help guide the development of new, safe somnogenic agents.

The broad objective of this proposal is to investigate the role of interleukin-1 (IL1) in the regulation of sleep/wake cycles. We hypothesize that IL1 plays a key role in the physiological regulation of sleep. Major evidence in support of this hypothesis includes: 1) IL1 and many substances that induce IL1 production enhance non-rapid-eye-movement sleep (NREMS), 2) substances that inhibit IL1 production or activity inhibit NREMS, 3) IL1B-mRNA and IL1 receptors are found in the normal brain, 4) IL1 immunoreactive hypothalamic neurons are in the normal brain, and 5) cerebrospinal fluid levels of IL1 are higher during NREMS than during wakefulness. Despite this recent knowledge, there remains a clear need to determine sleep-related molecular mechanisms of IL1 somnogenic actions. To address these issues, using rabbit and rat sleep assay models, we propose to: Investigate substances that are know to: a) mimic IL1, e.g. IL1 fragments; and b) alter IL1 activity and/or production, e.g. anti IL1 antibodies and anti IL1-receptor antibodies; such studies will help determine what part of the iL1 molecule is responsible for this somnogenic actions, provide experimental tools that later can be used in other sleep studies and reaffirm in two species often used in sleep research that IL1, indeed, enhances NREMS. We will also investigate a positive and a negative feedback loop involved in IL1 regulation. Substances to be investigated that form part of these feedback loops include interferon and the corticotropin-releasing factor-glucocorticoid axis. Using antibodies or surgical procedures, both feedback loops will be disrupted; the effects of opening these loops on sleep and on IL1 dose-response curves will be determined. Expected results will establish a role for IL1 in sleep regulation and provide information concerning the molecular events and their order, involved in IL1-altered sleep. Such information will help provide us with an understanding of one of the major enigmas of neurobiology, the function of sleep. It will also help us understand the adaptive role sleep may play in infectious disease and possibly help guide the development of new, safe somnogenic agents.

[unreadable] DESCRIPTION (provided by applicant): Sleep propensity varies across the day, increases after sleep deprivation (SD) or during the "flu." Changes in sleep propensity reflect the degree of activation of physiological sleep mechanisms including humoral sleep mechanisms. This application is focused on the broad hypothesis that tumor necrosis factor alpha (TNFa) is a key element in the molecular network regulating sleep. Much evidence implicates TNFa in physiological sleep mechanisms and in the sleep responses occurring in many pathologies. For instance, the soluble TNF receptor (sTNFR), reduces spontaneous sleep and SD-induced sleep responses in animal models and sleepiness in insomnia patients. TNFa is, however, but one member of a molecular sleep regulatory network. The experiments in this application are focused on that network, where it acts to produce sleep and the neural circuitry involved in TNFa-enhanced sleep and EEG slow-wave activity. Aim 1 is focused on the simultaneous measurement of multiple cytokines, all of which affect sleep, using multiplexed bead array technology. We test the hypothesis that the ratio of TNFa to other cytokines in the hypothalamus (hyp), thalamus (thal), cortex (ctx) and nucleus tractus solitarius (NTS) and plasma will dynamically change with sleep history and will predict sleep propensity. Aim 2 focuses on the hypothesis that pathology-associated increases in systemic TNFa promote sleep via vagal afferents to the NTS. We will determine by bead array and TNF immunohistochemistry, the responses to intraperitoneal TNFa before and after vagotomy. Aim 3 tests the hypothesis that TNFa promotes the functional state of cortical columns analogous to sleep. To test this hypothesis, cortical column functional state will be determined electrophysiologically using multi electrode arrays in conjunction with microinjection of TNFa onto the surface of columns. Functional states will be characterized by amplitudes of surface evoked potentials and several other measurements. Aim 4 tests the hypothesis that cytokine expression changes with sleep intensity throughout the sleep regulatory axis. TNFa and/or TNF inhibitors will be unilaterally injected onto the surface of the somatosensory cortex and fos- and cytokine immunoreactivity determined in affected circuits. Preliminary data provide a rationale for each specific aim and demonstrate feasibility. Successful completion of the experiments will help integrate the biochemical, neuroanatomical and electrophysiological sleep regulatory literature. [unreadable] [unreadable] [unreadable]

