Molecular Mechanisms Regulating Inhibitory Circuitry in the Spinal Cord

调节脊髓抑制电路的分子机制

• 批准号: 9093872
• 负责人: Julia Anna Kaltschmidt
• 金额: $ 36.85万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9093872
• 关键词: Adhesives; Adopted; Afferent Neurons; Binding; Biological Assay; Cell Adhesion; Cell Adhesion Molecules; Cells; Characteristics; Complex; Development; Disease; Electrophysiology (science); Embryo; Equilibrium; Foundations; Functional disorder; Genes; Genetic; Genetic Recombination; Glutamates; Goals; Health; Individual; Injection of therapeutic agent; Interneuron function; Interneurons; Intrinsic factor; Knowledge; Label; Mediating; Medical; Mission; Molecular; Molecular Genetics; Molecular Profiling; Motor Neurons; Mus; Neuraxis; Neurodegenerative Disorders; Neurologic; Neurons; Neurotransmitters; Output; Pattern; Process; Proprioceptor; Public Health; Reporter; Research; Role; Schizophrenia; Science; Sensory; Signal Transduction; Specific qualifier value; Specificity; Spinal; Spinal Cord; Spinal cord injury; Synapses; Tamoxifen; Testing; Time; Work; base; cholinergic; contactin; human disease; in vivo; innovation; knowledge base; molecular marker; nervous system disorder; neural information processing; neuronal circuitry; neuropsychiatry; novel; presynaptic; promoter; protein expression; receptor; regenerative therapy; research study; synaptogenesis; time use; transcription factor

DESCRIPTION (provided by applicant): The formation of specific synaptic connections by local interneurons is critical for the processing of neuronal information. However, little is known about the factors that regulate interneuronal connectivity in the central nervous system. Our long-term goal is to understand the genetic mechanisms that control interneuronal circuit formation. The objective of the proposed experiments is to describe how a cell-intrinsic factor and its downstream effectors determine GABAergic interneuronal identity and circuit connectivity. We focus our analysis on an identified and molecularly characterized subclass of spinal GABAergic inhibitory interneurons that form direct axo-axonic contacts on sensory afferent terminals, thereby inhibiting them presynaptically. We will test the hypothesis that the transcription factor Ptf1a controls synaptic targeting and differentiation of a class of spinal GABAergic interneurons, and that a transcriptional target of Ptf1a, NrCAM, contributes with Contactin-5 and Caspr4 to an adhesive signaling complex that directs specific synaptic connectivity. We test our hypothesis with the following three aims: #1) Characterize distinct GABAergic interneuron subtypes based on the timing of Ptf1a expression in neuronal precursors; #2) Define the role of Ptf1a in directing connectivity of GABApre interneurons; and #3) Assess the role of the Ptf1a effector gene NrCAM and the potential NrCAM receptor complex Contactin-5/CASPR4 in specifying GABApre target selection. In the first aim, we use timed tamoxifen injections to label and characterize single Ptf1a-expressing interneurons. In the second aim, we use mouse genetics to assess whether Ptf1a is necessary and sufficient for the targeting and differentiation of GABApre synapses. In the third aim, we use mouse genetics to perturb cell adhesion signaling and we analyze the consequences of this both micro-anatomically and functionally, via a novel electrophysiological assay of presynaptic inhibition. Taken together, the proposed experiments will determine which aspects of spinal GABAergic interneuronal identity and connectivity are directed by Ptf1a, and will suggest a downstream molecular mechanism by which specific synaptic connectivity is conferred. Our proposed research is innovative both technically and conceptually. Technically, we will combine new mouse lines with novel in vivo molecular genetics and electrophysiological analyses to manipulate and functionally characterize spinal GABAergic circuits in an otherwise intact network in vivo. Conceptually, we will explore the necessity and sufficiency of an intrinsic transcription factor signal (Ptf1a) for determining specific GABAergic identity and connectivity. Our proposed work is significant in that we will demonstrate - for the first time - a transcriptionl mechanism mediating synaptic specificity of inhibitory central circuits in vivo, and a novel role for cell adhesion-based signaling in directing specific interneuronal connectivity. Our analysis will contribute to a basic scientific understanding of neuronal circuit formation and will provide foundation for regenerative therapies aimed at rebuilding GABAergic circuitry disrupted by human disease.

描述（由申请人提供）：局部中间神经元的特定突触连接形成对于处理神经元信息至关重要。但是，鲜为人知 关于调节中枢神经系统中神经元连通性的因素。我们的长期目标是了解控制神经元电路形成的遗传机制。提出的实验的目的是描述细胞中性因子及其下游效应子如何确定GABA能中的神经元身份和电路连接。我们将分析重点放在脊柱GABA能抑制性中间神经元的鉴定且分子表征的亚类上，该抑制性中间神经元在感觉传统末端形成直接的Axo轴突接触，从而在突触前抑制它们。我们将检验以下假设：转录因子PTF1A控制一类脊柱GABA能中间神经元的突触靶向和分化，并且PTF1A，NRCAM的转录靶标可对Contactin-5和CaspR4的粘附信号传导络合物有助于指导特定突触连接性。我们以以下三个目的检验我们的假设：＃1）根据神经元前体中PTF1A表达的时机来表征不同的GABA能中间的亚型； ＃2）定义PTF1A在指导Gabapre Internerons的连通性中的作用；和＃3）评估PTF1A效应子基因NRCAM和潜在的NRCAM受体复合物Contectin-5/CaspR4在指定Gabapre靶标的选择中的作用。在第一个目的中，我们使用定时的他莫昔芬注射来标记和表征单个PTF1A表达的中间神经元。在第二个目标中，我们使用小鼠遗传学来评估PTF1A是否需要且足以实现Gabapre突触的靶向和分化。在第三个目的中，我们使用小鼠遗传学来扰动细胞粘附信号传导，并通过突触前抑制作用的新型电生理测定法对微观和功能上的后果进行了分析。综上所述，提出的实验将确定PTF1A指导的脊柱GABA能的脊髓间神经元身份和连通性的哪些方面，并提出一种下游分子机制，通过赋予特定的突触连通性。我们提出的研究在技术上和概念上都是创新的。从技术上讲，我们将将新的小鼠系与新型的体内分子遗传学和电生理分析相结合，以操纵和功能表征体内原本完整的网络中的脊柱GABA能回路。从概念上讲，我们将探讨固有转录因子信号（PTF1A）的必要性和充分性，以确定特定的GABA能认同和连通性。我们提出的工作很重要，因为我们将首次证明一种转录​​机制，介导了体内抑制性中心回路的突触特异性，以及基于细胞粘附信号传导在指导特定特定神经元连接性中的新作用。我们的分析将有助于对神经元电路形成的基本科学理解，并将为旨在重建受人类疾病破坏的GABA能回路的再生疗法提供基础。