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导睡眠图（fPSG），这是一种新型的、非 允许对神经系统功能进行神经遗传学和药理学询问的侵入性方法 通过全脑和全身成像，fPSG 将定制光片显微镜与新型技术相结合。 斑马鱼系“zPSG”携带四个在大脑中表达 GCaMP7a [Tg(5xUAS:GCaMP7a)] 的转基因 [Tg(α-微管蛋白:nls-Kal4FF)] 和躯干肌肉 [Et(gSAIzGFFD109A)]，以及心脏中的 GFP [Tg(cmlc2:GFP)] 为了捕获全脑 Ca2+ 活动（fEEG、荧光脑电图）、肌肉 Ca2+ 活动 （fEMG，荧光肌电图），心率（fECG，荧光心电图）以及眼睛 运动（fEOG，荧光多导睡眠图（PSG））是一种经典的方法。 表征睡眠并诊断神经和精神方面的睡眠障碍和睡眠异常 慢波睡眠（SWS，非快速眼动睡眠）和快速眼动睡眠（快速眼动睡眠，又名矛盾睡眠） 睡眠 (PS) 由基于表面记录的特定电生理 PSG 特征定义 新皮质 (EEG) 和随意或自主肌 (EMG+ECG+EOG) 具有。 迄今为止，仅在进化较晚的羊膜脊椎动物中报道过这种现象：哺乳动物、鸟类和爬行动物。 目前尚不清楚这种神经和肌肉动力学是否存在于非羊膜脊椎动物中，例如 在第一项研究中，我们发现鱼类和两栖动物有缓慢的同步神经活动和行波。 我们创造了这些新颖的特征：缓慢的突发睡眠。 (SBS) 和传播波睡眠 (PWS) 与 SWS 和 PS/REM 有显着的共同点 我们建议开发并应用 fPSG 来充分表征 SBS（目标 1）和 PWS。 （目标 2）在整个大脑、身体层面进行全面表征后，我们接下来将进行研究。 通过询问不同的神经发生背景来研究这些动力学的分子和电路基础 黑色素浓缩激素 (MCH) 信号传导是一种保守的神经肽能系统，参与 哺乳动物的睡眠，但其在鱼类睡眠中的作用已经争论了 30 多年（目标 3）。 该提案将 (i) 开发一种新的 PSG 方法，具有全脑-单细胞分辨率成像和身体 尺度理解也可用于其他鱼类模型 [例如穴居鱼、danionella、青鳉]，(ii) 建立鱼类睡眠的第一个神经定义，(iii) 揭示 MCH 在鱼类睡眠中的作用，最后 (iv) 揭示睡眠的常见神经特征是否在非羊膜脊椎动物大脑中出现 4.5 亿年前，fPSG 工具和方法可以扩展到任何神经科学。 清醒或睡眠动物的问题需要对心血管、眼部和眼部进行全脑成像 随意肌肉读数（例如自主和非自主系统的研究）。