Growth hormone (GH)-releasing hormone (GHRH) is a hypothalamic neurocrine which stimulates pituitary GH secretion. GHRH has become one of the best documented sleep promoting substances; much of this evidence was generated under the auspices of the previous grant. The aims of the present proposal are to study the possible humoral mechanisms involved in the sleep promoting activity of GHRH, and the contribution of GHRH to enhanced sleep elicited by heat exposure, interleukin-1 (IL1) (the key cytokine responsible for the acute phase response to infections), and by sleep deprivation. l) The role of GH in sleep has been controversial ever since a sleep-associated secretion of GH was first reported more than 20 years ago. The discovery of the sleep promoting activity of GHRH reopened this discussion. The effects on sleep of exogenous species specific GH and immunoneutralization of endogenous GH will be studied. The sleep promoting activity of GHRH will be tested in hypophysectomized rats and in rats pretreated with antiserum to GH. Many GH effects are mediated by insulin-like growth factor-I (IGF-I). Alterations in sleep in response to exogenous IGF-I will be studied in comparison with the sleep effects of the structurally homologous IGF-II and insulin. These experiments will help clarify the role of GH and IGF-I in sleep regulation and in the sleep enhancing actions of GHRH. 2) Models of sleep regulation distinguish between stimuli facilitating sleep by lowering the "sleep threshold" and stimuli acting on the "homeostatic mechanisms of sleep regulation". Heat and IL1 are putative inputs for "homeostatic sleep mechanisms", and they are also known to release GHRH. The involvement of GHRH in the sleep response to heat and IL1 will be studied in rats after immunoneutralization of GHRH. These experiments may contribute to an identification of the theoretically postulated "homeostatic sleep mechanisms". 3) Sleep pressure increases as the function of wakefulness and discharges in an enhanced recovery sleep after sleep loss. Previous studies indicate that both GHRH and IL1 are involved in the mechanisms of recovery sleep. To determine whether GHRH is one of the substances responsible for the increased sleep pressure elicited by sleep loss, hypothalamic GHRH concentration will be measured during and after sleep deprivation in normal rats and in rats pretreated with antibodies to IL1. The latter experiments will decide whether IL1 is an input for GHRH. All the proposed experiments will be carried out on rats except that the effects of IGF-I on sleep will also be tested in rabbits.

DESCRIPTION (adapted from applicant's abstract): The central hypothesis of this proposal is that interleukin-1-beta is a key regulatory component of sleep. Much data support this hypothesis; administration of exogenous IL-1-beta enhances non-rapid eye movement sleep (NREMS); inhibition of IL-1-beta inhibits spontaneous NREMS and NREMS - induced by sleep deprivation; IL-1-beta mRNA has a diurnal rhythm in brain and increases after sleep deprivation. In Specific Aim #1, the investigators will test the specific hypothesis that the IL-1 type I receptor (IL-1RI) is necessary for IL-1-mediated changes in sleep regulation. They will use a knockout mouse lacking this receptor; preliminary data suggest that these mice sleep less than strain controls. Further, they do not respond to IL-1-beta. In Specific Aim #2 the investigators will test the hypothesis that the IL-1-beta and tumor necrosis factor-alpha (TNF) systems act in concert to regulate NREMS and are part of a larger functional gene group involved in sleep regulation. For these experiments, the investigators will use the IL--1RI knockout mouse and double knockout mouse which lacks both the IL-1RI and the TNF55-kD receptor. Specific Aim #3 tests the hypothesis that somnogenic stimuli will induce changes in specific areas of brain in the levels of one or more of the members of the IL-1 receptor antagonist will be measured using reverse transcriptase polymerase chain reaction, Northern blot analyses and RNASE protection assays. The effects of various somnogens (sleep deprivation, muramyl dipeptide and acute mild increases in ambient temperature) on the levels and distribution of these mRNAs in brain will be determined. Anticipated results will help our understanding of how the brain is organized to produce sleep. They will also help our understanding of the cellular and molecular mechanisms of sleep regulation. Knowledge of such mechanisms is likely to be a necessary step toward our understanding of sleep function.

