Preoptic/hypothalamic mechanisms of sleep-wake regulation

描述(由申请人提供)： 本研究旨在研究一氧化氮(NO)和腺苷(AD)在调节穹隆周围-外侧下丘脑区(PF-LHA)的下克汀能(HCR)能(又称食欲素能)睡眠-觉醒依赖活动中的作用。这一设想的中心假设是，在自发和持续的觉醒过程中，HCRT神经元的激活有助于AD的局部产生；AD的产生是通过NO产生介导的；随后对HCRT神经元的抑制有助于自发睡眠和稳态睡眠。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。特定的Aim-1将测试在觉醒过程中HCRT神经元的激活是否对NO的产生起中心作用，而NO的产生反过来又有助于自发唤醒过程中局部AD的释放和持续唤醒过程中AD的积累。我们将测量从PF-LHA收集的透析液中NO代谢产物(NOx/)和AD的水平，以确定：a)自然觉醒期间NOx/和AD水平是否高于睡眠，并在睡眠剥夺期间进一步积累；b)睡眠剥夺期间阻断PF-LHA中的HCRT信号是否会减少NOx/和AD的产生；c)NO是否参与AD的产生。特异性Aim-2将测试在觉醒/持续唤醒过程中产生的NO是否通过抑制HCRT神经元而促进睡眠，并且这种作用是通过A1受体依赖的腺苷能机制介导的。A)我们将通过反向微透析将局部药物输送到PF-LHA，以确定PF-LHA中的NO是否有助于睡眠剥夺后的自然睡眠和恢复睡眠，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释放来确定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微透析探针给药到PF-LHA，以确定局部释放的NO在降低清醒动物HCRT神经元c-Fos表达的能力，以及A1受体依赖的腺苷能机制在介导这一效应中的作用。特定的Aim-3将在食欲素前体基因敲除(HCRT KO)小鼠中确认所提出的假说的关键成分，并确定这些小鼠中HCRT信号的缺乏是否会导致自发/长时间唤醒过程中NO和AD的产生减少，从而影响它们的睡眠调节。A)我们将测定在自然觉醒和睡眠剥夺期间，从HCRT KO及其野生型仔鼠的PF-LHA中收集的透析液中的NOx/和腺苷水平。B)我们还将在HCRT KO和野生型小鼠的PF-LHA中测定NO和AD的促进睡眠的能力。在拟议的研究中使用的三种互补的方法，即邻近微透析探针的单位记录，邻近微透析探针的双标记免疫组织化学，以及用于局部药物操作的微透析探针扩散场中释放的神经递质/神经调节剂的分析，为了解睡眠和清醒时PF-LHA内硝能、腺苷和HCR能系统之间的局部相互作用提供了独特的途径。我们相信，对拟议的相互作用及其对睡眠-觉醒行为的综合影响的更好理解可能有助于新的治疗策略，包括对药物设计和/或开发更好的组合疗法的新见解，以管理睡眠障碍以及可能与HCR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.