Thomas S. Kilduff - US grants

DESCRIPTION (provided by applicant): The Two Process model of homeostatic sleep regulation posits that the timing of sleep results from an interaction between a circadian process (Process C) and a homeostatically regulated sleep process (Process S). Process C is linked to neural systems that generate circadian rhythms, but the neural basis of Process S is currently unknown. During the previous funding period, we identified a population of GABAergic cells in the cerebral cortex that express neuronal nitric oxide synthase (nNOS) and also express the transcription factor FOS specifically during sleep and after sleep deprivation (SD). Activation of nNOS neurons is negatively correlated with time awake, unrelated to REM sleep, and most closely linked to NREM bout duration and NREM delta energy. In the absence of nNOS, mice have altered slow wave activity (SWA) during both wakefulness and sleep, are unable to sustain long bouts of NREM sleep, and are unable to respond to a homeostatic sleep challenge despite being sleepier than wild type (WT) mice. Based on these results, we hypothesize that cortical nNOS neurons are involved in the homeostatic regulation of sleep and are a neuroanatomical substrate of Process S. To test this hypothesis, we will first ask whether these sleep-active cortical nNOS neurons are projection neurons and use state-of-the-art viral tracers to identify afferents to these neurons. To determine the role of different afferent inputs in regulating corticl nNOS neurons, we will conduct in vitro patch clamp electrophysiology recordings to determine which neurotransmitters and neuromodulators previously implicated in the control of sleep and wakefulness excite or inhibit sleep-active cortical nNOS cells. We will address whether cortical nNOS neurons are causally involved in sleep homeostasis using optogenetic and pharmacogenetic approaches to activate these cells and characterize the effects on sleep/wake architecture and EEG spectra. Lastly, we will assess the role of nitric oxide derived from cortical nNOS neurons in sleep homeostasis by optogenetic and pharmacogenetic stimulation in the presence and absence of nitric oxide inhibitors. These experiments will elucidate the neurobiology of cortical nNOS neurons and allow us to determine whether the activity of cortical sleep-active nNOS cells is indeed related to Process S. The results will not only enhance our understanding of sleep/wake regulation, but may also have implications for understanding the role of sleep in neurological and psychiatric diseases involving the cortex such as epilepsy, anxiety, and schizophrenia.

Cigarette smoking represents the most commonly used drug of abuse in American society. Despite extensive public education campaigns regarding the deleterious consequences of smoking, the proportion of women who smoke in North America has been estimated to be between 18-28%. In addition to active smoking, one-third to one-half of pregnant women are exposed to passive smoke. Identification of nicotine and its metabolite cotinine in hair samples of neonates indicates that human fetuses are exposed to significant levels of nicotine both through active smoking by their mothers and through maternal exposure to passive smoke. Among other abnormalities, an increased incidence of Attention Deficit Disorders and sudden infant death syndrome (SIDS) occurs in the offspring of maternal smokers. The purpose of this proposal is to evaluate the post-natal consequences of prenatal nicotine exposure in a rodent model, with particular emphasis on the development of sleep-wake and circadian systems. An underlying hypotheses of our work is that impaired or delayed maturation of the circadian and sleep/wake systems plays a role in the occurrence of SIDS. We have found that prenatal nicotine exposure in rat pups induces expression of the immediate early gene (IEG) c-fos in the fetal, but not the maternal, suprachiasmatic nucleus (SCN). Since induction of c-fos mRNA in the adult SCN by light exposure has been associated with phase-shifts of the circadian system, our results suggest that nicotine exposure may induce a desynchrony between the circadian timekeeping system of the mother and the fetus. To test this hypothesis and to further elucidate the signal transduction pathway in the SCN, we will determine the molecular basis for the differential response of the fetal vs. maternal SCN to prenatal nicotine exposure. We hypothesize that this differential response may be due to differential composition of the nicotinic cholinergic receptor in the fetal and adult SCN. To further understand the signal transduction pathway underlying nicotine-induced c- fos expression in the fetal SCN, we will determine whether prenatal nicotine exposure causes phosphorylation of the CREB and/or SRF proteins in the fetal SCN and identify other IEGs induced in the fetal SCN by prenatal nicotine exposure.

DESCRIPTION (provided by applicant): Since the initial funding of this grant proposal, the hypocretin/orexin system has been recognized to be a hypothalamic neuropeptide system with widespread excitatory activity. Defects in this system, either presynaptically or postsynaptically, result in the sleep disorder narcolepsy in both animal models and in humans. Based on these and other observations, hypocretin (Hcrt) is now viewed as a central neurotransmitter system in the maintenance of wakefulness. During the next funding period, we propose to examine specific inputs to and outputs from the Hcrt neurons at both the cellular and behavioral levels. We hypothesize that there is an interaction between the Hcrt and the corticotropin releasing factor (CRF) systems. To further understand this relationship, we will (1) determine the origin of CRF-containing afferent innervation of the Hcrt neurons using anterograde tracing from potential CRF afferent sources combined with Hcrt immunohistochemistry and retrograde tracing from the perifornical hypothalamus with CRF-immunohistochemistry; (2) identify the mechanism of action of the excitatory effect of CRF on Hcrt cells; and (3) determine whether an intact Hcrt system is necessary for the anxiogenic effects of CRF. We will identify whether CRF is anxiogenic in hypocretin-ataxin cell knockout mice and evaluate the effect of hypocretin peptides in four anxiety models in wildtype mice. These studies will determine whether the Hcrt system is "downstream" from CRF. We will also determine the response of Hcrt neurons to stimulation by other neurotransmitters that may provide modulatory input to the Hcrt system. We will examine the mechanisms underlying GABAergic inhibition of the Hcrt neurons and determine whether endogenous inhibitory tone exists in this system. We will also examine other potential sources of modulatory input to the Hcrt cells, such as that from the hypothalamic ghrelin and galanin systems. Lastly, we will determine whether subpopulations of Hcrt neurons can be differentiated based on their efferent projections to sleep-related monoaminergic and cholinergic nuclei. To address this question, we will use dual retrograde tracing from different sleep-related regions to determine the extent of co-localization of retrograde markers within Hcrt neurons and the distribution of these markers within the Hcrt neuronal population. The information obtained will elucidate the neural networks related to the Hcrt system and thereby further our understanding of the neurobiological bases of sleep, wakefulness, and the sleep disorder narcolepsy.