DESCRIPTION (applicant's abstract): Fatigue, excessive sleepiness, excess sleep, and sleep disturbances are presenting symptoms in nearly all infectious diseases. The broad objective of this proposal is to characterize the molecular mechanisms responsible for changes in sleep induced by influenza virus. We hypothesize that viral double-stranded (ds) RNA is produced in infected cells and it, in turn, induces an upregulation of cytokines including interferons (IFN). The cytokines then induce growth hormone releasing hormone (GHRH) release and it, via nitric oxide (NO), enhances sleep. Substantial preliminary data support this hypothesis. The model used in the proposed studies is A/PR/8/34-HIN1 influenza virus infection in the mouse. PR8 causes a pneumonitis accompanied by early onset of sleep responses. In Specific Aim #1, a comparison will be made using gene arrays of the time courses of cytokines induced by pure influenza virus and dsRNA in lung and brain. We expect a similar cytokine profile after both stimuli. In Specific Aim #2 the role of the GHRH receptor in viral-induced sleep responses will be determined. Preliminary data indicate that mice lacking a functional GHRH receptor sleep less, rather than more, after viral challenge. We anticipate that that finding will be confirmed and that GH replacement therapy will not alter the virus-induced sleep responses, but may reduce mortality. In Specific Aim #3, nitric oxide synthase knockout mice will be used to investigate the role of NO in viral-induced sleep. Preliminary data indicate an attenuated sleep response after host challenge in NOS-2 (inducible NOS) knockout mice. In Specific Aim #4, IFN receptor (types I and II) knockout mice will be used to investigate the role of IFNs in viral-induced sleep. In Specific Aims 2, 3, and 4 we anticipate that the cytokine gene profiles induced by virus in the mutant strain will be different from controls and will reflect the different sleep responses induced by virus in these mutant strains of mice. In Specific Aim #5, we will investigate, in vitro, the role of virus-associated dsRNA in cytokine induction by influenza. Since we hypothesize that dsRNA upregulates cytokines via nuclear factor kappa B (NFKB) we will determine what other activators of NFKB, e.g., free radicals, do to NFKB activation in murine macrophages and how pharmacological blockers affect viral-induced activation of NFKB and the cytokine cascade. The anticipated results will greatly aid our understanding of the molecular mechanisms involved in viral-induced sleep responses and other facets of the acute phase response.

This study will determine whether sleep parameters are altered in hyperthyroid patients and, if so, whether the abnormalities return to normal during the treated, euthyroid state."

DESCRIPTION (provided by applicant): Growth hormone (GH) releasing hormone (GHRH) is a hypothalamic neurocrine that stimulates pituitary GH release and that has become one of the best documented sleep-regulatory substances. The fundamental hypothesis of this proposal is that promotion of non-rapid eye movement sleep (NREMS) and GH secretions are independent, albeit synchronized, functions of hypothalamic GHRH-containing neurons. Evidence in support of this hypothesis includes; 1) deep NREMS is associated with GH secretion in several species; 2) GHRH promotes NREMS in rats, mice, rabbits, and humans; 3) inhibition of endogenous GHRH inhibits spontaneous NREMS and GHRH-enhanced NREMS; 4) inhibition of GH does not block GHRH enhanced sleep; 5) GHRH enhances sleep when injected into the medial preoptic region; 6) hypothalamic GHRH and GHRH levels vary with sleep propensity; and 7) negative feedback mechanisms of the somatotropic system inhibit GH secretion and NREMS simultaneously. The broad objective of the current proposal is to determine the role of GHRH in sleep changes associated with chronic or acute alterations within the somatotropic system and the localization and regulation of the hypothalamic sleep-promoting GHRHergic system. In Specific Aim #1, spontaneous sleep and sleep responses to sleep deprivation will be studied in mutant and transgenic strains of rats and mice with various alterations to the somatotropic system. In Specific Aim #2 we will determine the role of GHRH in the sleep effects of ghrelin (a newly discovered somatotropic hormone) and of somatostatin (using octreotide, a synthetic somatostatin analog) and will distinguish the mechanisms of the various behavioral and endocrine responses to both peptides. In Specific Aim #3 we will study the hypothalamic GHRHergic network involved in sleep regulation. We will determine the importance of the connections between