Luis de Lecea - US grants

DESCRIPTION (provided by applicant): The hypocretins, also known as orexins, are excitatory neuropeptides produced in the lateral hypothalamus, which have a key role in the regulation of arousal. Hypocretin deficiency in dogs, mice and humans causes narcolepsy, a sleep disorder characterized by excessive daytime sleepiness and cataplexy. Hypocretin-containing neurons project to key structures involved in brain reward, most notably the nucleus accumbens, central amygdala and bed nucleus of stria terminalis. Since stress may represent an overactivation of the body's normal activational system and therefore is linked to the construct of arousal, we hypothesize that hypocretins may mediate at least in part, the activation of stress-induced signals that precipitate addiction and relapse. Under this proposal we will test three hypotheses: 1) that hypocretin neurons activate a "stress-like" response that results in reinstatement of cocaine seeking behavior;2) that hypocretin signaling is important for stress and cue-induced reinstatement of cocaine seeking behavior, and 3) that hypocretin deficient mice are resistant to drug abuse. In a first specific aim, mice with a history of cocaine self-administration and extinction will be infused with hypocretin and tested for reinstatement of cocaine seeking behavior. To determine whether stress signals are involved in hypocretin-induced reinstatement, mice deficient in corticotrophin release factor (CRF) receptor 1 will be tested for reinstatement of cocaine seeking behavior after hypocretinl infusion. Hypocretin-induced reinstatement will be blocked with hypocretin receptor antagonists and (CRF) receptor antagonists. In a second specific aim, the role of endogenous hypocretin on reinstatement will be tested by using a hypocretin receptor antagonist to block stress or cue- induced reinstatement. Accumulated clinical data has shown that hypocretin-deficient narcoleptic patients rarely abuse psychostimulants that are used to treat excessive daytime sleepiness. In specific aim 3, we will further examine whether hypocretin-deficient animals are resistant to stimulant abuse by amphetamine conditioned place preference test and by amphetamine and cocaine self administration. Finally, hypocretin-deficient mice will be tested for stress- and cue- induced reinstatement of cocaine or amphetamine seeking behavior. The results from these experiments will give us important clues about the role of the hypocretinergic system in brain reward and may result in new therapeutic tools to prevent drug craving and relapse.

Project E: Isolation of Novel Transcripts &Proteins in Hypocretin-Containing Cells The etiology of narcolepsy-cataplexy is now known to involve a degeneration of hypocretin-containing neurons in the hypothalamus. The cause of the hypocretin cell loss is unknown but, considering the well known HLA-narcolepsy association, likely to be autoimmune. Preliminary studies have not identified any specific autoantibodies against hypocretin peptides themselves, suggesting the existence of other factors Simple western blots and immunochemical staining experiments have also failed to reveal evidence fo hypothalamic-directed autoimmunity. Novel experimental approaches are needed to identify additiona factors. The goal of our revised proposal is to systematically isolate and study genes and gene products preferentially expressed in hypocretin-producing neurons. We believe that the identification of these factors will further our understanding of the physiology of these cells and provide further candidate proteins for the presumed autoimmune origin of narcolepsy. To isolate and study these factors, we will (1) Use gene expression array studies in hypocretin neuron deficient versus wild-type littermate mouse brains to establish a collection of transcripts potentially expressed specifically in hypocretin neurons. Hypocretin knockout mice will be used as an additional control. (2) Use subtractive hybridization to generate a collection of low abundant transcripts enriched in mouse hypocretin neurons. (3) Use gene expression arrays and quantitative mmunohistochemistry with brain tissue obtained from narcoleptic patients with and without cataplexy and com control individuals to establish key differences among these groups. (4) Validate all possible ranscriptional candidates by quantitative real time (RT)-PCR and in situ hybridization and explore their nvolvement in the pathophysiology of narcolepsy. This will involve verification of primary expression in hypocretin cells and basic characterization of diurnal variation. Depending on the candidate genes, other ypes of functional validation such as immunological studies, generation of genetically modified animals or candidate gene analysis in human narcolepsy DMA samples may be carried out. Whether or not narcolepsy s an autoimmune disease, the identification of products specific for hypocretin-containing cells is likely to shed new light into the pathophysiology of narcolepsy. These studies are the logical next step toward understanding why hypocretin cell loss occurs in human narcolepsy.