DESCRIPTION (provided by applicant): Sleep in the elderly is generally recognized as being of poorer quality than in younger adults. Nocturnal sleep in seniors is characterized by frequent awakenings, decreases in the quantity of deep slow wave sleep (Stages 3 and 4), and a concomitant decrease in delta frequencies in the EEG. Daytime alertness is reduced and naps are common, indicating diminution of the diurnal rhythms of sleep and wakefulness. Many of these changes in sleep architecture also occur in aged laboratory rodents. Our long-term objective is to understand the neural basis of age-related sleep dysfunction. The hypocretin/orexin (H/O) neurotransmitter system has recently been identified as being important in arousal state regulation, and degeneration of the H/O neurons has been found in human narcolepsy, a sleep disorder characterized by excessive daytime sleepiness and cataplexy. Therefore, this system is an attractive target to study with respect to sleep and aging. The overall hypotheses of this proposal are that (1) the H/0 system is important in the maintenance of wakefulness; (2) a dysfunction of the H/O system occurs in the aged; and (3) this dysfunction is related to the sleep/wake disturbances characteristic of the elderly. Based on the literature, we conclude that aged rodents, like elderly humans, are a heterogenous population and differ with respect to their rate of physiological aging. Therefore, we will identify a subpopulation of aged rats having disrupted body temperature rhythms and sleep architecture and use these animals to test the following specific hypotheses: (1) an age-related decline occurs in the number of H/O cells in aged F344 rats that is correlated with disrupted sleep architecture; (2) an age-related decline occurs in the levels of H/O mRNA and/or peptides; (3) waking-related activation of H/O cells declines with age; (4) the release of H/O peptides decreases with age; (5) an age-related decline in the mRNA for the H/O receptor 1 and receptor 2 occurs in brain regions associated with wakefulness; and (6) an age-related decrease in binding to H/O receptor 1 and receptor 2 and/or G protein activation occurs in arousal-related brain regions. To address these questions, we will use a combination of in vivo physiological, neuroanatomical, molecular, and receptor pharmacological methods.

