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）： 睡眠对最佳健康和表现至关重要。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稳态睡眠反应。稳态睡眠反应的增加与嗜睡增加和警觉性降低有关。因此，对调节稳态睡眠反应的机制的清楚理解对于设计针对军事人员经历的嗜睡和相关神经行为缺陷的靶向治疗以及退伍军人睡眠障碍的治疗是重要的。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[AD]ex增加BF，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）作为共递质并在清醒时的神经元活动期间通过胶质传递释放到细胞外空间。[ATP]ex的神经调节作用是通过其对P2受体的直接作用或在其被局部外核苷酸酶快速分解为[AD]ex后发挥的，[AD]ex通过P1受体发挥作用。最近的证据表明，在BF中，但不是在皮质中的神经元外核苷酸酶的密集存在。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的增加通过外核苷酸酶对AD的局部catalysis介导嗜睡，然后抑制唤醒促进BF神经元。在具体目标1中，我们将检验以下假设：在SD期间，BF和额叶皮质中的[ATP]ex增加，并产生稳态睡眠反应的升高，如通过大鼠恢复NREM睡眠期间δ活动（1-4.5 Hz）的增加所确定的。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：对P2受体的直接作用与通过外核苷酸酶的选择性作用将[ATP]ex快速降解为[AD]ex，预测外核苷酸酶抑制剂而不是P2拮抗剂将减弱稳态睡眠反应。在具体目标2中，将使用其中[ATP]ex的星形胶质细胞释放被阻止的小鼠模型。该小鼠中的转基因（星形胶质细胞选择性显性负性SNARE（dnSNARE））用膳食强力霉素有条件地调节。我们将检验SD增加BF中[ATP]ex的胶质传递的假设。我们预测允许dnSNARE表达（-多西环素）将阻止SD期间[ATP]ex释放和[AD]ex增加，而抑制dnSNARE表达（+多西环素）将显示[AD]ex增加和稳态反应，这是由于[ATP]ex-> [AD]ex的胶质传递释放。在具体目标3中，使用GAD 67-GFP小鼠，我们将测试[ATP]ex将在体外引起皮质投射BF神经元的抑制的假设，这是由于其分解为AD和A1受体的激活。我们还将确定ATP分解到BF中[AD]ex的时间过程。在具体的目标4中，我们将检验以下假设：ATP衍生的AD对A1受体的作用介导SD后嗜睡增加和随之而来的神经行为表现下降。我们将使用两种新的行为测试（i）我们的啮齿类动物版本的人类多次睡眠潜伏期测试，提供了一个直接的测量嗜睡，和（ii）我们的啮齿类动物版本的人类精神警觉性测试，以测量持续的注意力（警觉性）后3小时和6小时的SD在光周期。在P2和A1受体拮抗剂的SD期间，通过反向微透析检测受体特异性。我们预测，A1受体拮抗剂将减少嗜睡（sleep laundry）和警惕性，而P2拮抗剂的效果要弱得多，表明主要是腺苷能介导的嗜睡。这项全面的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。