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: 10011920
• 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/10011920
• 关键词: Adult; 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 manipulation; genetic profiling; indexing; limb movement; motor behavior; neural network; 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.

项目3 -细胞表型：内在生理学和遗传特征 发育途径和神经元亚型标记的鉴定使对神经元的研究成为可能。 脊髓是研究神经网络如何控制行为的最易处理的CNS系统之一。 相关活动。虽然现在存在标记主要中间神经元和运动神经元的一般框架， 使用Cre-mouse线在腹侧脊髓内的群体，很明显，每个主要脊髓神经元 事实上，从细胞的角度来看，细胞群是许多异质细胞类型的复杂混合物。 输入、输出、点火特性和分子遗传属性。尽管有明确的证据表明 异质性，这些细胞特性之间的关系是非常零碎的。的目标 项目3是如何将细胞谱系（主要神经元身份），与运动池的连接，内在放电 分子遗传学定义细胞类型，以提供细胞身份的真实定义。这 细胞特征的相互连接的框架是至关重要的，因为它将允许建模来预测脊髓回路如何 调节运动的控制，它将作为遗传实验的基础， 函数，以测试模型的预测。 这个U19脊髓回路团队假设前运动中间神经元之间的异质性将 与不同运动池介导的运动功能的复杂性成比例。如果这个假设是正确的， 控制腕部的肌肉群将由更多样化的前运动中间神经元群控制 因为这两者之间的运动自由度不同 接头.有两种主要的方法，将用于定义中间神经元异质性：膜片钳 电生理学以定义输入/输出关系，以及单细胞测序转录组学 （scRNAseq）来定义分子异质性。这些方法将通过以下方式锚定到连接性和血统 记录和测序已被Cre标记以标记其起源谱系（即V1，V2 a，V2 b， V3）和逆行跨突触标记狂犬病，以确定汽车池连接。 细胞类型特异性内在放电模式和转录组的表征将如何应用于 尤其是对神经科学和肢体运动有更广泛的理解首先，每个主中间神经元 根据其独特的基因组合模式，该组将被分为许多其他亚型 表情然而，我们的目标并不是试图将主要的中间神经元群分裂成 尽可能的亚群，而是建立一套分子标志，可以用来可靠地 识别和遗传干扰具有已知放电模式和连通性的中间神经元的子集。只有 这些信息包括细胞数量、连接性、突触强度、放电特性和“外科”分子 干扰神经元亚型活动的工具可以创建颈椎回路的模型， 测试以了解前肢运动是如何调节的。