[unreadable] DESCRIPTION (provided by applicant): Optogenetic manipulation of brain reward. Direct electrical stimulation in the lateral hypothalamus (LH) is highly rewarding. The rewarding effects of LH stimulation are potentiated by drugs of abuse that directly or indirectly increase dopaminergic activity, whereas dopaminergic antagonists or lesions of dopaminergic neurons greatly attenuate such stimulation. However, different parametric studies have ruled out that the primary source of reward is direct depolarization of dopaminergic fibers at the tip of the electrode. Therefore, studies aimed at identifying the source of stimulation within the medial forebrain bundle will yield essential information about the components of the brain reward circuitry. The hypocretins (Hcrts), also known as orexins, are two neuropeptides derived from a single precursor, produced in a few thousand neurons in the LH. The hypocretins are essential to maintain arousal, as lack of hypocretin function results in narcolepsy. We and others have recently demonstrated that hypocretins may also be involved in brain reward function. Thus, Hcrt-1 can reinstate cocaine seeking behavior, and hypocretin receptor antagonists can block stress-induced reinstatement. Hypocretin neurons are also known to innervate dopaminergic neurons in the VTA and to induce glutamatergic plasticity in those synapses. We have recently developed a novel optogenetic method to selectively stimulate Hcrt neurons in vivo with millisecond precision by using a lentivirus expressing channel rhodopsin ChR2 under the control of the Hcrt gene promoter. Under this proposal we will use this optogenetic technology to determine whether the hypocretinergic system is an important component of the self-stimulation circuitry. In particular we will test: 1) whether rats press levers for optogenetic stimulation of Hcrt neurons (10 msec pulses, 20Hz, 10 sec, 20 mW) in the hypothalamus; 2) whether rats show preference for different photostimulation frequencies of Hcrt neurons (20 Hz vs 1 -5 Hz). These two behavioral paradigms will be tested in wild-type and Hcrt-deficient rats. By using a state-of-the-art technology, these experiments will determine whether Hcrt excitation is an important component of the brain reward system, and may lead to new ways to analyze individual components of this system with unprecedented cellular specificity and temporal resolution. This proposal is directly responsive to the call of NIDA for new technology development relevant to drug addiction. PUBLIC HEALTH RELEVANCE: This proposal will use state of the art methods to identify neuronal structures involved in brain reward function. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Optogenetic control of hypocretin neurons and stress The neural underpinnings of the response to acute stress involve activation of neurons producing corticotrophin releasing factor (CRF) and interactions between multiple homeostatic circuits. However, the details of these interactions are poorly understood. Hypocretin (Hcrt, also known as Orexin)-producing neurons in the lateral hypothalamus (LH) are important for maintaining arousal stability since loss of Hcrt function has been linked to narcolepsy in mouse, dog and human subjects. Hypocretin neurons are activated by acute stress, receive innervation from CRF terminals and are depolarized by CRF. Conversely, infusion of Hcrt-1 activates the hypothalamo-pituitary-adrenal (HPA) axis, and hypocretin receptor antagonists can block the release of ACTH induced by acute stress. Here we propose to use a newly developed optogenetic method to test whether the activity of hypocretin neurons is necessary and sufficient to activate the HPA axis. In the first aim, we will determine whether Hcrt neurons are necessary to activate the HPA axis by monitoring the acute stress response in Hcrt-deficient mice. In specific aim 2, we will determine whether the activity of Hcrt neurons is sufficient to induce a stress-like response by using an optogenetic approach. We will also test when is this activation required by using mice transduced with a lentivirus expressing a photoactivatable chloride channel in hypocretin cells. This technology will allow us to decipher the neural code of the hypocretin network that is associated with the stress response. In the third aim, we will test the functional connectivity of Hcrt. We will test whether the effects of photostimulation on the HPA axis are mediated directly by CRF signaling in the paraventricular hypothalamic nucleus, or whether the effect is indirect through other brain structures. The data collected in this revised proposal will enhance our understanding of the neural basis of the stress response with unprecedented temporal resolution and may lead to novel therapeutics for stress disorders and related diseases, as well as identify potential side effects for drugs that target the Hcrt system for other disorders. PUBLIC HEALTH RELEVANCE: The data collected in this research proposal will enhance our understanding of stress response and may lead to novel therapeutics for stress disorders and related diseases in the general public.