[unreadable] DESCRIPTION (provided by applicant): Narcolepsy, a Rapid Eye Movement (REM) sleep-related disorder, is characterized by excessive daytime sleepiness (EDS), cataplexy (a sudden loss of muscle tone triggered by emotional stimulation), and a cluster of other symptoms. Narcolepsy afflicts approximately 1 in 1000 Americans, and current treatments are symptomatic: cataplexy has classically been treated with anticholinergics, which have such undesirable side- effects that patients elect to live with cataplexy; and the debilitating sleepiness has been treated with amphetamine and other stimulant medications, which have abuse potential. More recently, modafinil has been used as a wakefulness-promoting therapeutic, and gamma-hydroxybutyrate, a controlled substance, has been approved to treat both cataplexy and EDS symptoms. Recently narcolepsy been shown to be a neurodegenerative disorder in which the hypocretin (orexin)-containing neurons of the hypothalamus specifically degenerate. The hypocretin peptides (Hcrt1 and Hcrt2) differentially bind two G protein-coupled receptors known as Hcrt receptor 1 and 2 (HcrtR1 and HcrtR2). The onset of narcolepsy symptoms is associated with a decline in Hcrt1 levels in cerebrospinal fluid, thought to be indicative of the ongoing loss of Hcrt-containing neurons in the PLH. Thus, although Hcrt replacement is a potential therapeutic regimen, no small molecule brain-penetrable HcrtR agonists currently exist. By screening a compound library synthesized at SRI International for potential HcrtR agonists, we have identified two hits, SRI-2757 and SRI-5653, that can serve as the basis for the iterative process of optimization using rational drug design and molecular modeling followed by drug synthesis and biological testing. Aim 1 will establish a structure-activity relationship for SRI-2757 and SRI-5653 and develop ligand-derived pharmacophore models and pharmacophore-based in silico screening of small-molecule libraries to identify other HcrtR agonists with novel scaffolds. Aim 2 will determine compound activity in vitro using FLIPR-based HcrtR functional assays and will evaluate the intestinal permeability and metabolic stability of selected analogs in models in vitro. Aim 3 will evaluate the efficacy of Hcrt agonists in vivo using behavioral assays in a mouse genetic model of narcolepsy. Since the Hcrt system is now recognized to be centrally involved in the maintenance of wakefulness, novel Hcrt agonists identified in this proposal may have broader indications than treating narcolepsy; for example, they may be used to prolong wakefulness in situations where sustained performance is necessary. Since Hcrt1 appears to have antinociceptive properties, Hcrt agonists may also be useful when analgesia is necessary, such as in acute injury, arthritis, and postoperative or neuropathic pain. In addition to these potential therapeutic roles, subtype-specific Hcrt agonists would be useful to help elucidate the biological functions of HcrtR1 and HcrtR2, since all reports to date have focused on the endogenous ligands, Hcrt1 and Hcrt2, and a few peptide analogs. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Gammahydroxybutyrate (GHB), a product of intermediary metabolism, has profound effects on the activity of the central nervous system (CNS), particularly on consciousness. Because of its soporific effects, GHB has become both a drug of abuse and, paradoxically, a clinically useful therapeutic for treatment of the sleep disorder narcolepsy. Narcolepsy, a Rapid Eye Movement (REM) sleep-related disorder that afflicts approximately 1 in 1000 Americans, is characterized by excessive daytime sleepiness (EDS), cataplexy (a sudden loss of muscle tone triggered by emotional stimulation), and a cluster of other symptoms. Xyrem, the sodium salt of GHB, has been approved by the U.S. Food and Drug Administration for the treatment of both the cataplexy and EDS symptoms of narcolepsy. GHB facilitates slow wave activity (SWA) in the EEG and slow wave sleep (SWS), thereby consolidating nocturnal sleep and resulting in increased alertness on the subsequent day. Despite its clinical utility, the mechanism of action of GHB remains controversial with evidence for action both through GABA-B receptors and through specific GHB binding sites in the CNS. The specific goals of this project are to identify the neural substrates of GHB-induced SWA and understand the mechanism(s) underlying the therapeutic effects of GHB on narcolepsy/cataplexy. To achieve these goals, we will exploit a mouse model of narcolepsy/cataplexy in which the hypocretin (Hcrt) neurons degenerate postnatally as they do in human narcoleptics. We will follow up on our preliminary results using these hcrt/ataxin-3 mice which indicate that GHB can reduce cataplexy-like symptoms as it does in humans and test the hypothesis that these therapeutic effects are mediated through the GABA-B receptor. We will conduct functional neuroanatomical studies to test the hypothesis that GHB differentially affects behavioral state regulatory regions in hcrt/ataxin-3 mice. Based on our preliminary results in which GHB induces Fos expression in the locus coeruleus (LC), we will use the neurotoxin DSP-4 to lesion noradrenergic cells to test the hypothesis that an intact LC is necessary for the therapeutic effect of GHB. We will also conduct cellular electrophysiological studies to determine whether the intrinsic properties of the neurons in the LC or the ventrolateral preoptic area (VLPO) are affected by acute or chronic exposure to GHB. Lastly, we will evaluate whether the therapeutic efficacy of GHB is associated with brain gene expression changes. The results of the studies proposed above will enhance our understanding of the neurobiology that underlies the therapeutic activity of GHB and may also provide insights into the cellular and molecular mechanisms that underlie cataplexy and EEG SWA. PUBLIC HEALTH RELEVANCE In patients with narcolepsy, a sleep disorder characterized by excessive daytime sleepiness and related symptoms, degeneration of hypocretin (Hcrt) neurons in the brain has been observed. Gammahydroxybutyrate (GHB) is a clinically useful therapeutic for treatment of the sleep disorder narcolepsy but the mechanism of action is unknown. We will exploit a mouse model of narcolepsy in which the Hcrt neurons degenerate postnatally as they do in human narcoleptics to understand how GHB is beneficial in human narcolepsy.

DESCRIPTION (provided by applicant): The hypocretin/orexin (Hcrt) system is a neuropeptide system involved in behavioral arousal, metabolism, addiction, stress-induced analgesia (SIA) and neuroendocrine function. Hcrt deficiency results in narcolepsy, a CNS disorder characterized by impaired alertness, excessive sleepiness, restricted social activities, and increased body mass index. Given the functional significance of this system, identification of the inputs that control the Hcrt system and functional Hcrt efferent pathways is of clinical relevance. We will use a combination of optogenetics, transgenic mice in which light-sensitive proteins are expressed specifically in Hcrt neurons, and conditional transgenic mouse strains to further understand the Hcrt system. Using a novel transgenic mouse that expresses the light-sensitive proton pump Archaerhodopsin-3 specifically in Hcrt neurons, we will test the hypotheses that: (1) optogenetic inactivation of the Hcrt neurons will induce changes in cortical activity, muscle tone and behavior indicative of sleep, and (2), that homeostatic sleep pressure modulates the efficacy of optogenetic inactivation of Hcrt neurons to induce sleep. Using a transgenic mouse in which the 5HT1A receptor is conditionally overexpressed in Hcrt neurons, we will test the hypothesis that the negative feedback loop between Hcrt neurons and the serotonergic dorsal raphe nucleus has functional significance for the regulation of sleep and wakefulness. Lastly, we will exploit a newly created transgenic mouse in which conditional expression of diphtheria toxin A (DTA) occurs specifically in Hcrt neurons to establish and validate a novel mouse model of human narcolepsy that more closely resembles the human disorder. The research proposed here represents an integrated systems neurobiology approach to elucidate control of a neurotransmitter system implicated in a number of behaviors and whose pathology is clearly linked to CNS dysfunction and may lead to novel pharmacotherapeutic approaches for the treatment of disorders of Hcrt insufficiency such as narcolepsy.