the mediobasal hypothalamus and the anterior hypothalamic/preoptic region for sleep by means of small semicircular cuts placed in front of the arcuate nucleus. We will also use in situ hybridization to map the distribution of GHRH-receptor expressing neurons and determine which transmitter localizes with the GHRH-receptor. Finally, in Specific Aim #4 cultured hypothalamic neurons will be used in conjunction with measurement of intracellular Ca++ to determine colocalization of GHRH receptors with neurotransmitters and interleukin-1 receptors within cells and to study the regulation of GHRH-receptor expression in these cells. Preliminary data are presented for each specific aim to demonstrate feasibility. Anticipated results are expected to support the hypothesis that GHRH is a key regulatory component of NREMS and provide cellular mechanistic explanations for the involvement of GHRH in sleep regulation.

DESCRIPTION (provided by applicant): Many of the symptoms associated with sleep loss, e.g. fatigue, performance impairments, metabolic syndrome, chronic inflammation, sleepiness, etc; can be elicited by administration of interleukin-1 beta (IL1) or related cytokines or can be prevented by blocking them. Nevertheless, the regulation of the IL1 family in the brain is mostly undetermined. Recently a new brain-specific IL1 receptor accessory protein (IL1 AcPb) was identified. Although its function remains unknown preliminary data show that sleep deprivation enhances its cortical expression. The function for another IL1 family member, IL36 (formerly ILF7) has very recently (unpublished) been identified; IL36 inhibits several pro-inflammatory somnogenic cytokines including IL1, while simultaneously promoting expression of anti-inflammatory anti-somnogenic cytokines. In Aim 1, we determine the roles that IL1 AcPb and IL36 have in sleep regulation. For over 100 years it has been known that prolonged wakefulness (W) enhances brain production and release of sleep regulatory substances (SRSs). Nevertheless, the property of W that is responsible for enhanced SRS activity remains to be identified. In Aim 2, we investigate the hypothesis that ATP, released during neurotransmission, is a signal that provides a measure of prior W activity. Specifically, ATP is translated, via purine P2 receptors, into a longer lasting index of prior brain usage through release of cytokines such as IL1 from glia. Preliminary data indicate that ATP agonists promote sleep while ATP antagonists inhibit sleep and P2X7 receptor expression varies with sleep propensity. In Aim 2, we test our model, the ATP-cytokine-adenosine hypothesis, by using mice lacking key model component genes such as the P2X7 receptor. These mice, for example have attenuated sleep responses to sleep loss and the inflammatory stimulus, lipopolysaccharide. In Aim 3, we focus on activity-dependency of SRS gene expression and EEG delta power. We make use of the light-sensitive channelrhodopsin 2 (ChR2) gene by expressing it in cell cultures, then activating the cells with various patterns of light and determine SRS and model gene expressions. We also use ChR2- transgenic mice for in vivo controlled activation of cortical neurons and subsequent manifestations on the EEG. Anticipated results will provide mechanistic answers to questions of how inflammation alters sleep and how cellular activity is translated into SRS mechanisms. Results will have practical application to IL1- associated brain pathologies including inflammation-associated sleep disturbances occurring in sleep apnea and metabolic syndrome. PUBLIC HEALTH RELEVANCE: The roles that interleukin-1 (IL1) and IL1 family (IL1F) members have in inflammation-associated sleep disturbances and physiological sleep regulation are investigated. The involvement of a brain-specific IL1 receptor accessory protein and IL36 in sleep and brain-inflammation processes is determined. Further, ATP released during neurotransmission is posited to be part of the mechanism by which the brain tracks prior activity during wakefulness. We test the hypothesis that extracellular ATP, via purine type 2 receptors- mediates release of IL1 and other cytokines from glia and plays a key role sleep regulation and sleep responses to inflammatory signals. Finally, we test whether different patterns of cell activation change gene expression of IL1 and other components of the ATP-cytokine-adenosine hypothesis and whether different cell activation patterns differentially alter EEG delta power state-specifically. Results will have practical application to IL1-associated brain pathologies including inflammation-associated sleep disturbances occurring in sleep apnea and metabolic syndrome.

