MECHANISMS OF CIRCADIAN CLOCK OUTPUT

在这个提案中，我概述了一组三个相关但独立的昼夜节律神经研究。 输出。成像和数据分析捕获有关网络信息的最新进展 空间和时间精确度不断提高的现象。昼夜节律起搏器系统 我们的研究的优势在于它既能自发地产生生理活动，又能 有节奏地。在前一个周期中，我们使用平面照明方法在体内进行了24小时的实验 对昼夜节律神经回路进行全脑扫描。这项工作引入了一个新的概念 昼夜节律网络如何编码时间的理论：我们证明了分子上 同步起搏器网络显示不同身份的顺序激活 一天中的心脏起搏器小组。此外，我们还发现了起搏器细胞之间的相互作用， 神经肽介导的延迟，是影响顺序起搏器的关键机制 激活。这个项目的科学前提是需要扩展这些 昼夜节律起搏器神经元可塑性的观察 图案被传输到下游中心。在这里，我建议继续实时工作 果蝇核心昼夜节律起搏器神经元活动模式的活体研究 神经元。它还继续对神经肽调节机制的重点分析， 严格控制起搏器活动的具体时间。 以扩大我们最初研究的范围，并提供对神经元的更好的了解 关于起搏器和起搏网络的特性，该计划将追求三个目标。目标 1将通过以下方式更好地定义起搏器中的每日钙动态：(I)进行深入、高度的 频率采样，以及(Ii)通过确定支持这些的亚细胞机制 波动。AIM 2将对PDF受体(PDFR)进行结构-功能分析， 尤其是它的C末端，以了解控制数量的调控机制 每日PDF信令的范围。它还试图确定关键的PDFR调节蛋白。目标3 寻求将我们的工作范围扩展到昼夜节律起搏器网络之外，以识别 下游电路元素：我们将重点关注中央复合体中神经元的子集 初步证据表明与日常节律生理学有关 有运动能力。 时差、倒班工作和睡眠活动周期的干扰都会导致智力和 身体健康。两个主要的死亡原因(中风和心脏骤停)显示出明确的时间- 然而，我们对生物钟之间的因果联系知之甚少。 功能和致病机制。这项研究计划致力于更好地 了解基本的昼夜节律输出机制。

In this proposal, I outline a set of three related yet independent studies of circadian neural output. Recent advances in imaging and data analysis capture information regarding network phenomena with increasing spatial and temporal precision. The circadian pacemaker system we study is advantageous in that it produces physiological activity both spontaneously and rhythmically. In the previous cycle, we used planar illumination methods to perform 24 hr in vivo brain-wide scans of the circadian neural circuit. That work introduced a new concept to theories of how the circadian network encodes time: we showed that the molecularly synchronous pacemaker network displays sequential activation by different identified pacemaker groups across the day. Further we found pacemaker cell interactions, in the form of neuropeptide-mediated delay, represents a key mechanism to effect sequential pacemaker activation. The scientific premise for this project rests on the need to extend those observations on circadian pacemaker neuronal plasticity and to understand how these activity patterns are transmitted to downstream centers. Here I propose work that continues real-time in vivo studies of neuronal activity patterns for the core Drosophila circadian pacemaker neurons. It also continues the focused analysis of neuropeptide modulatory mechanisms that critically regulate the specific timing of pacemaker activity. To extend the scope of our initial studies, and to provide a better understanding of neuronal properties of pacemakers and pacemaking networks, this program will pursue three Aims. Aim 1 will better define daily Ca2+ dynamics in pacemakers by (i) performing in-depth, high frequency sampling, and (ii) by determining the sub-cellular mechanisms underlying these fluctuations. Aim 2 will pursue a Structure-Function analysis of the PDF receptor (PDFR), especially its C-terminus, to understand the regulatory mechanisms that control the quantitative extent of daily PDF signaling. It also seeks to identify key PDFR regulatory proteins. Aim 3 seeks to extend the scope of our work beyond the circadian pacemaker network to identify downstream circuit elements: we will focus on subsets of neurons in the Central Complex for which preliminary evidence suggests an involvement in daily rhythmic physiology associated with locomotion. Jet-lag, shift-work and disturbances in sleep-activity cycles all contribute to degrade mental and physical well-being. Two major causes of death (stroke and cardiac arrest) display clear time- of-day variation, yet, we have little understanding of the causal links between circadian clock functions and disease mechanisms. This research program is dedicated to a better understanding of fundamental circadian output mechanisms.