DESCRIPTION (provided by applicant): Narcolepsy afflicts 0.025-0.05% of the population and is characterized by excessive daytime sleepiness, cataplexy (a sudden loss of muscle tone triggered by emotional stimulation), and increased propensity for rapid-eye-movement (REM) sleep. Although narcolepsy results from degeneration of neurons that produce hypocretin (Hcrt; also known as orexin), no small-molecule brain-penetrable Hcrt receptor agonists currently exist for hypocretin replacement therapy. Current treatments include controlled substances with abuse potential or drugs with other undesirable side effects. In papers just published with scientists from F. Hoffmann- LaRoche, we describe novel, brain-penetrable agonists for Trace Amine-associated Receptor 1 (TAAR1). These compounds cause a dose-dependent increase in wakefulness, reduce REM sleep, and have pro- cognitive, antidepressant- and antipsychotic-like properties, suggesting TAAR1 as a novel target for the treatment of pathological sleepiness in addition to neuropsychiatric disorders. In this proposal, we will determine the therapeutic efficacy of TAAR1 agonism as a treatment for narcolepsy in proof-of-concept studies using two murine narcolepsy models. First, we will determine whether full and partial TAAR1 agonists promote wakefulness, reduce cataplexy and normalize arousal states in the orexin/ataxin-3 mouse, in which Hcrt neurons have been genetically engineered to degenerate postnatally. Next, we will test these compounds in a novel, inducible model of murine narcolepsy-the orexin/tTA; Tet-O DTA mouse-in which ablation of Hcrt neurons is controlled through the tetracycline transactivator (Tet-off) system to recapitulate the post-pubertal onset of human narcolepsy. In each model, we will compare the efficacy of TAAR1 agonists against the known wake-promoting therapeutic modafinil and anti-cataplectic agent desipramine. We will also compare the dose- response effects of TAAR1 agonism in orexin/ataxin-3 mice with wild-type littermates, and in orexin/tTA; Tet-O DTA mice before and after narcolepsy induction, to test the hypothesis that TAAR1 agonism normalizes arousal states. Discovery of TAAR1 agonists for the treatment of narcolepsy will also advance the development of wake-promoting therapeutics based on modulation of trace amine signaling.

DESCRIPTION (provided by applicant): The trace amines (TAs), endogenous amino acid metabolites previously considered false neurotransmitters, have recently been shown to act as endogenous ligands for trace amine-associated receptor 1 (TAAR1). TAAR1 is a G protein-coupled receptor that modulates dopaminergic, serotonergic and, possibly, glutamatergic activity. In papers recently published in Molecular Psychiatry and Biological Psychiatry with scientists from F. Hoffmann-LaRoche, we describe novel, brain-penetrable TAAR1 agonists with pro-cognitive, antidepressant- and antipsychotic-like properties, suggesting TAAR1 as a novel target for the treatment of neuropathological disorders. We also show that TAAR1 partial agonism causes a dose- dependent increase in wakefulness and decreases in NREM and REM sleep, indicating that this receptor activates an endogenous wake-promoting system. In the present proposal, we will determine whether endogenous TAAR1 tone contributes to the normal distribution of sleep and wakefulness, the homeostatic response to sleep deprivation, and the response to endogenous and exogenous compounds known to promote wakefulness. First, we will determine whether TAAR1 signaling is involved in the maintenance of daily sleep- wake patterns and the homeostatic regulation of sleep in TAAR1 null mutant mice. In the context of these studies, we will determine whether the wake-promoting effects of TAAR1 agonists studied to date are absent in these mice. Next, we will determine the consequences of overexpression of TAAR1 on the normal distribution of sleep and wakefulness and the homeostatic response to sleep deprivation. Lastly, we will determine whether TAAR1 signaling is necessary for the wake-promoting effects of the stimulant caffeine and the wake- promoting therapeutic modafinil (Provigil(R)) and the endogenous wake-promoting neuropeptide, hypocretin-1 (orexin-A). The results obtained will advance our understanding of the interaction of TAAR1 with wakefulness- promoting systems in the brain, and will likely impact the development of pharmacotherapies directed toward this novel target for the treatment of sleep/wake and other neural disorders.