[unreadable] DESCRIPTION (provided by applicant): Growth hormone (GH) releasing hormone (GHRH) is a hypothalamic neurocrine that stimulates pituitary GH release and that has become one of the best documented sleep-regulatory substances; much of the sleep- related evidence was generated under the auspices of this grant. The fundamental hypothesis of this proposal is that promotion of non-rapid eye movement sleep (NREMS) and GH secretion are independent, albeit synchronized, functions of the hypothalamic GHRH-containing neurons. Evidence in support of this hypothesis includes: 1) deep NREMS is associated with GH secretion in several species; 2) GHRH promotes NREMS in rats, mice, rabbits, and humans; 3) inhibition of endogenous GHRH inhibits spontaneous NREMS and GHRH-enhanced NREMS; 4) inhibition of GH does not block GHRH-enhanced sleep; 5) GHRH enhances sleep when injected into the medial preoptic region; 6) hypothalamic GHRH and GHRH levels vary with sleep propensity; and 7) negative feedback mechanisms of the somatotropic system inhibit GH secretion and NREMS simultaneously. The broad objective of the current proposal is to determine the role of GHRH in altered sleep associated with perturbations of the somatotropic axis and identification of hypothalamic structures and neurons indicating the sleep-promoting activity of GHRH. We also extend our work to the cerebral cortex to determine if GHRH and its receptors are involved in the regulation of neuronal assembly functional state. In Aim 1 we will study the hypothalamic GHRHergic network involved in sleep regulation. We will determine the importance of the connections between the mediobasal hypothalamus and the anterior hypothalamic/preoptic region for sleep by means of small semicircular cuts placed in front of the arcuate nucleus. We will also us immunohistochemistry to map the distribution of GHRH-receptor expressing neurons and determine which transmitter localizes with the GHRH-receptor. In Aim 2, we focus on how ghrelin modifies sleep. We test the hypothesis that injection of ghrelin into the lateral hypothalamus will promote feeding, but inhibit sleep, whereas injection into areas around the periventricular nucleus may promote sleep. We also determine sleep in mice lacking ghrelin or its receptor. Aim 3 investigates the cortical involvement of GHRH in EEG synchronization. Preliminary data demonstrate regulated expression and functionality of both GHRH and the GHRH receptor in the cortex. We will determine whether the cortical GHRH-GHRH-R system plays a role in EEG synchronization and the cell types expressing these substances. Preliminary data are presented for each specific aim to demonstrate feasibility. Anticipated results are expected to support the hypothesis that GHRH is a key regulatory component of NREMS and provide cellular mechanistic explanations for the involvement of GHRH in sleep regulation. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Influenza is a respiratory viral infection often accompanied by severe symptoms such as high fevers and a profound need to sleep. Collectively, these symptoms are part of the acute phase response (APR). The understanding of the molecular mediators of the viral APR is in its infancy. Some of the progress made in deciphering centrally regulated APRs resulted from research supported by this grant. Our accomplishments include; showing that sleep changes over the course of an influenza virus infection; demonstrating that a toxic viral by-product, double-stranded (ds) RNA, is found in lungs of infected mice and that it induces the APR; characterizing the influenza APR in mice, and investigating APR molecular mechanisms through development of appropriate infection models and using genetically-deficient mice. Most importantly, we recently demonstrated that strains of human influenza .previously considered incapable of accessing the mouse brain, in fact do so within a few hours following intranasal infection. In brain, viral RNA and viral antigen are accompanied by induction of cytokines with the potential for mediating the APR. We expand upon these data in Aims 1 and 2. Aim 1 addresses the general hypothesis that virus, viral proteins and/or viral RNA pass from the nasal cavities to the olfactory bulb (OB) via the olfactory nerve and induce cytokine production. We will use immunohistochemistry, polymerase