Circadian Structural Plasticity in Central Pacemakers

中央起搏器的昼夜节律结构可塑性

基本信息

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
项目类别：
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

项目摘要

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.

总结昼夜节律取决于分子转录/翻译负反馈环（TTL）的运作时钟神经元，以及这些神经元的网络特性。在可以招募的房产中生物钟的变化是突触前/后伙伴的身份和/或突触强度的变化时钟神经元之间的连接性，这种特性统称为昼夜节律结构可塑性。我们的中央假设哺乳动物和果蝇的中央生物钟内的昼夜节律结构可塑性是时间编码机制的一部分。我们将采用小鼠和果蝇遗传学与最先进的技术相结合艺术定量 3D 光学和电子显微镜技术，用于解决内部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 将评估果蝇昼夜节律网络中结构可塑性的广泛程度以及这是这些结构性变化的功能后果。我们将探讨昼夜节律的范围使用 CM 和 SBEM 对 PDF 和非 PDF 起搏神经元子集进行神经元重塑（子目标 1A i 和ii)。我们将通过以下方式检查时钟神经元之间随时间变化的功能连接变化：化学遗传学 GCamP6 报告（子目标 1B）。子目标 1C 将检查以下行为的后果损害结构改造；子目标1D将进一步研究潜在的分子机制昼夜节律结构可塑性。具体目标 2 将检查 SCN VIPergic 神经元的昼夜节律结构重塑程度，这些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）（子目标 2A），或带有标记的串行块面扫描电子显微镜（SBEM），可以分析树突超微结构（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是否通过在 VIP 特异性 Bmal1-/- 小鼠中重复 1A 中的实验来依赖 TTL（子目标 2B）。具体目标 3 将探讨 VIPergic 神经元的连接性在整个 24 小时周期中是否发生变化。使用绿色荧光蛋白跨突触伙伴的重建（GRASP），我们将调查这些连接是否随昼夜节律变化通过固定组织（子目标 3A）以及离体 SCN 中的免疫细胞化学和 CM 分析来确定时间切片（子目标 3B）。我们还将确定 GRASP 检测到的节律是否依赖于规范 TTL 通过在 VIP 或 SCN 星形胶质细胞特异性 Bmal1-/- 小鼠中重复 2A 和 2B 实验（子目标 3C）。我们的实验测试了昼夜节律结构可塑性代表了一个决定性假设的预测。中枢神经元昼夜节律起搏器的特征。对这一假设的支持将提供一个关键的新的的角度来了解这些起搏器如何在网络级别编码时间。此外，我们提出的实验为阿根廷的研究能力建设提供了一个独特的机会，在那里外国首席研究员所在地，学生和博士后将接受以下技术培训：那个国家还没有完全发展起来。