DESCRIPTION (provided by applicant): EEG power in the theta frequency range (6-9 Hz), particularly during waking and REM sleep, is believed to primarily be due to synchronous firing of hippocampal pyramidal cells. To date, studies supporting this hypothesis have relied on extracellular unit recordings where a relatively small number of cells are monitored and the activity of individual cells can only be followed for short periods of time. Here, we will provide n unprecedented network level view of neural activity in the hippocampus across arousal states by monitoring the activity of hundreds of individually identifiable cells over weeks to months. We will combine telemetric recording of EEG and EMG activity with an exciting new technology that, when used in conjunction with genetically-encoded fluorescent calcium indicators (e.g., GCaMP5 and 6), enables imaging the activity of hundreds of neurons simultaneously from a local brain region in unanesthetized, freely-moving animals. The Inscopix nVista HD imaging system is comprised of a miniature (<2 g) fluorescence microscope that can be borne on the skull of a mouse, software that enables high-speed imaging (20-100 Hz) over a field of view up to ~0.5 mm2, and microendoscopes that allow imaging with micron-scale resolution in subcortical brain structures. Because of the rich history of behavioral studies conducted in the hippocampus and its laminar organization, this region is the ideal neural structure in which to utilize this technology to obtain novel insights into neural activity across arousal states. First,we will determine the activity of localized networks within CA1 across arousal states and during conditions when homeostatic sleep pressure can be expected to differ, such as early in the lights on period vs. early in the dark period and in response to sleep deprivation and subsequent recovery sleep. Concurrent measurement of the activity of hundreds of cells in conjunction with the EEG will enable us to not only follow the activity of individual cells across sleep and wakefulness, but also to evaluate synchrony within the local network with respect to EEG frequencies and specific EEG events. Having defined the parameters of the hippocampal network in wildtype mice, we will then determine the activity of local networks within CA1 in a mouse model of Huntington's disease (HD). We have recently characterized changes in sleep and wakefulness and in the EEG of the R6/2 mouse model of HD and found both tremendously increased power in the theta range and slowing of the theta peak frequency (TPF) as disease progresses. Using theta power and TPF as biomarkers, we will exploit the power of the Inscopix technology to enable long-term recordings of identified neurons to determine how the hippocampal network is reorganized as disease progresses in R6/2 mice. The ability to observe calcium dynamics of hundreds of cells while simultaneously recording EEG in freely-behaving mice may lead to new insights into the role of the hippocampus in behavioral states and the network changes that occur in the hippocampus in HD.

DESCRIPTION (provided by applicant): The goal of this proposal is to determine whether ligands for Trace Amine-associated Receptor 1 (TAAR1) are wake-promoting and pro-cognitive in non-human primates as we have found in laboratory rodents. Recent identification of selective agonists for TAAR1 that exhibit pro-cognitive, antidepressant- and antipsychotic-like properties in both rodent and non-human primates suggest TAAR1 is a novel target for the treatment of neuropsychiatric disorders. In collaboration with scientists from F. Hoffmann-La Roche, we have described brain-penetrable compounds with high potency and selectivity at mouse, rat, monkey and human TAAR1. Moreover, we showed that TAAR1 agonism causes a dose-dependent increase in wakefulness in rats and have replicated this effect in mice. Given the widespread occurrence of sleep disorders, we will further test the hypothesis that TAAR1 agonism is a novel therapeutic pathway to promote wake and enhance cognition. First, we will determine whether TAAR1 agonism promotes wakefulness in Cynomolgus macaques under conditions of both high and low sleep pressure. Although TAAR1 agonists increase vigilance in rodents, these animals are nocturnal and have polyphasic sleep/wake cycles in comparison to the consolidated periods of sleep and wakefulness characteristic of both humans and non-human primates. Implementation of EEG recording in these studies will enable us to determine the effects of TAAR1 agonism on NREM and REM sleep and to conduct quantitative EEG (qEEG) analysis to assess the effects of these compounds on EEG frequencies associated with cognition such as gamma oscillations. Next, we will test these compounds for their ability to improve working memory functions in macaques under baseline and sleep deprivation conditions. Working memory function is dependent on prior sleep history and subject to decline in aging, stress and in diseases such as schizophrenia and Alzheimer's disease. We have shown previously that TAAR1 agonists improve executive functions (e.g., response inhibition, planning) in non-human primates and in a rodent PCP-induced deficit model, however, this represents only one cognitive domain that can be affected in sleep disorders. Simultaneous assessment of behavior and qEEG in macaques performing the working memory task will enable investigation of cognitive function at both behavioral and electrophysiological levels. The results of these studies will aid in establishing TAAR1 agonism as a novel mechanism to enhance wakefulness and cognition in humans.

DESCRIPTION (provided by applicant): The hypocretin/orexin (Hcrt) system is a neuropeptide system involved in behavioral arousal, metabolism, addiction, stress-induced analgesia (SIA) and neuroendocrine function. Hcrt deficiency results in narcolepsy, a CNS disorder characterized by impaired alertness, excessive sleepiness, restricted social activities, and increased body mass index. Given the functional significance of this system, identification of the inputs that control the Hcrt system and functional Hcrt efferent pathways is of clinical relevance. We will use a combination of optogenetics, transgenic mice in which light-sensitive proteins are expressed specifically in Hcrt neurons, and conditional transgenic mouse strains to further understand the Hcrt system. Using a novel transgenic mouse that expresses the light-sensitive proton pump Archaerhodopsin-3 specifically in Hcrt neurons, we will test the hypotheses that: (1) optogenetic inactivation of the Hcrt neurons will induce changes in cortical activity, muscle tone and behavior indicative of sleep, and (2), that homeostatic sleep pressure modulates the efficacy of optogenetic inactivation of Hcrt neurons to induce sleep. Using a transgenic mouse in which the 5HT1A receptor is conditionally overexpressed in Hcrt neurons, we will test the hypothesis that the negative feedback loop between Hcrt neurons and the serotonergic dorsal raphe nucleus has functional significance for the regulation of sleep and wakefulness. Lastly, we will exploit a newly created transgenic mouse in which conditional expression of diphtheria toxin A (DTA) occurs specifically in Hcrt neurons to establish and validate a novel mouse model of human narcolepsy that more closely resembles the human disorder. The research proposed here represents an integrated systems neurobiology approach to elucidate control of a neurotransmitter system implicated in a number of behaviors and whose pathology is clearly linked to CNS dysfunction and may lead to novel pharmacotherapeutic approaches for the treatment of disorders of Hcrt insufficiency such as narcolepsy.

