Fluorescent polysomnography and MCH neurogenetics

摘要 我们建议开发基于荧光的多导睡眠图(FPSG)并将其应用于斑马鱼，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结合了定制的光片显微镜和一种新的 在脑内表达GCaMP7a[TG(5xUAS：GCaMP7a)]的四个转基因斑马鱼“zPSG” [TG(α-微管蛋白：NLS-Kal4Ff)]和躯干肌[ET(GSAIzGFFD109A)]，心脏中的绿色荧光蛋白[TG(cmlc2：gfp)] 为了捕捉脑组织全钙活性(fEEG，荧光脑电)、肌肉钙活性 (fEMG，荧光肌电)，心率(fecg，荧光心电图)以及眼睛 运动(fEOG、荧光眼电)。多导睡眠图(PSG)是一种经典的 确定睡眠特征并诊断神经科和精神科的睡眠障碍和睡眠异常 精神错乱。慢波睡眠(SWS，非快速眼动睡眠)和快速眼动睡眠(REM，又名自相矛盾 睡眠，PS)由基于来自表面的记录的特定电生理PSG信号来定义 新皮质(EEG)和自主或自主肌肉(EMG+CG+EOG)。SWS-REM/PS具有 到目前为止，只有在进化程度更高的羊膜脊椎动物：哺乳动物、鸟类和爬行动物中才有报道。 目前尚不清楚这种神经元和肌肉动力学是否存在于非羊膜脊椎动物中，如 鱼和两栖动物。在第一项研究中，我们发现了缓慢的同步神经活动和行波 沉睡的鱼大脑中的神经活动。我们创造了这些新奇的标志：慢速睡眠 (SBS)和传播波睡眠(PWS)，它们与SWS和PS/REM有显著的共同点 分别是各州。我们建议开发和应用FPSG来全面表征SBS(目标1)和PWS (目标2)在整个大脑、身体的尺度水平。在此充分描述之后，我们接下来将调查 通过询问不同的神经遗传学背景来支持这些动力学的分子和电路 黑色素浓缩激素(MCH)信号，一种保守的神经肽能系统，参与 哺乳动物睡眠，但其在鱼类睡眠中的作用已经争论了30多年(目标3)。总体而言，这 提案将(I)开发一种新的具有全脑-单细胞分辨率成像和身体的PSG方法 尺度理解，也可用于其他鱼类模型[例如，洞鱼、丹尼奥拉、青海]，(2) 建立第一个鱼类睡眠的神经定义，(Iii)揭示MCH在鱼类睡眠中的作用，最后(Iv) 阐明在非羊膜脊椎动物的大脑中是否出现了共同的睡眠神经信号 4.5亿年前。重要的是，FPSG的工具和方法可以扩展到任何神经科学 清醒或睡眠动物中的问题需要进行心血管、眼部和脑部的全脑成像 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).