RP3: Cell Phenotyping: Intrinsic physiology and genetic characteristics

RP3：细胞表型：内在生理学和遗传特征

• 批准号: 9815389
• 负责人: SAMUEL L. PFAFF
• 金额: $ 70.9万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9815389
• 关键词: Adult; Behavior; Behavior Control; Bioinformatics; Biophysics; Categories; Cell Count; Cell Lineage; Cell Separation; Cells; Cervical; Characteristics; Code; Complex; Complex Mixtures; Data; Development; Elbow; Electric Capacitance; Electrophysiology (science); Forelimb; Foundations; Freedom; Gene Expression Profile; Genes; Genetic; Genetic Heterogeneity; Genetic Variation; Goals; Heterogeneity; Individual; Interneurons; Joints; Label; Link; Location; Maps; Mediating; Membrane; Methods; Modeling; Molecular; Molecular Genetics; Motor; Motor Neurons; Movement; Mus; Muscle; Neurons; Neurosciences; Operative Surgical Procedures; Output; Pathway interactions; Pattern; Phenotype; Physiological; Physiology; Population; Population Heterogeneity; Positioning Attribute; Process; Property; Rabies; Reporter; Resistance; Slice; Spinal; Spinal Cord; Subcellular Anatomy; Synapses; System; Testing; Wrist; base; biophysical properties; cell type; combinatorial; experimental study; genetic profiling; indexing; limb movement; neural network; off-label use; patch clamp; predictive modeling; protein expression; single cell sequencing; single-cell RNA sequencing; tool; transcription factor; transcriptome; transcriptomics

Summary: Project 3 – Cell Phenotyping: Intrinsic Physiology and Genetic Characteristics The identification of developmental pathways and neuronal subtype markers has made circuit studies of the spinal cord one of the most tractable CNS systems to investigate how neural networks control behaviorally relevant activity. Although a general framework now exists for labeling cardinal interneuron and motor neuron populations within the ventral spinal cord using Cre-mouse lines, it is apparent that each cardinal spinal neuron population is in fact a complex mixture of many heterogeneous cell types when viewed from the perspective of inputs, outputs, firing properties, and molecular-genetic attributes. Despite clear evidence for this heterogeneity, the relationship between each of these cellular properties is very fragmentary. The goal of Project 3 is to interrelate how cell lineage (cardinal neuron identity), connectivity to motor pools, intrinsic firing properties, and molecular genetics define cell types to provide a true definition of cell identity. This interconnected framework of cell features is critical because it will allow modeling to predict how spinal circuitry modulates the control of movement, and it will serve as the basis for genetic experiments that perturb neuronal function in order to test predictions of the model. This U19 Spinal Cord Circuit Team hypothesizes that the heterogeneity among premotor interneurons will scale with the complexity of motor functions mediated by different motor pools. If this hypothesis is correct, muscle groups controlling the wrist will be controlled by a more diverse population of premotor interneurons than the subset controlling the elbow because the degrees of freedom in movement differ between these two joints. There are two main approaches that will be employed to define interneuron heterogeneity: patch clamp electrophysiology in order to define input/output relationships, and single cell sequencing transcriptomics (scRNAseq) to define molecular heterogeneity. These methods will be anchored to connectivity and lineage by recording and sequencing cells that have been Cre-tagged to mark their lineage of origin (i.e. V1, V2a, V2b, V3) and retrograde trans-synaptically labeled with rabies to identify motor pool connectivity. How will the characterization of cell type-specific intrinsic firing patterns and transcriptome be applied to the broader understanding of neuroscience and limb movements in particular? First, each cardinal interneuron group will be divided into many additional subtypes based on their unique combinatorial patterns of gene expression. However, the goal is not to attempt to fractionate the cardinal interneuron groups into as many subpopulations as possible, rather it is to establish a set of molecular landmarks that can be used to reliably identify and genetically perturb subsets of interneurons with known firing patterns and connectivity. It is only with information about cell numbers, connectivity, synaptic strength, firing properties, and “surgical” molecular tools to perturb neuronal subtype activity can models of cervical spinal circuitry be created and functionally tested to understand how forelimb movements are regulated.

摘要：项目3 - 细胞表型：内在生理和遗传特征 发育途径和神经元亚型标记的鉴定已使回路研究 脊髓是研究神经网络如何在行为上控制的最容易理解的CNS系统之一 相关活动。尽管现在有一个通用框架用于标记红衣主教中间神经元和运动神经元 使用CRE小鼠线在腹侧脊髓内的种群，显然每个主要的脊柱神经元 实际上，当从 输入，输出，发射特性和分子遗传属性。尽管有明确的证据 异质性，这些细胞特性中的每一种之间的关系都是非常零散的。目标 项目3是为了相互关联细胞谱系（主要神经元身份），与电机池的连通性，内在的射击 性质，分子遗传学定义细胞类型，以提供细胞身份的真实定义。这 单元格特征的互连框架至关重要，因为它将允许建模能够预测脊柱电路 调节运动的控制，它将作为扰动神经元的基因实验的基础 功能以测试模型的预测。 这个U19脊髓电路团队假设，前神经元之间的异质性将 与不同电动机池介导的电机功能的复杂性相比。如果这个假设是正确的， 控制手腕的肌肉组将由更多潜水的前神经元的人群控制 比控制肘部的子集，因为这两个运动的自由度不同 关节。有两种主要的方法将被雇用以定义中间神经元异质性：斑块夹 电生理学以定义输入/输出关系和单细胞测序转录组学 （SCRNASEQ）定义分子异质性。这些方法将通过 记录和测序细胞已被标记为标记其原点谱系（即V1，V2A，V2B， v3）和逆行反射式突触，用狂犬病标记，以识别运动池连接性。 如何将特定细胞类型的内在触发模式和转录组的表征应用于 特别了解神经科学和肢体运动？首先，每个主要神经元 根据基因的独特组合模式，将组分为许多其他亚型 表达。但是，目标不是试图将红衣主教中间神经元组分为多数 亚群，而是建立一组可用于可靠的分子地标 识别具有已知触发模式和连接性的中间神经元中的和普遍的扰动子集。只是 有关细胞数量，连通性，突触强度，发射特性和“手术”分子的信息 扰动神经元亚型活性的工具可以创建宫颈脊柱回路的模型并在功能上 经过测试以了解如何调节前肢运动。