Purinergic Mechanisms in Homeostatic Sleep Control

稳态睡眠控制中的嘌呤能机制

• 批准号: 8598055
• 负责人: RADHIKA BASHEER
• 金额: --
• 依托单位: VA BOSTON HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-10-01 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598055
• 关键词: Adenosine; Adenosine A1 Receptor; Adenosine Triphosphate; Astrocytes; Attention; Attenuated; Bathing; Behavior; Bilateral; Binding; Brain; Catabolism; Cell physiology; Cells; Cognition; Diet; Dominant-Negative Mutation; Doxycycline; Electroencephalography; Extracellular Space; General Population; Health; Homeostasis; Human; In Vitro; Investigation; Laboratories; Light; Link; Mammals; Measures; Mediating; Mediation; Membrane Potentials; Methods; Microdialysis; Military Personnel; Molecular; Mus; Neurons; Pathway interactions; Pattern; Performance; Perfusion; Physiological; Proteins; Purine Nucleotides; Rattus; Recovery; Relative (related person); Rodent; SNAP receptor; Signal Transduction; Sleep; Sleep Deprivation; Sleep Disorders; Slice; Specificity; Testing; Time; Transgenes; Veterans; Wakefulness; Work; alertness; basal forebrain; base; behavior test; behavioral impairment; cholinergic; design; direct application; experience; extracellular; frontal lobe; inhibitor/antagonist; mouse model; multidisciplinary; neurobehavioral; novel; postsynaptic; pressure; prevent; receptor; response; sleep regulation; transmission process; vigilance; voltage clamp

DESCRIPTION (provided by applicant): Sleep is essential for optimal health and performance. Prolonged waking beyond its natural duration leads to a homeostatic sleep response based on the duration of prior wakefulness. Increases in homeostatic sleep response are associated with increased sleepiness and decreased alertness. Thus, a clear understanding of the mechanisms regulating the homeostatic sleep response is important in designing targeted treatments for sleepiness and associated neurobehavioral deficits experienced by military personnel and for the treatment of sleep disorders in veterans. Previous work from our laboratory demonstrated the importance of extracellular adenosine ([AD]ex) in sleep homeostasis within the basal forebrain (BF) wakefulness center. While there is ample evidence demonstrating a sleep deprivation (SD)-induced [AD]ex increase in BF, the mechanisms for this regional and localized increase is yet unknown. The purine nucleotide adenosine triphosphate (ATP) is released into extracellular space as a co-transmitter and via gliotransmission during neuronal activity in wakefulness. The neuromodulatory effects of [ATP]ex is exerted either by its direct action on P2 receptors or after its rapid breakdown by localized ectonucleotidase to [AD]ex, which acts via P1 receptors. Recent evidence suggests a dense presence of a neuronal ectonucleotidase in BF but not in cortex. Using state-of-the-art, multidisciplinary methods we will test the overarching hypothesis that an increase in [ATP]ex in the BF mediates sleepiness via its localized catabolism by ectonucleotidase to AD, which then inhibits wake promoting BF neurons. In specific aim 1 we will test the hypothesis that during SD [ATP]ex increases in BF and frontal cortex, and produce an elevation of the homeostatic sleep response as determined by an increase in the delta activity (1-4.5 Hz) during recovery NREM sleep in rats. We will examine the time course of SD-induced changes in [ATP]ex and determine the relative effect of two mechanisms of [ATP]ex on the homeostatic sleep response: direct action on P2 receptor versus rapid degradation of [ATP]ex to [AD]ex by selective actions of ectonucleotidase with the prediction that ectonucleotidase inhibitors, but not P2 antagonists, will attenuate homeostatic sleep response. In specific aim 2, will use a mouse model in which astrocytic release of [ATP]ex is prevented. The transgene in this mice (astrocyte-selective dominant negative SNARE (dnSNARE))is conditionally regulated with dietary doxycycline. , We will test the hypothesis that SD increases gliotransmission of [ATP]ex in BF. We predict allowing dnSNARE expression (-doxycycline) will prevent [ATP]ex release and the increase in [AD]ex during SD, whereas suppressing dnSNARE expression (+doxycycline) will show increases in [AD]ex and a homeostatic response, due to gliotrasmission release of [ATP]ex-> [AD]ex. In specific aim 3, using GAD67-GFP mice, we will test the hypothesis that [ATP]ex will cause an inhibition of cortically projecting BF neurons in vitro, due to its breakdown to AD and activation of A1 receptor. We will also determine the time course of ATP breakdown to [AD]ex in the BF. In specific aim 4, we will test the hypothesis that ATP-derived AD's action on the A1 receptor mediates increased sleepiness and consequent neurobehavioral performance decrements following SD. We will use two novel behavioral tests (i) Our rodent version of the human multiple sleep latencies test that provides a direct measure of sleepiness, and (ii) our rodent version of the human psychomotor vigilance test to measure sustained attention (vigilance) following 3h and 6h of SD during the light period. Receptor specificity will be tested by reverse microdialysis during SD of antagonists of the P2 and, separately, to the A1 receptor. We predict that A1 receptor antagonists will decrease sleepiness (sleep latencies) and vigilance whereas the P2 antagonists will be much weaker effect, indicating a predominantly adenosinergic mediation of sleepiness. The successful completion of this comprehensive investigation will shed light on purinergic mechanisms involved in homeostatic sleep controls.

