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 – 细胞表型分析：内在生理学和遗传特征 发育途径和神经亚型标记的鉴定已经对神经系统进行了回路研究 脊髓是研究神经网络如何控制行为的最容易处理的中枢神经系统之一 尽管现在存在标记主要中间神经元和运动神经元的通用框架。 使用Cre-小鼠系对腹侧脊髓内的群体进行观察，很明显，每个主要脊髓神经 从以下角度来看，群体实际上是许多异质细胞类型的复杂混合物 尽管有明确的证据表明这一点，但输入、输出、点火特性和分子遗传属性。 异质性，这些细胞特性之间的关系非常零散。 项目 3 旨在将细胞谱系（主要神经元身份）、与运动池的连接、内在放电相互关联 属性和分子遗传学定义细胞类型，以提供细胞身份的真实定义。 细胞特征的互连框架至关重要，因为它将允许建模来预测脊髓电路如何 调节运动的控制，它将作为扰乱神经元的基因实验的基础 函数来测试模型的预测。 这个 U19 脊髓回路团队竞争运动前中间神经元之间的异质性 如果这个假设是正确的，则与不同运动池介导的运动功能的复杂性成比例。 控制手腕的肌肉群将由更多样化的前运动中间神经元群体控制 与控制肘部的子集相比，因为这两者之间的运动自由度不同 有两种主要方法可用于定义中间神经元异质性：膜片钳。 电生理学以确定输入/输出关系，以及单细胞测序转录组学 (scRNAseq) 来定义分子异质性，这些方法将通过连接性和谱系进行锚定。 记录和测序已被 Cre 标记的细胞，以标记其起源谱系（即 V1、V2a、V2b、 V3）和逆行跨突触标记狂犬病以识别运动池连接。 细胞类型特异性内在放电模式和转录组的表征如何应用于 尤其是对神经科学和肢体运动的更广泛理解？ 根据其独特的基因组合模式，群体将被分为许多额外的亚型 然而，我们的目标不是试图将主要的中间神经元组分成尽可能多的组。 尽可能的亚群，而是建立一组可用于可靠地 识别并从基因上扰乱具有已知放电模式和连接性的中间神经元子集。 包含有关细胞数量、连接性、突触强度、放电特性和“外科”分子的信息 扰乱神经亚型活动的工具可以创建颈椎回路模型并发挥功能 进行测试以了解如何调节前肢运动。