Circadian Structural Plasticity in Central Pacemakers

中央起搏器的昼夜节律结构可塑性

• 批准号: 10266132
• 负责人: Maria Fernanda Ceriani
• 金额: $ 52.82万
• 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/10266132
• 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.

sumary 昼夜节律取决于分子转录/翻译负反馈回路（TTL） 时钟神经元以及这些神经元的网络属性。在可以招募的物业中 昼夜节律的时钟是突触前/突触伴侣的身份和/或强度的变化 时钟神经元之间的连通性，该属性统称为昼夜节律可塑性。我们的中心 假设是哺乳动物和果蝇中央昼夜节律中的昼夜节律可塑性 是时间编码机制的一部分。我们将采用鼠标和飞行遗传学，并结合使用 艺术定量3D光和电子显微镜技术，以解决结构可塑性的程度 小鼠上核核（SCN）和果蝇昼夜节律网络的特定神经元。 特定目标1将评估果蝇昼夜节律中的结构可塑性以及 这是这些结构变化的功能后果。我们将探索昼夜节律的程度 使用CM和SBEM（sub-aims 1a I），PDF和非PDF起搏器神经元子集的神经元重塑 和II）。我们将检查时钟神经元之间的依赖时间的功能连接性变化 化学发生GCAMP6报告（子-IAM 1B）。 Sub-aim 1C将检查的行为后果 损害结构重塑； Sub-aim 1D将进一步研究基础的分子机制 昼夜节律可塑性。 特定的目标2将检查SCN染色神经元中的昼夜节律结构重塑的程度， 通过病毒介导的稀疏标签（CM）（sub-- AIM 2A），或带有标记物的串行块扫描电子显微镜（SBEM），可实现分析 树突超微结构（Sub-aim 2c）。最后，我们将评估VIP神经元过程中的昼夜节律振荡是否 通过在VIP特异性BMAL1 - / - 小鼠（Sub-Aim 2b）中重复1A中重复实验来依靠TTL。 如果在整个24小时周期内发生变化，那么特定的目标3将探索是否发生抗毒牙神经元的连通性。使用GFP 跨突触伙伴的重建（抓取），我们将调查这些联系是否与昼夜节律发生变化 通过固定组织（Sub-Aim 3a）中的免疫细胞化学和CM分析的时间以及SCN中的离体 切片（亚目标3B）。我们还将确定掌握的节奏是否取决于规范的TTL 通过在VIP或SCN星形胶质细胞特异性BMAL1 - / - 小鼠（Sub-Aim 3C）中重复2A和2B中的实验。 我们的实验测试了昼夜节律可塑性的假设的预测 中央神经元昼夜节奏起搏器的特征。对这一假设的支持将提供一个关键的新 了解这些起搏器如何在网络级别编码时间。此外， 我们提出的实验代表了阿根廷研究能力建设的独特机会， 外国首席调查员所在，学生和博士后将接受有关技术的培训 在那个国家仍然没有完全发展。