Circadian Structural Plasticity in Central Pacemakers

中央起搏器的昼夜节律结构可塑性

• 批准号: 10409826
• 负责人: Maria Fernanda Ceriani
• 金额: $ 51.45万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10409826
• 关键词: 3-Dimensional; ARNTL gene; Address; Adhesions; Adult; Animals; Argentina; Astrocytes; Automobile Driving; Behavior; Behavioral; Biological Models; Brain; Calcium; Cell Communication; Cells; Circadian Rhythms; Confocal Microscopy; Country; Coupled; Cues; Drosophila genus; Electron Microscopy; Enterobacteria phage P1 Cre recombinase; Face; Feedback; Fiber; Flavoproteins; Genetic; Genetic Transcription; Hypothalamic structure; Immunohistochemistry; Impairment; Injections; Label; Mammals; Mediating; Molecular; Motor Activity; Mus; Neurites; Neuroglia; Neurons; Neuropeptides; Neurotransmitters; Organism; Pacemakers; Periodicity; Photoperiod; Physiological; Pigments; Postdoctoral Fellow; Principal Investigator; Process; Property; Reporter; Reporting; Research; Scanning Electron Microscopy; Signal Transduction; Sleep Wake Cycle; Slice; Structure; Students; Synapses; Synaptophysin; System; Techniques; Testing; Time; Tissues; Training; Translations; Vasoactive Intestinal Peptide; Viral; Virus; Visualization; circadian; circadian pacemaker; density; ex vivo imaging; experience; experimental study; fly; gene product; immunocytochemistry; knock-down; light microscopy; longitudinal analysis; mouse Cre recombinase; neuronal circuitry; polymerization; postsynaptic; presynaptic; promoter; receptor; reconstitution; recruit; response; suprachiasmatic nucleus; tool; vector

SUMARY Circadian rhythms depend on the molecular transcription/translation negative feedback loop (TTL) operating in clock neurons, and on the network properties of these neurons. Among the properties that could be recruited by the circadian clock are changes in the identity of pre/post synaptic partners and/or strength of the connectivity between clock neurons, a property collectively termed as circadian structural plasticity. Our central hypothesis is that circadian structural plasticity within the central circadian clocks of mammals and Drosophila are part of the time-encoding mechanisms. We will employ mouse and fly genetics combined with state-of-the- art quantitative 3D light and electron microscopy techniques to address the extent of structural plasticity within specific neurons of the mouse suprachiasmatic nucleus (SCN) and the Drosophila circadian network. Specific aim 1 will assess how widespread structural plasticity is in the Drosophila circadian network as well as which are the functional consequences of those structural changes. We will explore the extent of circadian neuronal remodeling of subsets of PDF and non-PDF pacemaker neurons using CM and SBEM (sub-aims 1A i and ii). We will examine time-of-day dependent functional connectivity changes among clock neurons through chemogenetic GCamP6-reporting (sub-aim 1B). Sub-aim 1C will examine the behavioral consequences of impairing structural remodeling; sub-aim 1D will further investigate the molecular mechanisms underlying circadian structural plasticity. Specific aim 2 will examine the degree of circadian structural remodeling in SCN VIPergic neurons, which are an essential component of the timekeeping mechanism, through virally mediated sparse-labeling (CM) (sub- aim 2A), or serial block-face scanning electron microscopy (SBEM) with a marker that enables the analysis of dendritic ultrastructure (sub-aim 2C). Finally, we will assess if circadian oscillations in VIP neuronal processes rely on the TTL by repeating experiments in 1A in VIP-specific Bmal1-/- mice (sub-aim 2B). Specific aim 3 will explore if connectivity of VIPergic neurons changes throughout the 24-h cycle. Using GFP reconstitution across synaptic partners (GRASP), we will investigate if these connections change with circadian time through immunocytochemistry and CM analysis in fixed tissue (sub-aim 3A) as well as ex vivo in SCN slices (sub- aim 3B). We will also determine whether GRASP-detected rhythms depend on the canonical TTL by repeating experiments in 2A and 2B in VIP- or SCN astrocyte-specific Bmal1-/- mice (sub-aim 3C). Our experiments test predictions of the hypothesis that circadian structural plasticity represents a defining feature of central neuronal circadian pacemakers. Support for this hypothesis would provide a critical new perspective to understand how these pacemakers encode time at the network level. Furthermore, the experiments we propose represent a unique opportunity for research capacity building in Argentina, where the foreign principal investigator is located, and where students and postdocs will be trained in techniques that are still not fully developed in that country.

总结 昼夜节律依赖于分子转录/翻译负反馈环（TTL）， 时钟神经元，以及这些神经元的网络特性。在可以招募的财产中， 由昼夜节律钟是在前/后突触伴侣的身份和/或强度的变化， 生物钟神经元之间的连接，一个属性统称为昼夜结构可塑性。我们的中央 一个假说是哺乳动物和果蝇中枢生物钟内的生物钟结构可塑性 是时间编码机制的一部分我们将采用老鼠和苍蝇遗传学结合国家的- 艺术定量三维光学和电子显微镜技术，以解决结构可塑性的程度， 小鼠视交叉上核（SCN）和果蝇昼夜节律网络的特定神经元。 具体目标1将评估如何广泛的结构可塑性是在果蝇昼夜节律网络，以及 这是这些结构变化的功能性后果。我们将探索昼夜节律 使用CM和SBEM的PDF和非PDF起搏神经元的子集的神经元重构（子目标1A是 和ii）。我们将通过以下方式研究时钟神经元之间的时间依赖性功能连接变化： 化学发生GCamP 6报告（子目标1B）。子目标1C将检查以下行为的后果： 损害结构重塑;子目标1D将进一步研究 昼夜结构可塑性 具体目标2将检查SCN VIPergic神经元中的昼夜节律结构重塑的程度，所述神经元是 计时机制的重要组成部分，通过病毒介导的稀疏标记（CM）（亚 aim 2A），或带有标记的连续块面扫描电子显微镜（SBEM），可以分析 树突状超微结构（子目标2C）。最后，我们将评估VIP神经元过程中的昼夜节律振荡， 依靠TTL，在VIP特异性Bmal 1-/-小鼠中重复1A中的实验（子目标2B）。 具体目标3将探索VIPergic神经元的连接性是否在整个24小时周期中发生变化。使用GFP 通过研究跨突触伙伴重建（GRASP），我们将研究这些连接是否随昼夜节律而变化。 固定组织（子目标3A）以及SCN离体免疫细胞化学和CM分析的时间 切片（子目标3B）。我们还将确定GRASP检测到的节律是否依赖于典型的TTL 通过在VIP-或SCN星形胶质细胞特异性Bmal 1-/-小鼠中重复2A和2B中的实验（子目标3C）。 我们的实验测试了昼夜结构可塑性代表了一种定义性的假设的预测。 中枢神经元昼夜节律起搏器的特征。支持这一假设将提供一个关键的新的 从这个角度来理解这些起搏器如何在网络层面上编码时间。而且 我们提出的实验代表了阿根廷研究能力建设的独特机会， 外国首席研究员的所在地，学生和博士后将接受技术培训， 在这个国家还没有完全发展起来。