Melanin-concentrating hormone (MCH) and hypocretin/orexin (HCRT)-expressing neurons are intermingled populations in the tuberal hypothalamus that project widely throughout the brain to many of the same terminal fields. Whereas the HCRT system has been implicated in the control of wakefulness because the sleep disorder narcolepsy results when these cells degenerate, this system is also involved in energy metabolism. Conversely, the MCH system has primarily been associated with food intake and energy metabolism, but recent studies have established that MCH neurons also participate in the regulation of sleep and wakefulness. The hypothesis underlying this proposal is that the HCRT system is wake-stabilizing and REM-inhibiting whereas the MCH system is sleep-facilitating and REM-stabilizing. We will test this hypothesis by determining the phenotype of mice in which either the HCRT or MCH neurons have been partially ablated by removal of doxycycline in the diet of two conditional mouse models. We will then evaluate whether partial ablation of the HCRT neurons results in a phenotype of narcolepsy without cataplexy and whether cataplexy is exacerbated by simultaneously eliminating both neuronal populations. We will also assess whether direct connectivity exists between these cell groups using optogenetically-assisted neuroanatomical tracing and whole-cell patch- clamp electrophysiology in the presence and absence of selective HCRT and MCH receptor antagonists. To assess what occurs in the brain when the HCRT neurons degenerate as in human narcolepsy, we will use the conditional HCRT neuron ablation model to determine how the excitability of the MCH population is affected by chronic loss of HCRT input. We will also use conditional MCH neuron ablation to assess the converse effect of MCH loss on HCRT neuron excitability. Based on recordings from a limited number of cells in head-fixed animals, the HCRT and MCH neurons have been reported to have reciprocal activity across the sleep-wake cycle with HCRT neurons having their highest firing rates during active wakefulness and MCH neurons being primarily active during REM sleep. To determine the accuracy of this conclusion, we will use genetically- encoded Ca2+ indicators and microendoscopic imaging to measure the activity of hundreds of HCRT and MCH neurons across the sleep/wake cycle in unrestrained, freely-moving animals. To evaluate whether the HCRT and MCH neurons are functionally interconnected, we will pharmacogenetically activate one population while imaging Ca2+ fluorescence in the other population in the presence of selective HCRT and MCH receptor antagonists. Lastly, since these two populations project to many of the same brain regions, we will assess their relative input to brain areas known to be involved in arousal state control, specifically, the locus coeruleus, tuberomammillary nucleus, medial septum, and the amygdala. Together, these experiments should provide a more complete picture of the anatomical and functional connectivity of these two populations and the consequences of selective loss of one population or the other.

PROJECT SUMMARY Mammalian hibernation is an integrative organismal adaptation involving a coordinated orchestration of systems physiology that results in a seasonally expressed change in behavioral state. As body temperature declines from euthermic values of 36-38°C to 2°C or lower in the ground squirrel, heart rate drops from 200- 300 beats/min to 7-10 beats/min, respiration rates fall from 100-150 breaths/min to 1-2 breaths/min, and metabolism declines to 1/30 to 1/100 of euthermic values. The mechanisms that underlie the entrance into, and arousal from, hibernation are poorly understood. However, the molecular, neurobiological, and endocrine adaptations underlying these dramatic arousal state changes are likely to have significance for human health in understanding metabolic homeostasis and aging, as well as for strategies to enhance organ preservation. Transcriptomic and proteomic studies have begun to describe the molecular landscape that underlies these changes in arousal state. To date, published studies have primarily focused on one organ system at a time, with little effort at integration across systems. In this proposal, we will exploit a bank of 12 dissected brain regions and 10 peripheral tissues collected from the golden-mantled ground squirrel (Callospermophilus lateralis) at 12 time points across the 5 major phases of the hibernation cycle to create a public database of transcriptomic information from the same individuals. In Aim 1, genome sequencing and de novo assembly of the C. lateralis genome will be conducted by a contractor who has previously assembled the genome of the 13- lined ground squirrel; the resultant genome will be annotated in collaboration with colleagues at the National Library of Medicine. These efforts will provide the necessary scaffold to enable Aim 2, whose goal is to conduct RNAseq analyses of multiple brain regions sampled across the hibernation cycle and to establish a public database of transcriptomic information. Our studies will initially focus on basal forebrain, hypothalamus, pons, and cerebral cortex because of their distinct roles in arousal state regulation. This RNAseq data will enrich the genome annotation conducted in Aim 1. The RNAseq data will be visualized and made available for mining through a publicly available database similar to CircaDB (http://circadb.hogeneschlab.org), a gene expression database widely used in circadian biology, that we will create in Aim 2b. The database will be extensible, enabling addition of transcriptomic and proteomic data from other brain regions and peripheral organs by our group as well as others. Collectively, this information will fuel hypothesis testing and organism-wide interrogation of the molecular bases of the dynamic changes in physiology that occur across the hibernation cycle and may provide insights into the adaptations that enable metabolic suppression and those that prevent hibernators from experiencing stroke, apnea, muscle atrophy and memory loss.

