Fluorescent polysomnography and MCH neurogenetics

荧光多导睡眠图和 MCH 神经遗传学

• 批准号: 10400045
• 负责人: Philippe Mourrain
• 金额: $ 75.23万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10400045
• 关键词: Amphibia; Animal Model; Animals; Behavioral; Birds; Brain; Brain Stem; Brain imaging; Brain region; Cardiovascular system; Cell Nucleus; Cells; Cognition; Coin; Complex; Comprehension; Coupled; Custom; DNA Repair; Data; Defect; Development; Diagnosis; Dorsal; Ecology; Electrocardiogram; Electroencephalogram; Electroencephalography; Electrooculogram; Electrophysiology (science); Equilibrium; Evolution; Eye Movements; Fishes; Fluorescence; Genes; Genetic; Goals; Heart; Heart Rate; Human; Hypothalamic structure; Image; Imaging Device; Light; Lizards; Maintenance; Mammals; Measures; Medical; Mental disorders; Metabolic; Methodology; Methods; Microscope; Microscopy; Modeling; Molecular; Molecular Genetics; Muscle; Muscle Tonus; Muscle relaxation phase; Neocortex; Nervous System Physiology; Neurobiology; Neurons; Neuropharmacology; Neurosciences; Oryziinae; Outcome; Pathology; Pharmaceutical Preparations; Pharmacology; Physiological; Plague; Polysomnography; Protocols documentation; REM Sleep; Reporting; Reptiles; Resolution; Role; Skeletal Muscle; Sleep; Sleep Deprivation; Sleep Disorders; Sleep disturbances; Slow-Wave Sleep; Societies; Speed; Structure; Surface; System; Testing; Time; Transgenes; Transgenic Organisms; Travel; Vertebrates; Work; Zebrafish; alpha Tubulin; analog; awake; base; genetic approach; habituation; hindbrain; hormonal signals; hypnotic; imaging approach; imaging platform; melanin-concentrating hormone; memory consolidation; molecular imaging; mutant; nervous system disorder; neurogenetics; neurotransmission; non rapid eye movement; novel; receptor; relating to nervous system; sleep abnormalities; synaptic pruning; tool; trait; whole body imaging

Abstract We propose to develop and apply fluorescence-based polysomnography (fPSG) in zebrafish, a novel, non- invasive method allowing neurogenetic and pharmacological interrogations of nervous system function through whole brain and whole body imaging. fPSG combines custom light sheet microscopy with a new zebrafish line “zPSG” carrying four transgenes expressing GCaMP7a [Tg(5xUAS:GCaMP7a)] in the brain [Tg(α-tubulin:nls-Kal4FF)] and trunk muscles [Et(gSAIzGFFD109A)], and GFP in the heart [Tg(cmlc2:GFP)] in order to capture brain wide Ca2+ activity (fEEG, fluorescent electroencephalogram), muscle Ca2+ activity (fEMG, fluorescent electromyogram), heart rate (fECG, fluorescent electrocardiogram) as well as eye movement (fEOG, fluorescent electrooculogram). Polysomnography (PSG) is a classic method used to characterize sleep and diagnose sleep disorders and sleep abnormalities in neurological and psychiatric disorders. Slow wave sleep (SWS, non-REM) and rapid eye movement sleep (REM, a.k.a. paradoxical sleep, PS) are defined by specific electrophysiological PSG signatures based on recordings from the surface of the neocortex (EEG), and voluntary or autonomous muscles (EMG+ECG+EOG). SWS-REM/PS have only been reported so far in the more evolutionary-recent amniotic vertebrates: mammals, birds and reptiles. It is unclear whether such neuronal and muscular dynamics are found in non-amniotic vertebrates such as fishes and amphibians. In a first study we have found slow synchronous neural activity and traveling waves of neural activity in the sleeping fish brain. We have coined these novel signatures: Slow Bursting Sleep (SBS) and Propagating Wave Sleep (PWS) which share remarkable commonalities with SWS and PS/REM states, respectively. We propose to develop and apply fPSG to fully characterize SBS (Aim 1) and PWS (Aim 2) at the whole brain, body scale levels. After this full characterization, we will next investigate the molecular and circuit underpinning of these dynamics by interrogating different neurogenetic contexts of melanin-concentrating hormone (MCH) signaling, a conserved neuropeptidergic system which is involved in mammalian sleep but whose role in fish sleep has been debated for over 30 years (Aim 3). Overall, this proposal will (i) develop a new PSG methodology with whole brain-single cell resolution imaging and body scale comprehension that could also be used with other fish models [e.g. cavefish, danionella, medaka], (ii) establish the first neural definition of sleep in fish, (iii) uncover the role of MCH in fish sleep, and finally (iv) shed light on whether common neural signatures of sleep emerged in the non-amniotic vertebrate brain over 450 million years ago. Importantly, fPSG tools and methodology can be extended to any neuroscience question in the awake or asleep animal requiring whole brain imaging with cardiovascular, ocular and voluntary muscles readouts (e.g. studies of the autonomic and non-autonomic systems).

