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快速眼动睡眠调节的脑干细胞、分子和网络机制。具体来说，本次更新申请的目标是研究桥脚被盖 (PPT) 细胞内信号传导机制在快速眼动睡眠调节中的作用。该提案的中心假设是，快速眼动睡眠是由 PPT 中细胞机制的平衡调节的；具体地，在PPT中，快速眼动睡眠是通过cAMP依赖性蛋白激酶A(PKA)的激活来诱导的，而快速眼动睡眠是通过Ca2+依赖性蛋白激酶II(CaMKII)和/或丝裂原激活蛋白激酶(MAPK)的激活引起的觉醒的诱导来抑制的。我们设计了三个具体目标来系统地检验这一假设： 1. 识别 PPT 中细胞的神经递质表型，这些细胞的激活会诱导 REM 睡眠。为了实现这一目标，状态依赖性激活的 PPT 细胞将通过 c-fos 和 pCREB ​​表达进行识别，其神经化学特性将通过胆碱乙酰转移酶 (ChAT) 和 GABA 的免疫细胞化学标记来确定。 2. 检验 PPT 中 PKA 的激活诱导 REM 睡眠的假设。这一目标将通过量化不同时间的 REM 睡眠后 PPT PKA 活性水平，以及通过将选择性 cAMP-PKA 激活抑制剂显微注射到 PPT 中以阻止 REM 睡眠的稳态驱动来实现。 3. 检验 PPT 中 CaMKII 和 MAPK 激活通过诱导觉醒终止 REM 睡眠的假设。这一目标将通过测量不同觉醒和快速眼动睡眠时 PPT CaMKII 和 MAPK 活性水平，以及将 CaMKII 和 MAPK 激活抑制剂应用到 PPT 中，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成年、自由活动的老鼠身上进行。初步数据为每个具体目标提供了理由并证明了可行性。该提案从机制层面解决了基础神经生物学中的普遍问题：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有助于设计下一代药物，用于治疗快速眼动睡眠障碍以及因快速眼动睡眠中断而加剧的各种严重疾病，例如发作性睡病/猝倒症、不宁腿综合征、内源性抑郁症、精神分裂症、阿尔茨海默病和亨廷顿病。公共健康相关性 本研究的目标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型化合物具有治疗内源性抑郁症、癌症、体温过低和血小板病理性聚集的潜力。同样，识别参与快速眼动睡眠正常调节的细胞内分子可能有助于设计下一代药物，用于治疗快速眼动睡眠障碍以及因快速眼动睡眠中断而加剧的各种严重疾病，例如发作性睡病/猝倒症、不宁腿综合征、内源性抑郁症、精神分裂症、阿尔茨海默病和亨廷顿病。

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.