PROJECT SUMMARY Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor involved in the regulation of dopaminergic, serotonergic and glutamatergic activity. TAAR1 agonists have anxiolytic, antidepressant-, and antipsychotic-like properties in both rodent and non-human primates; TAAR1 agonists are in clinical trials for schizophrenia and Parkinson?s disease psychosis. We have previously shown that TAAR1 agonists are wake- promoting in mice, rats and, most recently, non-human primates, characterized the sleep/wake phenotype of Taar1 knockout (KO) and overexpressing (OE) mice, and evaluated the effects of TAAR1 agonists on sleep/wake in wildtype (WT), KO and OE mice. We also showed that two different TAAR1 agonists suppressed REM sleep and reduced cataplexy in mouse models of narcolepsy, precisely the properties desirable in a narcolepsy therapeutic. Having established TAAR1 agonists as potential novel treatments for narcolepsy, we will now investigate the underlying in vivo neurobiology. In Taar1-LacZ mice, we will determine whether TAAR1 is expressed in monoaminergic, glutamatergic or other cell types and use the RNAscope technology to determine endogenous Taar1 mRNA expression in WT and KO mice and rats. Since TAAR1 negatively regulates dopaminergic (DA) neuronal activity in vitro, we will test the hypothesis that TAAR1 partial agonism promotes wakefulness by modulating DA arousal systems. To address this hypothesis, we will assess neuronal activity in the ventral tegmental area and dorsal raphe nuclei of DAT-ires-Cre mice using in vivo Ca2+ microendoscopy, and determine whether pretreatment with DA D1- and D2-receptor antagonists attenuates TAAR1-mediated wake-promotion. Since serotonergic neurons are wake-active and REM-inactive and TAAR1 negatively regulates serotonergic neuronal activity in vitro, we will also test the hypothesis that TAAR1 partial agonism promotes wakefulness by modulating serotonergic arousal systems. We will determine whether TAAR1 partial agonists modulate the activity of DRN serotonergic neurons using in vivo Ca2+ microendoscopy in Fev-Cre mice and assess whether blockade of serotonergic signaling attenuates the wake-promoting effects of TAAR1 partial agonists. We have found that TAAR1 deletion elevates high-frequency gamma EEG activity, suggesting that TAAR1 modulates cortical function. To determine whether TAAR1-mediated elevation of gamma activity is conserved across species and specific to TAAR1, we will investigate basal sleep/wake physiology and conduct quantitative EEG analyses in Taar1 KO and OE rats and Taar2-9 KO mice. Together, these Aims will begin to establish the neural circuitry and mechanisms that underlie the efficacy of TAAR1 agonists.

PROJECT SUMMARY The hypocretin/orexin (Hcrt) system is a hypothalamic neuropeptide system that is involved in behavioral arousal, metabolism, addiction, stress and neuroendocrine function. Defects in this system, either presynaptically or postsynaptically, result in narcolepsy in both humans and animals. A convergence of results led to the recognition that human narcolepsy is a neurodegenerative disorder in which the Hcrt neurons, located exclusively in the perifornical and lateral hypothalamus, specifically degenerate. Thus, narcolepsy is due to Hcrt insufficiency. The Hcrts are a pair of peptides that differentially bind the G protein-coupled receptors Hcrt receptor 1 (HcrtR1 or OX1R) and HcrtR2 or OX2R. Based in part upon the excessive daytime sleepiness (EDS) that characterizes narcolepsy, it was recognized that the Hcrt system is centrally involved in the maintenance of wakefulness, which led to the development of HcrtR antagonists for the treatment of insomnia and FDA approval of suvorexant (Belsomra?) in 2015. In contrast, current narcolepsy treatments are purely symptomatic: cataplexy is treated with antidepressants or anti-cholinergics which have undesirable side- effects, and the debilitating EDS is often treated with amphetamine or other stimulants which have abuse potential. The goal of our research is to develop HcrtR/OXR-directed narcolepsy therapeutics that are both wake-promoting and cataplexy-inhibiting. Based on the screening of UT Southwestern's ~220,000-compound library, we have developed new HcrtR/OXR agonists with novel scaffolds and carried out structure-activity relationship studies to improve their potencies. Having optimized these initial HcrtR/OXR agonist hits using an iterative process of analog synthesis, biological testing, and molecular modeling using HcrtR/OXR crystal structures, we will now determine the pharmacokinetic (PK) properties of drug-like small molecule HcrtR/OXR agonists to select the best candidates for in vivo efficacy experiments. Thus, we will conduct PK studies on each molecule for metabolic stability, intestinal permeability, plasma concentration over time, and blood-brain- barrier (BBB) permeability using a combination of in vitro and in vivo assays. We will then test the most promising compounds in series of in vivo screens, culminating in evaluation in orexin/tTA; Tet-O DTA mice, a state-of-the-art narcolepsy model in which ablation of Hcrt neurons is controlled through the tetracycline transactivator (Tet-off) system. Using this model, the effects of HcrtR/OXR agonists on arousal state will be evaluated before and after induction of Hcrt cell loss to determine whether HcrtR/OXR agonism normalizes the narcoleptic phenotype; dose-response tests will also be conducted in the DTA model. Given that the Hcrt system is centrally involved in the maintenance of wakefulness, Hcrt agonists may have a broader indication than narcolepsy, e.g., to prolong wakefulness in civilian, industrial or military situations where sustained alertness is required. In addition to these and other potential therapeutic indications, subtype-specific Hcrt agonists would be useful to help elucidate the biological functions of HcrtR1/OX1R and HcrtR2/OX2R.