抽象的 我们建议在斑马鱼中开发和应用基于荧光的多聚疗法（FPSG），这是一种新颖的，非 - 侵入性方法允许对神经系统功能的神经遗传学和药物询问 通过整个大脑和全身成像。 FPSG结合了自定义轻型显微镜和新的 斑马鱼线“ ZPSG”带有四个表达gcamp7a的转基因[tg（5xuas：gcamp7a）] [tg（α-微管蛋白：nls-kal4ff）]和树干肌肉[et（gsaizgffd109a）]和心脏中的GFP [tg（cmlc2：gfp）] 为了捕获大脑广泛的Ca2+活性（FEEG，荧光脑电图），肌肉Ca2+活性 （FEMG，荧光肌电图），心率（FECG，荧光心电图）以及眼睛 运动（Feog，荧光电解图）。多摄影术（PSG）是一种经典的方法 表征睡眠和诊断睡眠障碍以及神经和精神病学的睡眠异常 疾病。慢波睡眠（SWS，非REM）和快速眼动睡眠（REM，又名悖论 睡眠，ps）由基于表面的记录的特定电生理PSG特征定义 新皮层（EEG）以及自愿或自主肌肉（EMG+ECG+EOG）。 SWS-REM/PS有 到目前为止，据报道，在进化更具进化的羊水脊椎动物中：哺乳动物，鸟类和爬行动物。 目前尚不清楚在非释放脊椎动物中是否发现这种神经元和肌肉动力学 鱼类和两栖动物。在第一项研究中，我们发现了缓慢的同步神经活动和行进波 熟睡中的鱼大脑中的神经活动。我们创造了这些新颖的签名：缓慢的睡眠 （SBS）和传播波睡眠（PWS），与SWS和PS/REM共享非凡的共同点 国家分别。我们建议开发和应用FPSG以完全表征SB（AIM 1）和PWS （AIM 2）在整个大脑，体重尺度上。完整的表征之后，我们接下来将调查 通过审问不同神经遗传环境的分子和电路基础 黑色素浓缩马酮（MCH）信号传导，一种涉及的保守神经肽系统 哺乳动物的睡眠，但其在鱼睡眠中的作用已经辩论了30多年（AIM 3）。总体而言，这 提案将（i）使用全脑单个细胞分辨率成像和身体开发新的PSG方法论 比例理解也可以与其他鱼类模型一起使用[例如Cavefish，Danionella，Medaka]，（ii） 建立鱼类睡眠的第一个神经定义，（iii）发现MCH在鱼睡眠中的作用，最后（iv） 阐明了在非注射脊椎动物大脑中是否出现了常见的睡眠神经信号 4.5亿年前。重要的是，FPSG工具和方法可以扩展到任何神经科学 在清醒或入睡的动物中，需要用心血管，眼部和眼睛进行整个大脑成像 自愿肌肉读数（例如，自主和非自主系统的研究）。