Probing Neural Circuits of Zebrafish Sleep with Electrophysiology and Calcium Imaging

摘要 斑马鱼已经成为发现和表征遗传和神经元回路的有用模型系统 调节脊椎动物的睡眠然而，该模型的局限性在于，睡眠是使用行为特征来确定的。 标准，而不是脑电图（EEG）和肌电图（EMG）的措施，用于定义 哺乳动物的睡眠和清醒状态。使用类似EEG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脑活动慢波， 在行为活动期间，大脑活动的振幅快波，类似于在 哺乳动物的NREM睡眠和觉醒。在目标1中，我们将进行一系列实验来测试 这一假说认为这些大脑活动模式反映了斑马鱼的睡眠和清醒状态。验证该 这一假设将增加斑马鱼作为睡眠模型的有用性，因为EEG记录可以更精确地 睡眠和清醒状态的分类以及EEG节律的分类将有助于比较 斑马鱼和哺乳动物睡眠表型之间的关系。睡眠脑电图节律也可以揭示不同的睡眠状态 无法通过行为分析捕捉到的信息在目标2中，我们将识别产生慢动作的神经元。 我们在斑马鱼行为静止期间观察到的波， 记录和全脑GCaMP 6 f神经元活动成像。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。 神经元这一假设是基于对皮层单个神经元的细胞内记录的组合 和丘脑，以及脑电图记录，捕捉表面附近皮层神经元的集体活动模式 的大脑。然而，无法全面确定个人的贡献， 神经元到慢波在全脑规模。因此，目前还不清楚观察到的慢波是否 在哺乳动物的NREM睡眠中，由相对较小或较大的神经元群体产生，以及是否 类似的神经元活动模式存在于除皮质和丘脑之外的脑区域中。我们将 解决这些问题的慢波，我们观察到在斑马鱼行为静止， 同时进行全脑GCaMP 6 f成像，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的神经元，其活动以2-4 Hz振荡。结果 从这些实验可能产生新的假设，关于神经元的基础上慢波在NREM 睡眠可以通过有针对性的记录在哺乳动物中测试。这个项目有可能揭示一个新的 哺乳动物和斑马鱼睡眠之间的相似性层，并早于已知的哺乳动物的出现- 就像4.5亿年前大脑在睡眠中的活动模式一样。该项目还将建立和验证一个 该方法对于随后的靶向脑回路项目R 01应用程序至关重要，该应用程序旨在 确定在睡眠期间观察到的慢波的功能。

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.