PROJECT SUMMARY Melanin-concentrating hormone (MCH) and hypocretin/orexin (HCRT)-expressing neurons are intermingled populations in the tuberal hypothalamus that project widely throughout the brain to many of the same terminal fields. Whereas the HCRT system has been implicated in the control of wakefulness because the sleep disorder narcolepsy results when these cells degenerate, the HCRT system is also involved in energy metabolism. Conversely, the MCH system has primarily been associated with food intake and energy metabolism, but recent studies have established that MCH neurons also participate in the regulation of sleep and wakefulness. The overarching hypothesis of the Parent R01 grant (R01 NS098813 ?The Tuberal Hypothalamus and Arousal State Control?) is that the HCRT system is wake-stabilizing and REM-inhibiting whereas the MCH system is sleep-facilitating and REM-stabilizing. We will test this hypothesis by determining the phenotype of mice in which either the HCRT neurons, MCH neurons or both cell types have been ablated by removal of doxycycline from the diet of two conditional mouse models, orexin-tTA;TetO DTA mice and MCH-tTA;TetO DTA mice. Professor Yamanaka's lab at Nagoya University has recently created two novel mouse strains that would be quite valuable for us in executing the Aims 1b, 2 and 3 proposed in the Parent R01 grant to test the overarching hypothesis. Specifically, his lab has crossed the orexin-tTA;TetO DTA mice with MCH-tTA mice to create an orexin-tTA;MCH-tTA;TetO DTA mouse strain in which both the orexin and MCH neurons can be conditionally ablated by removal of doxycycline. This strain of double-ablated neuron mice is critical for Aim 1 of ?The Tuberal Hypothalamus and Arousal State Control? to determine whether cataplexy is exacerbated by simultaneously eliminating both neuronal populations. The second novel mouse strain that Professor Yamanaka's lab has generated is a knock-in (KI) mouse in which Flp recombinase is expressed exclusively in the orexin neurons (orexin-Flp mice), which will be extremely valuable for us to address Aims 2 and 3 of NIH R01 NS098813 ?The Tuberal Hypothalamus and Arousal State Control?. An Administrative Supplement from the US-Japan Brain Research Cooperative Program would also enable these two laboratories to establish collaboration on a new topic: investigation of the role of MCH neurons in memory consolidation during sleep. Together, these experiments should provide a more complete picture of the interaction of the HCRT and MCH neuronal populations, the consequences of simultaneous loss of these cells, and the hypothetical role of MCH neurons in memory.

PROJECT SUMMARY Epidemiological studies have consistently shown that chronic pain is a major factor contributing to insomnia. Chronic pain is typically treated with mu opioid receptor (MOP) agonists but the widespread misuse of prescription opioids has underscored the need to develop effective, non-addicting medications for pain. Agonists at the nociceptin/orphanin FQ (N/OFQ) receptor (NOPR) have shown considerable promise as modulators of the antinociceptive and rewarding effects of MOP agonists. Our preliminary studies with two different NOPR agonists demonstrate potent effects on non-Rapid Eye Movement (NREM) sleep and EEG delta power in rats and mice, suggesting that the N/OFQ-NOPR system may have a previously unrecognized role in sleep/wake regulation. Accordingly, we will begin to test the hypothesis that the N/OFQ-NOPR system is a component of the endogenous sleep/wake regulatory system. First, we will determine the basal sleep/wake characteristics and response to homeostatic sleep challenge in two strains of NOPR null mutant mice: a constitutive NOPR knockout and a conditional global NOPR knockout. If the N/OFQ-NOPR system is a component of the endogenous sleep/wake regulatory system, elimination of NOPRs would be expected to alter either the basal expression of sleep/wake or the homeostatic response to sleep deprivation. These studies will be complemented by intracerebroventricular injection of a NOPR agonist to demonstrate that the hypnotic effects are mediated centrally and are absent in NOPR knockout mice. We have also shown that N/OFQ terminals innervate hypocretin/orexin (Hcrt) neurons, that the N/OFQ peptide directly inhibits Hcrt neural activity in a dose-dependent manner, and that N/OFQ and corticotrophin releasing factor (CRF) coordinately regulate Hcrt cells in stress-induced analgesia (SIA). Given the critical role of the Hcrt system in maintaining wakefulness, we will test the hypothesis that the N/OFQ-induced increase in sleep is dependent upon an intact Hcrt system by assessing the efficacy of a NOP agonist on NREM sleep and EEG delta power in orexin/ataxin-3 mice, a strain in which the Hcrt neurons degenerate. This study will be complemented by one in which we will utilize the recently-described floxed NOPR mice to selectively eliminate NOPRs from the Hcrt neurons. Together, these experiments will provide an initial assessment of the hypothesis that the N/OFQ-NOPR system is a component of the endogenous sleep/wake regulatory system and will advance understanding of the neurobiology of the neuropeptidergic N/OFQ-NOPR system. 1