Cellular and Neurochemical Mechanisms of REM Sleep

描述（由申请人提供）：本研究的长期目标是进一步了解脑干细胞、分子和网络机制对快速眼动睡眠的调节。具体来说，本更新应用的目的是研究桥脚被盖层（PPT）细胞内信号机制在REM睡眠调节中的作用。该建议的中心假设是快速眼动睡眠是由PPT中的细胞机制平衡调节的；具体来说，在PPT中，REM睡眠是由cAMP依赖性蛋白激酶A （PKA）的激活诱导的，而REM睡眠是由Ca2激活引起的觉醒诱导抑制的？？？？依赖性蛋白激酶II （CaMKII）和/或丝裂原活化蛋白激酶（MAPK）。为了系统地检验这一假设，设计了三个具体目标：确定激活诱发快速眼动睡眠的PPT细胞的神经递质表型。为了实现这一目标，将通过c-fos和pCREB表达来鉴定状态依赖性激活的PPT细胞，并通过免疫细胞化学标记胆碱乙酰转移酶（ChAT）和GABA来确定其神经化学身份。2. 验证PPT中PKA激活诱发REM睡眠的假说。这一目标将通过量化不同REM睡眠时间后PPT PKA活性水平，并在PPT中微注射选择性cAMP-PKA激活抑制剂来阻断REM睡眠的稳态驱动来实现。3. 验证在PPT中激活CaMKII和MAPK通过诱导清醒来终止快速眼动睡眠的假设。这一目标将通过测量不同清醒和快速眼动睡眠量的PPT CaMKII和MAPK活性水平，以及将CaMKII和MAPK激活抑制剂应用于PPT，同时量化它们对自发和反弹快速眼动睡眠结构的影响来实现。所有这些实验都将在自由活动的成年大鼠身上进行。初步数据为每个具体目标提供了基本原理，并证明了可行性。这一提议在机制层面上解决了基础神经生物学中的一般问题：快速眼动睡眠是如何调节的？重要的是，识别参与快速眼动睡眠调节的细胞内信号分子可能会导致设计下一代药物来治疗快速眼动睡眠障碍以及各种因快速眼动睡眠中断而加剧的严重疾病，如发作性睡病/猝厥、不宁腿综合征、内源性抑郁症、精神分裂症、阿尔茨海默氏症和亨廷顿病。本研究的目的是确定参与快速眼动（REM）睡眠正常调节的细胞内信号分子。最近的证据表明，旨在改变细胞内转导途径的新化合物具有治疗内源性抑郁症、癌症、低体温和血小板病理聚集的潜力。同样，识别参与快速眼动睡眠正常调节的细胞内分子可能会导致设计下一代药物来治疗快速眼动睡眠障碍以及各种因快速眼动睡眠中断而加剧的严重疾病，如发作性睡病/猝厥、不宁腿综合征、内源性抑郁症、精神分裂症、阿尔茨海默氏症和亨廷顿氏病。

DESCRIPTION (provided by applicant): The long-term objective of this research is to further our understanding of brainstem cellular, molecular, and network mechanisms of REM sleep regulation. Specifically, the goal of this renewal application is to investigate the involvement of pedunculopontine tegmentum (PPT) intracellular signaling mechanisms in the regulation of REM sleep. The central hypothesis of this proposal is that REM sleep is regulated by a balance of cellular mechanisms in the PPT; specifically, in the PPT, REM sleep is induced by the activation of cAMP- dependent protein kinase A (PKA), and REM sleep is suppressed by the induction of wakefulness caused by activation of Ca2????dependent protein kinase II (CaMKII) and/or mitogen-activated protein kinase (MAPK). Three specific aims have been designed to systematically test this hypothesis: 1. Identify the neurotransmitter phenotype(s) of cells in the PPT whose activation induces REM sleep. To achieve this goal, state-dependently activated PPT cells will be identified by c-fos and pCREB expression, and their neurochemical identity will be determined by the immunocytochemical labeling of choline acetyltransferase (ChAT) and GABA. 2. Test the hypothesis that the activation of PKA in the PPT induces REM sleep. This goal will be achieved by quantifying the levels of PPT PKA activity after different amounts of REM sleep and by microinjecting a selective cAMP-PKA activation inhibitor into the PPT to block the homeostatic drive for REM sleep. 3. Test the hypothesis that CaMKII and MAPK activation in the PPT terminates REM sleep by inducing wakefulness. This goal will be achieved by measuring the levels of PPT CaMKII and MAPK activity at varying amounts of wakefulness and REM sleep and by applying inhibitors of CaMKII and MAPK activation into the PPT while quantifying their effects on the architecture of spontaneous as well as rebound REM sleep. All of these experiments will be performed on adult, freely moving rats. Preliminary data provide a rationale for each specific aim and demonstrate feasibility. This proposal addresses, at the mechanistic level, the general question in basic neurobiology: how is REM sleep regulated? Importantly, identification of the intracellular signaling molecules involved in the regulation of REM sleep may lead to the design of a future generation of drugs to treat REM sleep disorders as well as a variety of serious medical conditions that are exacerbated by disrupted REM sleep, such as narcolepsy/cataplexy, restless legs syndrome, endogenous depression, schizophrenia, Alzheimer's, and Huntington's disease. PUBLIC HEALTH RELEVANCE The goal of this research is to identify the intracellular signaling molecules involved in the normal regulation of rapid eye movement (REM) sleep. Recent evidence indicates that novel compounds designed to modify intracellular transduction pathways have therapeutic potential for endogenous depression, cancer, hypothermia, and pathological aggregation of platelets. Similarly, identification of the intracellular molecules involved in normal regulation of REM sleep may lead to the design of a future generation of drugs to treat REM sleep disorders as well as a variety of serious medical conditions that are exacerbated by disrupted REM sleep, such as narcolepsy/cataplexy, restless legs syndrome, endogenous depression, schizophrenia, Alzheimer's, and Huntington's disease.