Preoptic/hypothalamic mechanisms of sleep-wake regulation

描述（由申请人提供）： 本研究旨在探讨一氧化氮（NO）和腺苷（AD）在调节下丘脑穹窿周围外侧区（PF-LHA）下丘脑泌素能（HCRTergic，又称orexinergic）系统睡眠-觉醒依赖性活动中的作用。该提议的中心假设是HCRT神经元的激活有助于自发和持续唤醒期间AD的局部产生; AD产生通过NO产生介导;并且HCRT神经元的随后抑制有助于自发和稳态睡眠。设计了三个具体目标，以系统地评估上述总体假设的各个组成部分。 具体目标-1将测试唤醒期间HCRT神经元的激活是否是NO产生的核心，这反过来又有助于自发觉醒期间局部AD释放及其在持续唤醒期间的积累。我们将测量从PF-LHA收集的透析液中的NO代谢物（NOx/）和AD的水平，以确定：a）NOx/和AD水平是否在自发觉醒期间高于睡眠期间，并在睡眠剥夺期间进一步累积，B）睡眠剥夺期间PF-LHA中HCRT信号传导的局灶性阻断是否减弱NOx/和AD的产生，c）以及NO是否有助于AD的产生。 具体目标-2将测试在唤醒/持续唤醒期间产生的NO是否通过抑制HCRT神经元而有助于睡眠，并且该效应通过A1受体依赖性腺苷能机制介导。a）我们将通过反向微透析将局部药物递送到PF-LHA中，以确定PF-LHA中的NO是否有助于自发睡眠以及睡眠剥夺后的恢复睡眠，以及是否通过腺苷能机制介导。B）我们将使用细胞外单位记录结合药物递送来确定NO抑制PF-LHA神经元放电的能力，并确定这种作用是否通过腺苷能机制介导。c）我们将使用双标记免疫组织化学结合通过微透析探针到PF-LHA中的药物递送来确定局部释放的NO降低清醒动物HCRT神经元中c-Fos表达的能力以及A1受体依赖性腺苷能机制在介导该效应中的功效。 具体目标-3将确认前食欲素原敲除（HCRT KO）小鼠中提出的假设的关键组成部分，并确定这些小鼠中缺乏HCRT信号传导是否会导致自发/长时间唤醒期间NO和AD的产生减弱，从而影响其睡眠调节。a）我们将测量在自发觉醒以及睡眠剥夺期间从HCRT KO及其野生型同窝仔的PF-LHA收集的透析液中的NOx/和腺苷水平。B）我们还将确定HCRT KO与野生型小鼠中PF-LHA中NO和AD的睡眠促进能力。 拟议研究中使用的三种互补方法，即，单位记录相邻的微透析探针，双标记免疫组织化学相邻的微透析探针，和用于局部药理学操作的微透析探针的扩散场中释放的神经递质/神经调质的测定提供了一种独特的方法来了解在睡眠和清醒期间PF-LHA内的氮能，腺苷能和HCRTergic系统之间的局部相互作用。我们相信，对所提出的相互作用及其对睡眠-觉醒行为的综合影响的更好理解将可能有助于新的治疗策略，包括对药物设计的新见解和/或更好的联合疗法的开发，用于管理睡眠障碍以及可能与HCRTergic系统相关的疾病，这些疾病在普通人群和退伍军人中很常见。

DESCRIPTION (provided by applicant): This proposal is intended to characterize the contributions of nitric oxide (NO) and adenosine (AD) in regulating sleep-wake dependent activity of hypocretinergic (HCRTergic also called orexinergic) system in the perifornical-lateral hypothalamic area (PF-LHA). The central hypothesis of this proposal is that the activation of HCRT neurons contributes to the local production of AD during spontaneous and sustained arousal; that AD production is mediated via NO production; and that a subsequent inhibition of HCRT neurons contributes to spontaneous and as well as homeostatic sleep. Three specific aims have been designed to systematically evaluate various components of the above overarching hypotheses. Specific aim-1 will test whether activation of HCRT neurons during arousal is central to NO production, which in turn contributes to local AD release during spontaneous waking and its accumulation during sustained arousal. We will measure levels of NO metabolites (NOx/ ) and AD in the dialysates collected from the PF-LHA to determine: a) whether NOx/ and AD levels are higher during spontaneous arousal vs. sleep and further accumulates during sleep deprivation, b) whether focal blockade of HCRT signaling in the PF-LHA during sleep deprivation attenuates production of NOx/ and AD, c) and whether NO contributes to the production of AD. Specific aim-2 will test whether NO produced during arousal/sustained arousal contributes to sleep by inhibiting HCRT neurons and that this effect is mediated via an A1 receptor- dependent adenosinergic mechanism. a) We will use local drug delivery via reverse microdialysis into the PF-LHA to determine whether NO in the PF-LHA contributes to spontaneous sleep as well as recovery sleep subsequent to sleep deprivation, and if it is mediated via adenosinergic mechanism. b) We will use extracellular unit recording in combination with drug delivery to determine the ability of NO to suppress the discharge of PF-LHA neurons and determine if this effect is mediated via adenosinergic mechanism. c) We will use double label immunohistochemistry in combination with drug delivery via a microdialysis probe into the PF-LHA to determine the ability of locally released NO in decreasing c-Fos expression in HCRT neurons in awake animals and the efficacy of A1 receptor-dependent adenosinergic mechanism in mediating this effect. Specific aim-3 will confirm the key components of proposed hypotheses in prepro-orexin knockout (HCRT KO) mice and determine whether a lack of HCRT signaling in these mice causes attenuated production of NO and AD during spontaneous/prolonged arousal, thus affecting their sleep regulation. a) We will measure NOx/ and adenosine levels in the dialysates collected from the PF-LHA of HCRT KO and its wild type littermate in spontaneous arousal as well as during sleep deprivation. b) We will also determine the sleep-promoting abilities of NO and AD in the PF-LHA in HCRT KO vs. wild type mice. The three complementary methods used in proposed studies, i.e., unit recording adjacent to the microdialysis probe, double labeling immunohistochemistry adjacent to the microdialysis probe, and the assay of the neurotransmitters/neuromodulators released in the diffusion field of the microdialysis probe used for local pharmacological manipulations provide a unique approach to understanding local interactions among nitrergic, adenosinergic, and HCRTergic systems within the PF-LHA during sleep and waking. We believe that an improved understanding of the proposed interactions and their integrated influences on sleep-wake behavior will likely contribute to new therapeutic strategies including novel insights into drug designing and/or the development of better combination therapies for the management of sleep disorders as well as diseases potentially related to the HCRTergic system that are common in general population as well as in Veterans.