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: 8692038
• 负责人: Julia Anna Kaltschmidt
• 金额: $ 36.04万
• 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 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8692038
• 关键词: Adhesives; Adopted; Afferent Neurons; Binding; Biological Assay; Cell Adhesion; Cell Adhesion Molecules; Cells; Characteristics; Complex; Development; Disease; Embryo; Equilibrium; Foundations; Functional disorder; Genes; Genetic; Genetic Recombination; Glutamates; Goals; 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; public health relevance; 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）：局部中间神经元形成特定突触连接对于神经元信息的处理至关重要。然而，鲜为人知 关于调节中枢神经系统中神经元间连接的因素。我们的长期目标是了解控制神经元间回路形成的遗传机制。所提出的实验的目的是描述细胞内在因子及其下游效应器如何确定 GABA 能神经元间身份和电路连接。我们的分析重点是一个已鉴定和分子特征的脊髓 GABA 能抑制性中间神经元亚类，这些中间神经元在感觉传入末梢上形成直接的轴突接触，从而在突触前抑制它们。我们将测试以下假设：转录因子 Ptf1a 控制一类脊髓 GABA 能中间神经元的突触靶向和分化，并且 Ptf1a 的转录靶标 NrCAM 与 Contactin-5 和 Caspr4 一起形成粘附信号复合物，指导特定的突触连接。我们通过以下三个目标来检验我们的假设：#1）根据神经元前体中 Ptf1a 表达的时间来表征不同的 GABA 能中间神经元亚型； #2) 定义 Ptf1a 在指导 GABA 前中间神经元连接中的作用； #3) 评估 Ptf1a 效应基因 NrCAM 和潜在的 NrCAM 受体复合物 Contactin-5/CASPR4 在指定 GABApre 靶标选择中的作用。在第一个目标中，我们使用定时他莫昔芬注射来标记和表征单个表达 Ptf1a 的中间神经元。在第二个目标中，我们利用小鼠遗传学来评估 Ptf1a 对于 GABApre 突触的靶向和分化是否是必要和充分的。在第三个目标中，我们利用小鼠遗传学来扰乱细胞粘附信号，并通过突触前抑制的新型电生理学测定，从微观解剖学和功能上分析其后果。总而言之，所提出的实验将确定脊髓 GABA 能中间神经元身份和连接的哪些方面是由 Ptf1a 指导的，并将提出赋予特定突触连接的下游分子机制。我们提出的研究在技术和概念上都是创新的。从技术上讲，我们将新的小鼠品系与新颖的体内分子遗传学和电生理学分析相结合，以在体内完整的网络中操纵和功能表征脊髓 GABA 能回路。从概念上讲，我们将探讨内在转录因子信号 (Ptf1a) 的必要性和充分性，以确定特定的 GABA 能身份和连接性。我们提出的工作具有重要意义，因为我们将首次证明介导体内抑制性中枢回路突触特异性的转录机制，以及基于细胞粘附的信号传导在指导特定神经元间连接方面的新作用。我们的分析将有助于对神经元回路形成的基本科学理解，并将为旨在重建被人类疾病破坏的 GABA 能回路的再生疗法提供基础。