Circadian Structural Plasticity in Central Pacemakers

汇总表 昼夜节律取决于分子转录/翻译负反馈环(TTL)在体内的作用 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 生物钟是突触前/突触后伙伴身份和/或突触强度的变化 时钟神经元之间的连通性，这一特性统称为昼夜结构可塑性。我们的中央 假说是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。我们将利用老鼠和苍蝇的遗传学与最新进展相结合 ART定量3D光学和电子显微镜技术，以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将评估结构可塑性在果蝇昼夜节律网络中的广泛程度以及 这是这些结构性变化的功能后果。我们将探索昼夜节律的范围 使用CM和SBEM对PDF和非PDF起搏神经元亚群的神经元重构(次级目标1AI 和ii)。我们将通过以下方式研究时钟神经元之间随时间变化的功能连接变化 化学遗传GCamP6--报告(次级目标1B)。次级目标1C将检查以下行为后果 损害结构重塑；子目标1D将进一步研究 昼夜节律结构可塑性。 特定目标2将检查SCN Viper能神经元的昼夜节律结构重构的程度，这些神经元是 计时机制的基本组成部分，通过病毒介导的稀疏标记(CM)(亚 目标2a)，或连续的块面扫描电子显微镜(SBEM)，具有能够分析 树枝状超微结构(亚目的2C)。最后，我们将评估VIP神经元突起中的昼夜节律振荡 依赖于TTL，在VIP特异的BMal1/-小鼠中重复1A中的实验(亚目标2B)。 具体目标3将探索Viper能神经元的连接性是否在24小时周期中发生变化。使用GFP 跨突触伙伴的重建(GRAP)，我们将调查这些连接是否会随着昼夜节律的变化而变化 免疫细胞化学和CM分析在固定组织中的时间(亚目的3A)以及体外培养的SCN 切片(分目标3B)。我们还将确定GRAPH检测到的节律是否取决于典型的TTL 通过在VIP或SCN星形胶质细胞特异性BMal1/-小鼠(亚目标3C)中重复2A和2B中的实验。 我们的实验测试了以下假设的预测：昼夜节律结构可塑性代表着一个定义 中枢神经元昼夜节律起搏器的特点。对这一假设的支持将提供一个关键的新 透视以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， 外国首席调查员所在地，学生和博士后将在那里接受技术培训 在那个国家还没有完全发展起来。

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.