Synaptic Basis of Sleep Cycle Control

描述（由申请人提供）：为什么我们醒来后会昏昏欲睡？为什么睡眠驱力会对行为和表现产生如此先发制人的影响？对这些简单的问题提出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稳态睡眠驱动（HSD）的机制，这种驱动调节睡眠丧失或睡眠剥夺（SD）后的嗜睡。我们在动物模型中的早期发现指出，神经化学物质腺苷（AD）作为一种稳态睡眠因子，在胆碱能基底前脑（CBF）中有效地发挥作用，在睡眠剥夺（短期SD）3至6小时后增加嗜睡。我们现在认为，随着SD持续时间的延长（SD持续6小时或更长时间），HSD效应越来越多地由非CBF皮质区域介导。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搜索显示仅在过去一年就有31篇文章。为了帮助推进我们的理解，我们提出了以下研究相关的分子，细胞和行为机制的HSD。具体目标1）。我们将测试AD产生和细胞外水平的变化是否首先发生在CBF中，然后，随着SD的延长，在皮质区。我们预测腺苷A1受体结合（活性受体的数量）与24小时SD增加。我们将测试我们的初步数据来源的假设表明，诱导型一氧化氮合酶（iNOS）产生的一氧化氮（NO）是一个直接介质AD增加SD期间，我们预测，iNOS和NO活性将显示相同的时空分布AD。具体目标2）。使用在GABA能神经元中表达绿色荧光蛋白（GFP）的新型遗传修饰小鼠的体外切片，我们将研究NO的细胞作用以证实NO最初兴奋皮质投射CBF胆碱能和GABA能神经元的初步数据，然后产生依赖于NO产生的AD的抑制作用。我们将进一步研究鉴定的CBF的细胞成分。具体目标3）。我们将调查的影响和相互作用的SD，AD和NO在CBF嗜睡和警惕使用一种新的啮齿类动物版本的人类多次睡眠潜伏期测试（rMPERT）和啮齿类动物版本的人类精神警觉任务（rPVT）。总之，该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。这些方法包括使用一种新的染料（Dye）来识别NO产生的细胞来源;使用基因修饰的GFP小鼠，其允许在电生理记录之前识别GABA能神经元，最后使用啮齿动物类似物测量睡眠（rMptl）和警惕性（rPVT）。因此，我们认为这个应用程序是使用最先进的技术来回答有关稳态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）导致的睡眠不足被认为是生活质量下降，事故以及工作和学校效率下降的主要原因。该提案使用动物模型，以使人们能够理解在睡眠丧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.