Probing Neural Circuits of Zebrafish Sleep with Electrophysiology and Calcium Imaging

用电生理学和钙成像探测斑马鱼睡眠的神经回路

• 批准号: 10436734
• 负责人: David Aaron Prober
• 金额: $ 76.34万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10436734
• 关键词: Address; Adult; Area; Attention; Behavioral; Biological Models; Birds; Brain; Brain region; Calcium; Classification; Cognition; Coupling; Deltastab; Electroencephalogram; Electrophysiology (science); Exhibits; Eye Movements; Fertilization; Frequencies; Genes; Genetic; Image; Individual; Lizards; Mammals; Measurement; Measures; Methods; Modeling; Monitor; Neurons; Pattern; Phenotype; Physiological; Population; Preparation; Resolution; Role; Series; Sleep; Surface; Techniques; Testing; Thalamic structure; Validation; Wakefulness; Work; Zebrafish; base; design; experimental study; mathematical model; neural circuit; neuronal circuitry; neuronal patterning; novel

ABSTRACT The zebrafish has emerged as a useful model system to discover and characterize genetic and neuronal circuits that regulate vertebrate sleep. However, a limitation of this model is that sleep is determined using behavioral criteria and not the electroencephalogram (EEG) and electromyogram (EMG) measures that are used to define mammalian sleep and wake states. Using EEG-like electrophysiological recordings, we have observed that zebrafish exhibit large amplitude slow waves of brain activity during periods of behavioral quiescence, and small amplitude fast waves of brain activity during periods of behavioral activity, similar to those observed during mammalian NREM sleep and wakefulness, respectively. In Aim 1, we will perform a series of experiments to test the hypothesis that these patterns of brain activity reflect zebrafish sleep and wake states. Validation of this hypothesis will increase the usefulness of zebrafish as a sleep model since EEG recordings enable more precise categorization of sleep and wake states, and the classification of EEG rhythms will facilitate comparisons between zebrafish and mammalian sleep phenotypes. Sleep EEG rhythms may also reveal distinct sleep states that cannot be captured using behavioral analyses. In Aim 2, we will identify the neurons that generate the slow waves that we observe during zebrafish behavioral quiescence by performing simultaneous electrophysiological recordings and whole-brain GCaMP6f neuronal activity imaging. The slow waves that are observed during mammalian NREM sleep are thought to be largely generated by synchronous firing of cortical and thalamic neurons. This hypothesis is based on a combination of intracellular recordings of single neurons in the cortex and thalamus, and EEG recordings that capture the collective activity pattern of cortical neurons near the surface of the brain. However, it has not been possible to comprehensively determine the contribution of individual neurons to the slow waves at whole-brain scale. As a result, it is unclear whether the slow waves observed during mammalian NREM sleep are generated by relatively small or large populations of neurons, and whether similar patterns of neuronal activity are present in brain regions in addition to the cortex and thalamus. We will address these questions for the slow waves that we observe during zebrafish behavioral quiescence by performing simultaneous whole-brain GCaMP6f imaging, with single neuron resolution, and electrophysiological recordings, in order to directly visualize the neurons in the entire brain whose activity oscillates at 2-4 Hz. Results from these experiments may generate new hypotheses regarding the neuronal basis of slow waves during NREM sleep that can be tested using targeted recordings in mammals. This project has the potential to reveal a new layer of similarity between mammalian and zebrafish sleep, and to predate the known emergence of mammalian- like patterns of brain activity during sleep by >450 million years. This project will also establish and validate a method that is essential for a subsequent Targeted Brain Circuits Project R01 application that will aim to determine the function of the slow waves that are observed during sleep.

摘要 斑马鱼已经成为发现和描述遗传和神经元回路的有用模型系统 来调节脊椎动物的睡眠。然而，该模型的局限性在于，睡眠是由行为决定的 标准，而不是用于定义的脑电(EEG)和肌电(EMG)测量 哺乳动物的睡眠和清醒状态。使用类似脑电的电生理记录，我们观察到 斑马鱼在行为静止期表现出大幅度的大脑活动慢波，而小的 在行为活动期的大脑活动的幅度快波，与在 哺乳动物的NREM睡眠和清醒分别。在目标1中，我们将执行一系列实验来测试 一种假设，认为这些大脑活动模式反映了斑马鱼的睡眠和清醒状态。对此进行验证 假设将增加斑马鱼作为睡眠模型的有用性，因为脑电记录使 睡眠和清醒状态的分类以及脑电节律的分类将有助于比较 斑马鱼和哺乳动物的睡眠表型之间。睡眠脑电节律也可能显示不同的睡眠状态 这是行为分析无法捕捉到的。在目标2中，我们将识别产生慢波的神经元 我们在斑马鱼行为静止期间通过同时进行电生理观察到的波 记录和全脑GCaMP6f神经元活动成像。在此过程中观察到的慢波 哺乳动物的NREM睡眠被认为主要是由皮层和丘脑的同步放电产生的 神经元。这一假说是基于大脑皮层单个神经元的细胞内记录的组合。 和丘脑，以及捕捉地表附近皮质神经元集体活动模式的脑电记录 大脑的一部分。然而，还不可能全面确定个人的贡献 神经元对全脑尺度的慢波的反应。因此，目前还不清楚观察到的慢波 在哺乳动物中，NREM睡眠是由相对较少或较多的神经元群体产生的，以及 除皮质和丘脑外，大脑区域也存在类似的神经元活动模式。我们会 解决我们在斑马鱼行为静止期间观察到的慢波的这些问题 同时进行全脑GCaMP6f成像，具有单神经元分辨率和电生理 记录，以便直接可视化整个大脑中活动以2-4赫兹振荡的神经元。结果 这些实验可能会产生关于NREM过程中慢波的神经元基础的新假说 可以使用哺乳动物的目标记录来测试的睡眠。这个项目有可能揭示一种新的 哺乳动物和斑马鱼睡眠的相似之处，并早于哺乳动物的已知出现- 比如4.5亿年前睡眠时大脑活动的模式。该项目还将建立和验证 该方法对于后续针对大脑电路项目R01的应用至关重要，该项目将旨在 确定睡眠期间观察到的慢波的功能。