? DESCRIPTION (provided by applicant): The neuronal underpinnings of the sleep/wake cycle and the possible functions of sleep in cognition remain to be fully characterized. Neuromodulators such as dopamine (DA), norepinephrine (NE) and acetylcholine (ACh) have been implicated in arousal in numerous behavioral settings. Work from many laboratories, including ours, has demonstrated that the neuropeptide Hypocretin (Hcrt), also known as orexin, is essential for arousal stability, possibly by orchestrating the activity of these neuromodulators however, the neuronal mechanisms underlying this coordination are still unknown. Here, we will use transgenic, anatomical, electrophysiological, chemo and optogenetic approaches to test functional connectivity between neuronal circuits associated with sleep to wake transitions. In the first aim, we will study which hypothalamic inhibitory circuits are sensitive t external factors that affect arousal and sleep, such as acute stress and metabolic challenges, and whether these factors induce sleep by inhibiting Hcrt neurons. In aim 2 we will test the hypothesis is that DA, NE, Ach have different and specific roles on the dynamic of sleep and wakefulness and that their functional connectivity with Hcrt is critical for a healthy sleep/wake cycle. The necessity of DA or ACh transmission for Hcrt-mediated awakenings will be interrogated by photoinhibition of the neuromodulators with simultaneous photostimulation of Hcrt. In aim 3, we will test whether manipulation of individual arousal circuits during sleep has consequences on the consolidation of different types of memories. Together, these experiments will significantly increase our understanding of how Hcrt neurotransmission is modulated by external factors and how Hcrt neurons integrate and transmit this information into effector systems. Our experiments will also shed light into the mechanisms that underlie memory consolidation during sleep, with consequences in cognitive performance.

DESCRIPTION (provided by applicant): Panic disorder is a common psychiatric illness with a lifetime prevalence of about 4.5%. The hallmark of the disorder is recurring panic attacks, which can appear suddenly and unexpectedly, consisting of pronounced fear, as well as cardiovascular and respiratory responses. The initial pathology in these patients appears to be an alteration somewhere in the central neural pathways regulating normal panic responses, thus rendering the patients susceptible to unprovoked panic symptoms when exposed to ordinarily mild interoceptive stressors. Understanding the neuronal underpinnings of panic attacks would significantly improve the outcomes and treatment. The neurotransmitter hypocretin (Hcrt), also known as orexin, has been recently linked to hyperarousal, anxiety and panic, but the specific effector circuits are unknown. Anatomical and functional evidence suggests that one of the possible Hcrt/orexin targets are norepinephrine (NE)-containing neurons in the brainstem. Here, we will test the overall hypothesis that Hcrt/orexin acts through ventral norepinephrine A2 neurons and their efferents to produce different components of the anxiety and panic responses. We will use anatomical tracing and optogenetic tools to obtain a functional map of the neuronal circuitry connecting A2 neurons with several of its anatomical targets, namely the paraventricular hypothalamic nucleus (PVN) and the bed nucleus of the stria terminalis (BNST). We will also determine the connectivity between these circuits and the arousal promoting Hcrt/orexin neurons in the lateral hypothalamus. Our experiments will increase our understanding of the circuitry associated with anxiety with cellular specificity and may lead to potentially more selective treatments for anxiety and panic disorders.

DESCRIPTION (provided by applicant): Aging leads to a functional deterioration of multiple physiological systems, including those underlying sleep and wakefulness. Older individuals tend to have reduced sleep amounts and changes in sleep composition and distribution, decreased alertness, increased fragmentation, reduced nocturnal sleep and amplitude of delta EEG frequency. Sleep disruptions in the elderly also severely affect the health and well-being of their caregivers to the point that, in pathologies such as Alzheimer's disease, sleep fragmentation is the main cause of institutionalization. The underlying mechanisms of these changes in sleep architecture and efficiency during aging are unknown. In this proposal, we will use optogenetics to interrogate the role of specific neuronal cell types and circuits in the decline of sleep qualit and cognition during aging. Optogenetics is an ideal method to study sleep/wake mechanisms in rodents because pharmacological approaches far exceed the short time scale of sleep/wake cycles (in the order of minutes), and electrical stimulations cannot provide cellular specificity. n particular we propose to determine whether the ability of three neurotransmitters (Hcrt/orexin, norepinephrine and acetylcholine) to facilitate wakefulness is reduced in old mice. In aim 2, we will determine whether specific features of sleep quality driven by these three transmitters are relevant for the cognitive decline observed during aging. In a third aim we will test whether a young systemic environment is able to improve sleep composition and distribution in an old mouse using plasma transfer and parabiosis experiments. The data obtained from these experiments may lead to selective therapeutic interventions to improve the quality of life of the elderly and their caregivers.