描述（由申请人提供）： 睡眠对于最佳健康和表现至关重要。长时间醒来的持续时间超出其自然持续时间会导致基于先前清醒的持续时间的稳态睡眠反应。稳态睡眠反应的增加与嗜睡和警报降低有关。因此，对调节体内稳态睡眠反应的机制有清晰的了解对于设计有针对性的嗜睡和相关的神经行为缺陷很重要。我们实验室的先前工作表明，细胞外腺苷（[AD] EX）在基础前脑（BF）觉醒中心内的睡眠体内平衡中的重要性。尽管有足够的证据表明剥夺睡眠（SD）诱导的BF的[AD] Ex升高，但这种区域和局部增加的机制尚不清楚。嘌呤核苷酸三磷酸腺苷（ATP）被释放到细胞外空间中，作为共磷脂，并通过神经元活性在觉醒中通过胶质传递。 [ATP] EX的神经调节作用是通过其直接作用对P2受体的直接作用，或者在其局部核苷酸酶快速分解后，通过P1受体起作用。最近的证据表明，在BF中存在神经元外核苷酸酶，但在皮质中不存在。使用最先进的多学科方法，我们将测试总体假设，即BF中[ATP] EX的增加通过其局部核苷酸酶通过其局部分解代谢介导AD，然后抑制唤醒促进BF神经元的唤醒。在特定目标1中，我们将测试以下假设：在SD [ATP] EX期间，BF和额叶皮层的增加，并产生稳态睡眠反应的升高，这取决于大鼠恢复NREM睡眠期间的增长Delta活性（1-4.5 Hz）所确定的。我们将检查[ATP] EX的[ATP] EX的两种机制对体内稳态睡眠响应的相对效应的时间过程：直接对P2受体对[ATP] EX ex ex ex ex ex ex extrication contriation the Ectonucleticase intectistation intectositation noteants Intibal intiby intiby intiby pantiber and-pantstion and-pantstions in Not and pantst and-pant and tand and tand and 2的直接作用与[AD] Ex的快速降解。在特定的目标2中，将使用小鼠模型，在该模型中，可以防止[ATP] EX的星形细胞释放。该小鼠中的转基因（星形胶质选择性显性阴性SNARE（DNSNARE））有条件地受到饮食强力霉素的条件调节。 ，我们将检验以下假设：SD增加了BF中[ATP] EX的胶质传递。我们预测，允许DNSNARE表达（-DoxyCycline）可以防止[ATP] EX释放和SD期间[AD] EX的增加，而抑制DNSNARE表达（+DOXYCYCLINE）将显示[AD] EX和稳态响应的增加，并且由于[ATP] EX-> [AD] EX- [AD] EX- [AD] EX-EX-[AD]。在使用GAD67-GFP小鼠的特定目标3中，我们将检验以下假设：[ATP] EX将在体外抑制皮层投射BF神经元，这是由于其对AD的细分和A1受体的激活。我们还将确定BF中ATP分解的时间过程。在特定的目标4中，我们将测试以下假设：ATP衍生的AD对A1受体的作用介导了SD后的嗜睡增加和随之而来的神经行为性能降低。我们将使用两个新型的行为测试（i）人类多重睡眠潜伏期测试的啮齿动物版本，可直接衡量嗜睡，（ii）我们的人类精神运动警惕性测试的啮齿动物版本在光周期内进行3H和6H后的持续注意力（警惕）。受体特异性将通过P2拮抗剂的SD和分别对A1受体的反向微透析进行测试。我们预测A1受体拮抗剂将降低嗜睡（睡眠潜伏期）和警惕性，而P2拮抗剂的效果将弱得多，表明主要是腺苷能的嗜睡介导。这项全面调查的成功完成将阐明掌握稳态睡眠控制涉及的嘌呤能机制。