Project 4: Whole-brain and body characterization of sleep disturbances and interventions in Fmr1, Shank3 and Cntnap2 knockout zebrafish

项目4：项目摘要/摘要 睡眠对正常的突触连接和大脑发育至关重要。我们的小组之前已经确定 像其他物种一样，斑马鱼的睡眠障碍会阻碍正常的结构突触可塑性。相反， 适当的睡眠和褪黑激素的催眠/昼夜节律治疗可以改善这些突触缺陷。动物模型 的ASD，像ASD患者一样，在发育过程中睡眠中断，并显示突触和 行为缺陷。在这里，我们假设发育过程中的睡眠中断是有原因的 和/或ASD突触和行为缺陷的加重因素，睡眠干预可能 缓解这些问题。而人类(项目1和2)和鼠标(项目3)的方法允许精致 研究社会互动、重复行为和相关的皮质突触缺陷，斑马鱼是一种 在发育生物学中流行的透明脊椎动物，可以对整个大脑和身体进行研究。 重要的是，像Fmr1、Shank3和cntnap2这样的asd风险基因是泛神经元的，它们的丢失可能会影响 睡眠时的整个中枢神经系统。我们最近开发了基于荧光的 斑马鱼多导睡眠图(FPSG)--一种允许全脑和全身的新的非侵入性方法 睡眠时使用单细胞分辨率进行成像。利用FPSG，我们已经证明斑马鱼有睡眠的大脑 动力学类似于哺乳动物，包括我们创造的一种状态-慢爆睡眠(SBS)，它分享了许多 非快速眼动慢波睡眠(SWS)的共性。我们的初步数据表明，SBS是支离破碎的 开发Fmr1斑马鱼突变体。此外，来自其他组织的研究表明，基于运动测量， 斑马鱼cntnapt2ab和shank3ab突变体的睡眠/觉醒模式也被打乱。然而，他们的大脑 睡眠中的活动还没有被调查。因此，在目标1中，我们将对这三种基因类型应用FPSG (FMR1、shank3ab和cntnap2ab突变体)和对照组，以全面描述他们的睡眠神经和肌肉 发展过程中的动态。接下来，我们将应用相同的药物干预措施(H1R抗组胺药， GABAA激动剂和下丘脑/食欲素受体拮抗剂)用于人(项目2)和小鼠(项目2) 3)，改善ASD危险基因突变体的入睡潜伏期和睡眠/SBS巩固。然后，在Aim 2，我们将调查这些NREM/SWS/SBS睡眠干预对以下各项的有益影响 端脑、下丘脑和脊髓环路结构突触密度的纵向成像 表达与PSD95-EGFP、突触素-EGFP等荧光标记融合的突触蛋白 吉普林-绿色荧光蛋白。同时，将评估经过处理的鱼在重复和社会行为方面的改善情况。 就像老鼠(项目3)和人类(项目2)一样。与后者相辅相成的是斑马鱼的透明度 模型将揭示睡眠动力学是如何在整个大脑中被破坏的，以及睡眠干预是如何 也有利于整个中枢神经系统的突触正常化，进一步建立 睡眠障碍在自闭症特征发展中的因果/加重作用。

Project 4: Project Summary/Abstract Sleep is critical for proper synaptic connections and brain development. Our group previously established that sleep disruptions in zebrafish, like in other species, prevent normal structural synapse plasticity. Conversely, proper sleep and melatonin hypnotic/circadian treatment can improve these synaptic defects. Animal models of ASD, like ASD patients, suffer from sleep disruptions during development and display synaptic and behavioral deficiencies. Here, we hypothesize that sleep disruptions during development are causal and/or aggravating factors of ASD synaptic and behavioral defects, and that sleep interventions could alleviate these issues. While human (Projects 1 & 2) and mouse (Project 3) approaches permit exquisite studies of social interactions, repetitive behaviors, and associated cortical synaptic defects, zebrafish is a transparent vertebrate popular in developmental biology allowing whole brain and body investigation. Importantly, ASD risk genes like Fmr1, Shank3, and Cntnap2 are pan-neuronal, and their loss likely impacts the entire central nervous system during sleep. We have recently developed fluorescence-based polysomnography (fPSG) in zebrafish, a novel, non-invasive method allowing whole-brain and whole-body imaging with single cell resolution during sleep. Using fPSG, we have shown that zebrafish have sleep brain dynamics analogous to mammals, including a state we coined slow bursting sleep (SBS) which shares many commonalities with Non-REM slow wave sleep (SWS). Our preliminary data indicates that SBS is fragmented in developing Fmr1 zebrafish mutants. Further, studies from other groups have shown that based on actimetry, sleep/wake pattern is also disrupted in zebrafish cntnapt2ab and shank3ab mutants. However, their brain activity during sleep has not yet been investigated. Thus, in Aim 1, we will apply fPSG to these three genotypes (fmr1, shank3ab, and cntnap2ab mutants) and controls to fully characterize their sleep neural and muscular dynamics during development. Next, we will apply the same pharmacological interventions (H1R antihistamine, GABAA agonist, and hypocretin/orexin receptors antagonist) used in human (Project 2) and mouse (Project 3), to improve sleep onset latency and sleep/SBS consolidation in these ASD risk gene mutants. Then, in Aim 2, we will investigate the respective beneficial effects of these NREM/SWS/SBS-sleep interventions on structural synapse density using longitudinal imaging of telencephalic, hypothalamic and spinal cord circuits expressing synaptic proteins fused to fluorescent markers such as PSD95-eGFP, Synaptophysin-eGFP or Gephyrin-eGFP. In parallel, treated fish will be assessed for improvement in repetitive and social behaviors like in mouse (Project 3) and human (Project 2). Complementing the latter, the transparency of the zebrafish model will reveal how sleep dynamics are disrupted throughout the entire brain and how sleep interventions can also be beneficial for synaptic normalization throughout the CNS, further establishing the causal/aggravating role of disrupted sleep in the development of autistic traits.