Fluorescent polysomnography and MCH neurogenetics

摘要 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），这是一种新的， 允许神经系统功能的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的 在脑中携带表达GCaMP 7a [Tg（5xUAS：GCaMP 7a）]的四个转基因的斑马鱼系“zPSG” [Tg(α-微管蛋白：nls-Kal 4FF）]和躯干肌[Et（gSAIzGFFD 109 A）]，以及心脏中的GFP [Tg（cmlc 2：GFP）] 为了捕获大脑范围内的Ca 2+活动（fEEG，荧光脑电图），肌肉Ca 2+活动 (fEMG荧光肌电图）、心率（fECG，荧光心电图）以及眼睛 运动（fEOG，荧光眼电图）。多导睡眠图（PSG）是用于 描述睡眠并诊断神经和精神疾病中的睡眠障碍和睡眠异常 紊乱慢波睡眠（SWS，非REM）和快速眼动睡眠（REM，a.k.a.矛盾 睡眠，PS）由基于来自表面的记录的特定电生理PSG特征来定义 以及随意或自主肌肉（EMG+ECG+EOG）。SWS-REM/PS具有 迄今为止，只有在进化较近的羊膜脊椎动物中才有报道：哺乳动物，鸟类和爬行动物。 目前还不清楚这种神经元和肌肉动力学是否在非羊膜脊椎动物中发现， 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波 睡眠中的鱼脑中的神经活动。我们创造了这些新颖的签名：缓慢爆发睡眠 (SBS)与SWS和PS/REM有显著的共同点 国，分别。我们建议开发和应用fPSG来充分表征SBS（目标1）和PWS (Aim 2）在整个大脑、身体规模水平上。在完成这一完整的表征之后，我们接下来将研究 这些动力学的分子和电路基础，通过询问不同的神经发生背景， 黑色素浓集激素（MCH）信号传导是一种保守的神经肽能系统， 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）。总的来说，这 建议将（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 规模的理解，也可以用于其他鱼类模型[例如洞穴鱼，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） 揭示了睡眠的共同神经特征是否出现在非羊膜脊椎动物的大脑中， 四亿五千万年前。重要的是，fPSG工具和方法可以扩展到任何神经科学 在清醒或睡眠的动物中的问题，需要全脑成像， 自主肌肉读数（例如，自主和非自主系统的研究）。

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).