Molecular Mechanisms Regulating Inhibitory Circuitry in the Spinal Cord

描述（由申请人提供）：局部中间神经元形成特异性突触连接对于神经元信息的处理至关重要。然而， 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。本实验的目的是描述细胞内因子及其下游效应子如何决定GABA能神经元间的身份和电路连接。我们的分析集中在一个确定的和分子特征的脊髓GABA能抑制性中间神经元，形成直接的轴-轴接触的感觉传入末梢，从而抑制他们突触前的亚类。我们将测试的假设，即转录因子Ptf 1a控制一类脊髓GABA能中间神经元的突触靶向和分化，以及Ptf 1a的转录靶点，NrCAM，有助于与Contactin-5和Caspr 4的粘附信号复合物，指导特定的突触连接。我们用以下三个目标来检验我们的假设：#1）基于Ptf 1a在神经元前体中表达的时间来表征不同的GABA能中间神经元亚型; #2）定义Ptf 1a在指导GABApre中间神经元的连接中的作用;以及#3）评估Ptf 1a效应基因NrCAM和潜在的NrCAM受体复合物Contactin-5/CASPR 4在指定GABApre靶选择中的作用。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Ptf 1a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Ptf 1a对于GABApre突触的靶向和分化是否是必要的和足够的。在第三个目标中，我们使用小鼠遗传学来干扰细胞粘附信号传导，并通过一种新的突触前抑制电生理检测，从微观解剖学和功能上分析其后果。两者合计，拟议的实验将确定哪些方面的脊髓GABA能神经元间的身份和连接的Ptf 1a的指导，并将建议一个下游的分子机制，其中特定的突触连接被赋予。我们提出的研究在技术和概念上都是创新的。从技术上讲，我们将联合收割机新的小鼠品系与新的体内分子遗传学和电生理学分析相结合，以操纵和功能性地表征体内完整网络中的脊髓GABA能回路。从概念上讲，我们将探索内在转录因子信号（Ptf 1a）用于确定特定GABA能身份和连接性的必要性和充分性。我们提出的工作是重要的，因为我们将证明-第一次-转录机制介导的突触特异性抑制性中枢电路在体内，和一个新的作用，细胞粘附为基础的信号在指导特定的神经元间的连接。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。

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.