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

项目 4：Fmr1、Shank3 和 Cntnap2 敲除斑马鱼睡眠障碍的全脑和身体特征及干预措施

• 批准号: 10698080
• 负责人: Philippe Mourrain
• 金额: $ 39.66万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-06 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10698080
• 关键词: ASD patient; Agonist; Animal Model; Antihistamines; Behavior; Behavioral; Brain; Brain Stem; Brain region; Cardiovascular system; Cell Nucleus; Cells; Central Nervous System; Chimeric Proteins; Coin; Complement; Coupled; DLG4 gene; Data; Defect; Development; Developmental Biology; Etiology; Eye Movements; FMR1; Fishes; Fluorescence; Fragile X Syndrome; Future; Genetic; Genotype; Goals; Heart Rate; Human; Hyperactivity; Hypothalamic structure; Impairment; Intervention; Investigation; Knock-out; Maintenance; Mammals; Melatonin; Methods; Modeling; Motor Activity; Mus; Muscle; Muscle Tonus; Nervous System; Neurons; Optics; Pattern; Physiological; Polysomnography; Proteins; Pyrilamine; Resolution; Role; Schools; Siblings; Skeletal Muscle; Sleep; Sleep Wake Cycle; Sleep disturbances; Slow-Wave Sleep; Social Behavior; Social Interaction; Spinal Cord; Swimming; Synapses; Synaptophysin; Testing; Time; Zebrafish; antagonist; autism spectrum disorder; autistic; behavioral deficiency; circadian; density; gamma-Aminobutyric Acid; gephyrin; hypnotic; hypocretin; imaging approach; improved; improvement on sleep; mutant; neural; non rapid eye movement; novel; olfactory bulb; pharmacologic; pressure; prevent; receptor; repetitive behavior; risk variant; serial imaging; sleep abnormalities; sleep onset; social; tool; trait; whole body imaging; zolpidem

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.

项目4：项目概要/摘要 睡眠对于正确的突触连接和大脑发育至关重要。我们小组先前确定， 像其他物种一样，斑马鱼的睡眠中断会阻止正常的结构性突触可塑性。相反地， 适当的睡眠和褪黑激素催眠/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，在发育过程中遭受睡眠中断，并显示突触和 行为缺陷在这里，我们假设睡眠中断在发展过程中是因果关系， 和/或ASD突触和行为缺陷的加重因素，睡眠干预可以 缓解这些问题。虽然人类（项目1和2）和小鼠（项目3）的方法允许精致的 研究社会互动，重复行为，以及相关的皮层突触缺陷，斑马鱼是一种 透明脊椎动物，在发育生物学中很受欢迎，可以研究整个大脑和身体。 重要的是，ASD风险基因如Fmr 1，Shank 3和Cntnap 2是泛神经元的，它们的缺失可能影响 整个中枢神经系统在睡眠中。我们最近开发了基于荧光的 斑马鱼多导睡眠图（fPSG），一种新的，非侵入性的方法，允许全脑和全身 在睡眠期间以单细胞分辨率成像。使用fPSG，我们已经证明斑马鱼有睡眠脑 类似于哺乳动物的动态，包括我们创造的缓慢爆发睡眠（SBS）状态， 非REM慢波睡眠（Non-REM Slow Wave Sleep，SWS）我们的初步数据表明，SBS是分散的 在开发Fmr 1斑马鱼突变体方面。此外，来自其他团体的研究表明，基于活动测定法， 在斑马鱼cntnapt 2ab和shank 3ab突变体中睡眠/觉醒模式也被破坏。然而，他们的大脑 睡眠期间的活动尚未被研究。因此，在目标1中，我们将fPSG应用于这三种基因型 (fmr1，shank 3ab和cntnap 2ab突变体）和对照，以充分表征它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（H1 R抗组胺药， GABAA激动剂和下丘脑分泌素/食欲素受体拮抗剂）用于人（项目2）和小鼠（项目 3）改善ASD危险基因突变者的睡眠潜伏期和睡眠/SBS巩固。在Aim 2，我们将研究这些NREM/SWS/SBS睡眠干预对睡眠的各自有益影响。 利用端脑、下丘脑和脊髓回路纵向成像研究结构突触密度 表达与荧光标记物融合的突触蛋白，所述荧光标记物例如PSD 95-eGFP、突触素-eGFP或 Gephyrin-eGFP。同时，将评估经处理的鱼在重复和社会行为方面的改善 如小鼠（项目3）和人类（项目2）。与后者相辅相成的是，斑马鱼的透明度 该模型将揭示整个大脑的睡眠动力学是如何被破坏的，以及睡眠干预是如何被破坏的。 也有利于整个CNS的突触正常化，进一步建立 睡眠中断在自闭症特征发展中的因果/加重作用。