Functional heterogeneity of Hcrt neurons Complex neuronal circuits regulate arousal, and deficiencies in these circuits are a highly prevalent cause for sleep disturbances and anxiety disorders. The multi-functional hypocretin/orexin (Hcrt) neurons regulate arousal-related behavioral states including sleep, wakefulness, feeding, emotions, stress and reward. However, how a presumably uniform Hcrt population regulates such diverse functions, and how alteration in these circuitries result in sleep and anxiety disorders is not clear. We hypothesize that Hcrt neurons are genetically, anatomically and functionally heterogenous, and that subpopulations of Hcrt neurons projecting to different brain regions regulate specific features of sleep and arousal. Here, we will use a combination of viral monosynaptic circuit tracing, two-photon live imaging of single synapse and neuronal activity, optogenetics, fiber photometry and behavioral experiments in zebrafish and mice to identify and elucidate the function of subpopulations of Hcrt neurons in regulating sleep. In aim 1, we will use retrogradely transported viral vectors and live imaging of single pre- and post-synaptic structure to characterize anatomically distinct subpopulations of Hcrt neurons, innervating the VTA, LC and TMN regions. The transparent zebrafish model enables anatomical and functional live visualization of the relatively simple, but conserved, Hcrt system (20-40 cells). In aim 2, fiber photometry recording in behaving mice and real-time two-photon imaging of neuronal activity in single cell resolution will be performed to elucidate the differential neuronal activity of subpopulations of Hcrt neurons in regulating sleep and arousal. In aim 3, optogenetic, synaptic silencing and genetically induced neuron ablation will be used to manipulate subpopulations of Hcrt neurons. We will monitor the effect on neuronal activity in post synaptic dopaminergic and histaminergic target neurons using genetically encoded calcium indicators. These neuronal activity recordings will be complemented with behavioral experiments in mice. The results are expected to challenge the generalized assumption that neurons that secrete a specific neuropeptide share similar identities and functions. The combined strength of the two vertebrate models will uncover the role of evolutionary conserved sub neuronal circuitries and may provide new therapeutic targets for the treatment of sleep disorders.

Abstract Neuropeptides have a critical role in modulating sleep and wakefulness and offer unique opportunities to treat sleep disorders. Among them, Neuropeptide S shows outstanding features: i) Administration of NPS increases wakefulness and reduces anxiety; ii) Neuropeptide S knockout mice show display increased NREM and anxiety; iii) Mutations of the NPS receptor that give rise to overactive signaling result in short sleep in humans and mice; iv) Expression of NPS is restricted to a few thousand neurons distributed across five main clusters in the basomedial amygdala, dorsomedial thalamus, Kolliker-Fuse/parabrachial area, pericoerulear region and nucleus incertus. These regions have been directly or indirectly associated with arousal and anxiety, but the detailed mechanisms as to how the modulate sleep architecture are unknown. We have recently generated a new line of mice expressing cre recombinase under the control of the endogenous NPS gene promoter (NPS-IRES-cre mice). Here we propose to use these mice and a combination of circuit mapping tools to decipher the mechanisms by which NPS modulates sleep/wake cycle. First, we will use viral-mediated tracing to determine if the five clusters of NPS+ neurons are interconnected, and their anatomical relationship with known arousal circuits. In a second aim, we will use fiber photometry to determine the activity profiles of the five clusters of NPS cells across the sleep/wake cycle and in response to stress and positive emotional stimuli. We will also determine which arousal circuits are activated by optogenetic stimulation of NPS, and which circuits activate NPS neurons. We will also assess whether NPS stimulation affects locomotor activity, anxiety, core body temperature and other physiological variables that may confound the arousal effect. In aim 3, we will test whether individual clusters of NPS neurons are necessary for NPS?s effects on sleep by using opto and chemogenetic inhibition. Finally, we will use a CRISPR-based approach to introduce NPS gene mutations in individual NPS+ cell clusters and determine whether NPS release in these brain regions is essential to control sleep/wake architecture and anxiety behaviors. The results from these experiments will shed new light into the function of NPS and NPS+ neurons, as well as the interconnection between them and arousal circuits. These data may lead to improved treatments of neuropsychiatric disorders associated with imbalances in arousal systems.