Synaptic Basis of Sleep Cycle Control

描述（由申请人提供）：为什么我们醒来后会犯困？为什么睡眠驱动会对行为和表现产生这种先发制人的影响？为这些简单的问题制定更好的答案是本提案的目的。我们使用动物模型来了解调节体内平衡睡眠驱动（HSD）的机制，这种驱动在睡眠缺失或睡眠剥夺（SD）后调节嗜睡。我们在动物模型中的早期发现表明，细胞外神经化学物质腺苷（AD）水平升高是一种体内平衡睡眠因子，在胆碱能基底前脑（CBF）中起着强有力的作用，在睡眠剥夺3至6小时（短期SD）后增加嗜睡。我们现在认为，随着SD持续时间的逐渐延长（6小时或更长），HSD效应越来越多地由非脑皮质区介导。我们注意到，“腺苷和睡眠”是一个科学热点话题，2008年2月的PubMed搜索显示，仅去年一年就有31篇文章。为了促进我们的理解，我们提出了以下与HSD相关的分子、细胞和行为机制的研究。具体目标1)。我们将测试AD生成和细胞外水平的变化是否首先发生在CBF，然后在更长的SD中发生在皮质区域。我们预测，随着24小时SD，腺苷A1受体结合（活性受体数量）增加。我们将验证我们的初步数据假设，即诱导型一氧化氮合酶（iNOS）产生的一氧化氮（NO）是SD期间AD增加的直接介质，我们预测iNOS和NO活性将呈现与AD相同的时空分布。具体目标2)。利用在gabaergergy神经元中表达绿色荧光蛋白（GFP）的新型转基因小鼠体外切片，我们将研究NO的细胞作用，以证实NO最初激活皮质投射CBF胆碱能和gabaergy神经元，然后产生依赖于NO产生的AD的抑制作用。我们将进一步研究CBF的内在神经生理特性和已确定的细胞成分的药理学。具体目标3)。我们将使用一种新的啮齿动物版本的人类多重睡眠潜伏期测试（rMSLT）和一种啮齿动物版本的人类精神运动警觉性任务（rPVT）来研究CBF中SD、AD和NO对嗜睡和警觉性的影响和相互作用。总之，这个项目建议使用我们认为最先进的分子、电生理和行为方法，包括我们实验室在睡眠领域独有的几种方法。这些包括使用一种新型染料（DAF）来识别NO生产的细胞来源；使用转基因GFP小鼠，可以在电生理记录之前识别gaba能神经元，最后使用啮齿动物模拟人类测试来测量嗜睡（rMSLT）和警惕性（rPVT）。因此，我们认为这项应用使用了最先进的技术来回答有关体内平衡睡眠驱动的关键问题。疾病（包括失眠和抑郁）和职业需求（轮班工作、医生、急救人员）造成的睡眠不足被认为是生活质量下降、事故发生以及工作和学校效率下降的主要原因。该建议使用动物模型来理解睡眠缺失或睡眠剥夺后介导嗜睡的生物学机制。通过这样做，我们希望为失眠和其他睡眠障碍的合理治疗奠定基础。

DESCRIPTION (provided by applicant): Why do we get sleepy after being awake? Why does the sleep drive have such preemptive effects on behavior and performance? Formulating better answers to these simple questions is the purpose of this proposal. We use an animal model in our quest to understand the mechanisms mediating the homeostatic sleep drive (HSD), the drive that mediates the sleepiness following sleep loss or sleep deprivation (SD). Our earlier findings in animal models pointed to elevated extracellular levels of the neurochemical adenosine (AD) as a homeostatic sleep factor acting potently in the cholinergic basal forebrain (CBF) to increase sleepiness after as little as 3 to 6h of sleep deprivation (short term SD). We now think that with progressively longer periods of SD (6 or more h of SD), the HSD effects are progressively more mediated by non-CBF cortical regions. We note that "adenosine and sleep" is a scientific hot topic, with a February 2008 PubMed search revealing some 31 articles in the past year alone. To help advance our understanding, we propose the following studies relevant to the molecular, cellular, and behavioral mechanisms underlying the HSD. Specific Aim 1). We will test whether changes in AD production and extracellular levels occur first in CBF and then, with longer SD, in cortical areas. We predict an increase in adenosine A1 receptor binding (number of active receptors) with 24h SD. We will test our preliminary data-derived hypothesis indicating that inducible nitric oxide synthase (iNOS)-produced nitric oxide (NO) is an immediate mediator of AD increases during SD, and we predict that iNOS and NO activity will show the same temporal-spatial distribution as AD. Specific Aim 2). Using in vitro slice in a novel genetically modified mouse that expresses green fluorescent protein (GFP) in GABAergic neurons, we will investigate the cellular actions of NO to confirm preliminary data that NO initially excites cortically projecting CBF cholinergic and GABAergic neurons, and then produces an inhibition that is dependent on AD produced by NO. We further will study the intrinsic neurophysiological properties and pharmacology of identified cellular components of the CBF. Specific Aim 3). We will investigate the effects and interaction of SD, AD and NO in the CBF on sleepiness and vigilance using a novel rodent version of the human multiple sleep latency test (rMSLT) and a rodent version of the human psychomotor vigilance task (rPVT). In summary, this project proposes to use what we view as state-of-the-art molecular, electrophysiological and behavioral methods, including several which are unique to our lab within the sleep field. These include the use of a novel dye (DAF) to identify the cellular sources of NO production; the use of genetically modified GFP mice which allow identification of GABAergic neurons prior to electrophysiological recordings and finally the use of rodent analogues of human tests measuring sleepiness (rMSLT) and vigilance (rPVT). We thus see this application as using state-of-the art technology to answer critical questions about the homeostatic sleep drive. PUBLIC HEALTH RELEVANCE Sleep loss from disease (including insomnia and depression) and vocational demands (shift work, doctors, emergency workers) is known as a major contributor to diminished quality of life, to accidents and to decreased work and school efficiency. This proposal uses an animal model to enable understanding of the biological mechanisms mediating the sleepiness following sleep loss or sleep deprivation. By doing this we hope to lay the foundation for a rational treatment of insomnia and other sleep disorders.