Purinergic Mechanisms in Homeostatic Sleep Control

稳态睡眠控制中的嘌呤能机制

• 批准号: 8244639
• 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/8244639
• 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)： 睡眠对于最佳的健康和表现是必不可少的。超过自然持续时间的长时间清醒会导致基于先前清醒持续时间的动态平衡睡眠反应。稳态睡眠反应的增加与睡意的增加和警觉性的降低有关。因此，清楚地了解调节稳态睡眠反应的机制对于设计针对军人经历的嗜睡和相关神经行为缺陷的有针对性的治疗以及治疗退伍军人的睡眠障碍非常重要。我们实验室以前的工作证明了细胞外腺苷([AD]EX)在基底前脑(BF)觉醒中心的睡眠稳态中的重要性。虽然有充分的证据表明睡眠剥夺(SD)诱导的BF[AD]EX增加，但这种区域性和局部性增加的机制尚不清楚。在清醒状态的神经元活动中，嘌呤核苷酸三磷酸腺苷(ATP)被释放到细胞外空间，作为一种共同递质，并通过神经胶质传递。[ATP]ex的神经调节作用可以通过直接作用于P2受体来实现，也可以通过定位于[AD]ex的胞外核苷酸酶迅速分解为[AD]ex，后者通过P1受体来发挥作用。最近的证据表明，在BF中存在密集的神经元型ectDNA酶，但在皮质中不存在。使用最先进的、多学科的方法，我们将检验最重要的假设，即BF中[ATP]EX的增加通过其通过胞外核苷酸酶向AD的局部分解代谢来调节嗜睡，后者随后抑制促进觉醒的BF神经元。在具体目标1中，我们将测试这一假设，即在SD[ATP]ex期间，BF和额叶皮质中的含量增加，并产生由恢复NREM睡眠期间Delta活动(1-4.5赫兹)增加所确定的动态平衡睡眠反应的升高。我们将研究SD诱导的[ATP]ex变化的时间过程，并确定[ATP]ex对稳态睡眠反应的两种机制的相对影响：直接作用于P2受体和通过选择性作用将[ATP]ex迅速降解为[AD]ex，并预测ecton核苷酸酶抑制剂，而不是P2拮抗剂，将减弱稳态睡眠反应。在特定目标2中，将使用阻止星形胶质细胞释放[ATP]ex的小鼠模型。这种转基因小鼠(星形胶质细胞选择显性负性陷阱(DnSNARE))受饮食中多西环素的条件性调控。，我们将检验SD增加BF中[ATP]EX神经胶质传递的假设。我们预测，允许dnSNARE表达(-多西环素)将阻止SD时[ATP]ex的释放和[AD]ex的增加，而抑制dnSNARE表达(+多西环素)将显示[AD]ex的增加和动态平衡反应，这是由于[ATP]ex-&gt；[AD]ex的胶质传递释放。在特定的目标3中，我们将使用GAD67-GFP小鼠来验证这样的假设，即[ATP]EX将在体外导致BF神经元皮质投射的抑制，这是由于它分解到AD和激活A1受体。我们还将确定ATP在高炉内分解到[AD]EX的时间进程。在特定的目标4中，我们将测试这一假设，即ATP衍生的AD对A1受体的作用介导了SD后嗜睡增加和随后的神经行为表现下降。我们将使用两个新的行为测试(I)我们的啮齿动物版本的人类多重睡眠潜伏期测试提供了一个直接测量困倦程度的方法，以及(Ii)我们的啮齿动物版本的人类精神运动警戒测试来测量在光照期间SD持续3小时和6小时后的持续注意力(警戒)。P2受体拮抗剂和A1受体拮抗剂在SD期间的受体特异性将通过反向微透析法进行测试。我们预测A1受体拮抗剂将减少嗜睡(睡眠潜伏期)和警觉性，而P2拮抗剂的作用将弱得多，表明主要是腺苷能调节嗜睡。这项全面研究的成功完成将有助于阐明参与稳态睡眠控制的嘌呤能机制。