Functional Mapping of the Suprachiasmatic Nucleus

项目摘要/摘要 这一建议旨在描绘视交叉上核(SCN)和 使用新型纳米线阵列为SCN神经元的受体表达亚型提供新的证据 单电池同时记录1024个触点。SCN是最强大和影响最深远的 大脑的网络。位于下丘脑腹侧的SCN整合了光输入，以保持24小时的 人体内几乎每一个细胞和器官系统的时间功能和转录的节律。 由于其离散的结构、强大的分子和电子节律以及可量化的行为输出， SCN是研究全面了解神经网络基本机制的理想网络。这个 该方案的目标是在单小区和全网络层面上理解SCN功能，使用新的 开发了提供长期电生理记录的技术。在指导阶段，第一个 实验将演示使用纳米线阵列在分散的SCN中进行长期的细胞内类记录 初级神经元，有昼夜节律的放电模式。我将确定激发特征，周期和多肽 具有1024个触点的阵列上的单个电池的响应性。然后，我们将研究一下 分子时钟对这些神经元的放电模式的干扰。最后，我们将收获这些单一的SCN 神经元的RNA测序将放电特性与转录组联系起来，并与已发表的比较 数据集。这些实验的目的是描述个体的功能和分子特性 获得SCN培养、转录学和大规模电生理学分析方面的培训。 在独立阶段，实验将使用纳米线阵列在SCN切片上进行长期记录 来自野生型和转基因动物。SCN的转录网络在切片制备中保持不变， 并具有分子时钟的独特时空模式；目前尚不清楚发射是否遵循类似的模式。我 我将使用这些数组创建SCN神经元放电类型、阶段和属性的功能图，并说明 光遗传学在受体表达群体沉默中的作用。我们将继续采用这种方法 来自突变小鼠的SCN切片，其受体表达神经元的条件时钟中断，并最终 描述这些动物的行为。这些实验的目标是了解阶段和 这些神经元之间的网络关系使用电生理数据，以及如何离散的受体- 表达群体对SCN网络和行为做出了贡献。归根结底，这些研究的数据 一起用来询问单个SCN神经元的转录组是如何通知其放电模式的 在SCN网络内部和外部，以及这些单个振荡器如何结合在一起形成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 这一关键的大脑区域。

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.