chain reaction techniques, and electron microscopy to determine the time course of viral invasion of the OB and its relationship to cytokine expression. Double-labeling techniques will be used to determine co-localization of virus protein with cytokines and the cell types expressing cytokines and containing virus proteins. Aim 2 tests the hypothesis that OB-expressed cytokines activate hypothalamic (HT) cytokines and subsequent HT-regulated APRs. The timing of HT cytokine expression relative to OB expression will be determined. The effects of TNFa injection into the OB and the effects of cutting OB-brain connections on HT-cytokine and sleep and fever responses will be determined. Aim 3 tests the general hypothesis that invasion of the CNS by influenza virus is affected by immune status of the host. We determine whether homologous immunity to influenza virus offers partial protection against viral invasion of the brain. The measures made in Aim 1 will be used in Aims 2 and 3 along with sleep, body temperature, locomotor activity and body weight determinations. Aim 4 is derived from our past work showing that viral dsRNA induces flu-like symptoms. Thus mice deficient in toll- like receptor 3 (TLR3), a receptor that binds dsRNA, will be challenged with virus. APRs including sleep and brain cytokine expression will be determined. There is an association between upper respiratory viral infections and sudden infant death syndrome (SIDS); SIDS occurs during sleep and is associated with sleep abnormalities. The proposed work will provide insights into how viruses induce disease symptoms as well as such complications as SIDS and viral encephalitis/encephalopathy. [unreadable] [unreadable] [unreadable]

Project Description: Sleep and sleep homeostasis are regulated by a network of sleep regulatory substances. A role for tumor necrosis factor ? (TNF) in sleep regulation is well characterized. However, TNF-TNF receptor interactions are complex; the mechanism involved in TNF-regulated sleep is unknown. Several electrophysiological parameters characterize sleep in animals; they include action potential burstiness (BI ? burstiness index), synchronization (SYN) of slow waves (SW) (0.5-3.5 Hz), SW power (µV2) and evoked response potential (ERP) amplitudes. We use homologous measures to characterize sleep-like states in neuronal/glial co- cultures. Over the course of neuronal/glial culture development these parameters emerge as the networks mature. If cultured networks are stimulated electrically, these parameters decrease suggesting a more wake- like state. In contrast, if cultures are treated with TNF, these parameters increase indicating a deeper sleep- like state. Cultures also exhibit sleep homeostasis; after electrical stimulation a rebound increase in BI, SYN and SW power occurs. The effects of TNF on ERPs are stunning; TNF greatly enhances ERP amplitude and synchronization. We will use our in vitro sleep model to determine how TNF interacts with its receptors to affect culture state. In Aim 1, four experiments are proposed to determine which of the three known methods of TNF-TNF receptor interactions is responsible for the TNF-sleep actions. The TNF-TNF receptor interactions are: a) soluble TNF (17kD) acting as ligand for one of its receptors; b) trans-membrane TNF (26kD) directly binding to a TNF receptor on an adjacent cell to initiate responses (direct cell-to-cell signaling); and c) a soluble TNF receptor binding to trans-membrane TNF to initiate responses. In Aim 2 we determine which TNF receptor is involved. The in vitro system, due to its simplicity and our ability to control the intensity of the emergent state properties, offers a novel experimental platform to determine the mechanisms of action of TNF- sleep regulation and of emergent network properties. We use the R21 mechanism because the approach used, in vitro glial/neuronal cultures, is exploratory and in development and the experiments involve some risk yet have revolutionary potential (e.g. local sleep-like states being a consequence of direct cell-to-cell communication). As such they could transform basic sleep research and approaches to sleep clinical issues by providing a new, bottom-up approach to network emergent properties that have a role in practical sleep medicine problems, epilepsy, traumatic brain injury and other CNS disorders impacted by sleep.