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定的突触连接对于神经元信息的处理至关重要。然而，人们对此知之甚少 关于调节中枢神经系统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。拟议实验的目的是描述细胞内在因素及其下游效应器如何决定GABA能神经元间特性和电路连接。我们的分析集中在一类识别的和分子特征的脊髓GABA能抑制中间神经元上，这些神经元在感觉传入终末形成直接的轴突接触，从而在突触前抑制它们。我们将检验这样一种假设，即转录因子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的转录靶点NrCAM与Contactin-5和Caspr4一起参与指导特定突触连接的粘连信号复合体。我们用以下三个目标来验证我们的假设：#1)根据Ptf1a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)确定Ptf1a在指导GABApre中间神经元连接中的作用；以及#3)评估Ptf1a效应基因NrCAM和潜在的NrCAM受体复合体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，我们使用小鼠遗传学来评估Ptf1a对于GABApre突触的靶向和分化是否必要和充分。在第三个目标中，我们利用小鼠的遗传学来干扰细胞黏附信号，并通过一种新的突触前抑制的电生理学方法，从微观解剖和功能两个方面分析了这种信号转导的后果。综上所述，拟议的实验将确定Ptf1a指导脊髓GABA能神经元间识别和连接的哪些方面，并将提出特定突触连接被授予的下游分子机制。我们提出的研究在技术和概念上都是创新的。从技术上讲，我们将把新的小鼠品系与新的体内分子遗传学和电生理分析相结合，以操纵和功能表征活体内原本完整的网络中的脊髓GABA能回路。从概念上讲，我们将探索内在转录因子信号(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，因为我们将首次展示一种转录机制，该机制介导体内抑制性中枢电路的突触特异性，以及基于细胞黏附的信号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。

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.