RP3: Cell Phenotyping: Intrinsic physiology and genetic characteristics

摘要：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物的识别已经对脑功能进行了电路研究。 脊髓是研究神经网络如何控制行为的最易处理的中枢神经系统之一 相关活动。虽然现在已经有了标记基数、中间神经元和运动神经元的通用框架 使用Cre-小鼠品系的腹侧脊髓内的群体，很明显，每个基本脊髓神经元 人口实际上是许多异质细胞类型的复杂混合体，从 输入、输出、激发特性和分子遗传属性。尽管有明确的证据证明这一点 异质性，这些细胞特性之间的关系是非常零散的。的目标是 项目3是如何将细胞谱系(主要神经元的特性)、运动池的连通性、内在放电 属性和分子遗传学定义了细胞类型，以提供对细胞身份的真实定义。这 细胞功能的互连框架至关重要，因为它将使建模能够预测脊髓电路如何 调节运动的控制，它将成为扰乱神经元的基因实验的基础 函数，以测试模型的预测。 这个U19脊髓回路研究小组假设运动前中间神经元之间的异质性将 根据不同运动池调节的运动功能的复杂性进行扩展。如果这个假设是正确的， 控制手腕的肌肉群将由更多样化的运动前中间神经元群控制 而不是控制肘部的子集，因为这两者的运动自由度不同 关节。有两种主要的方法可以用来定义神经元间异质性：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学 (ScRNAseq)来定义分子异质性。这些方法将通过以下方式与连接性和世系相联系 记录已经标记了Cre标签以标记其起源谱系(即V1、V2a、V2b， V3)和逆行突触标记狂犬病，以识别运动池连接。 细胞类型特定的内在激发模式和转录组的特征将如何应用于 对神经科学，特别是肢体运动有更广泛的了解？首先，每个主要的中间神经元 群体将根据其独特的基因组合模式分为许多额外的亚型 表情。然而，我们的目标不是试图将主要的中间神经元组细分成同样多的 更确切地说，它是为了建立一套可以可靠地用于 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亚群。它只是 关于细胞数量、连通性、突触强度、激发特性和“外科”分子的信息 扰乱神经元亚型活动的工具可以创建颈椎脊髓回路模型并在功能上 测试以了解前肢运动是如何调节的。

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.