Functional Mapping of the Suprachiasmatic Nucleus

项目总结/摘要 该建议旨在描绘视交叉上核（SCN）的电和分子多样性， 使用新型纳米线阵列为SCN神经元的受体表达亚型提供了新的证据， 单细胞同时记录1024次接触。SCN是世界上最强大和影响最深远的 大脑的网络位于腹侧下丘脑，SCN整合光输入，以保持24小时 节律告知体内几乎每个细胞和器官系统的一天中的时间功能和转录。 由于其离散的结构、稳健的分子和电节律以及可量化的行为输出， SCN是一种理想的网络，可以用来研究神经网络的基本机制。的 本提案的目标是了解SCN在单细胞和整个网络水平上的功能， 开发了一种技术，可以提供长期的电生理记录。在辅导阶段，第一个 实验将证明使用纳米线阵列在分散的SCN中进行长期的细胞内样记录 初级神经元，具有昼夜节律的放电模式。我将确定发射特征，周期，和肽 在具有1024个触点的阵列上的单个电池的响应性。然后，我们将研究 破坏了这些神经元的分子钟。最后，我们将收获这些单个SCN 神经元进行RNA测序，将发射特性与转录组相关联，并与已发表的 数据集。这些实验的目的是表征个体的功能和分子特性， SCN神经元，并获得SCN培养，转录组学和大规模电生理学分析的培训。 在独立阶段，实验将使用纳米线阵列在SCN切片中进行长期记录 从野生型和转基因动物。SCN的转录网络保持在切片制备物中， 并且具有独特的分子钟时空模式;不知道是否发射遵循类似的模式。我 我将使用这些阵列来创建SCN神经元放电类型、相位和属性的功能图，并说明 使用光遗传学的受体表达群体沉默的效果。我们将继续这一做法， 来自突变小鼠的SCN切片，在受体表达神经元中有条件的时钟中断， 描述这些动物的行为。这些实验的目的是了解相位， 这些神经元之间的网络关系，使用电生理数据，以及如何离散受体- 表达群体有助于SCN网络和行为。最终，这些研究的数据 将一起用于询问单个SCN神经元的转录组如何通知其放电模式 在SCN网络内部和外部，以及这些单独的振荡器如何联合起来形成SCN网络。 总之，这些实验将促进我们对神经网络和细胞组成的理解。 这个关键的大脑区域

Project Summary/Abstract This proposal aims to delineate the electrical and molecular diversity of the suprachiasmatic nucleus (SCN) and provide new evidence for receptor-expressing subtypes of SCN neurons using novel nanowire arrays that allow single cell recording at 1024 contacts simultaneously. The SCN is among the most robust and far-reaching networks of the brain. Located in the ventral hypothalamus, the SCN integrates light input to keep a 24-hour rhythm that informs time-of-day function and transcription in nearly every cell and organ system in the body. Because of its discrete structure, robust molecular and electrical rhythm, and quantifiable behavioral output, the SCN is an ideal network to study to understand the fundamental mechanisms in neural networks overall. The goal of this proposal is to understand SCN function at a single-cell and whole-network level, using newly developed technology that affords long-term electrophysiological recordings. In the mentored phase, the first experiments will demonstrate use nanowire arrays for long-term, intracellular-like recordings in dispersed SCN primary neurons, which have a circadian firing pattern. I will determine firing characteristics, period, and peptide responsiveness of the individual cells on the array with 1024 contacts. Then, we will examine the effect of disruption of the molecular clock on firing patterns in these neurons. Finally, we will harvest these single SCN neurons for RNA sequencing to associate firing properties with the transcriptome, and compare to published datasets. The goal of these experiments is to characterize the functional and molecular properties of individual SCN neurons, and to gain training in SCN culture, transcriptomics, and large-scale electrophysiology analysis. In the independent phase, the experiments will use the nanowire arrays for long-term recordings in SCN slices from wildtype and transgenic animals. The transcriptional network of the SCN is maintained in slice preparations, and has a unique spatiotemporal pattern of the molecular clock; it is unknown if firing follows a similar pattern. I will use the arrays to create a functional map of SCN neuron firing types, phases, and properties, and illustrate the effect of receptor-expressing population silencing using optogenetics. We will continue this approach with SCN slices from mutant mice with conditional clock disruptions in receptor-expressing neurons, and ultimately characterize the behavior of these animals. The goal of these experiments is to understand the phase and network relationship among these neurons using electrophysiological data, and how discrete receptor- expressing populations contribute to the SCN network and behavior. Ultimately, the data from these studies would be used together to interrogate how the transcriptome of a single SCN neuron informs its firing patterns both within and outside of the SCN network, and how these individual oscillators unite to form the SCN network. Together, these experiments will advance our understanding of neural networks and the cellular composition of this critical brain region.