Molecular Mechanisms Regulating Inhibitory Circuitry in the Spinal Cord

建立特定的神经元回路是产生协调肌肉功能的基础。 然而，神经元间与其靶点之间连接的特异性背后的信号 到目前为止，哺乳动物的中枢神经系统(CNS)仍然很大程度上是未知的。我们的长期研究目标是 了解神经元间电路连接的规则和控制它的分子机制。的目标是 这项拟议的研究旨在描述粘附性信号和神经营养信号的组合如何决定 GABA能神经元间回路连接。一类GABA能中间神经元，称为GAABApre，形成突触 与本体感觉传入的终末接触，从而直接控制本体感觉 通过一种称为突触前抑制的抑制策略进行输入。我们将检验这一假设 一类脊髓GABA能神经元在功能不同的感觉神经元和 这种连接是通过(Ii)肌源性神经营养因子(NT)-3和(Ii)a的差异表达来调节的 IgSF黏附蛋白基质。这项建议背后的理由是，通过理解 GABA能中间神经元回路形成的机制，我们将增进我们对-和 最终控制体内过度抑制神经元回路的发育和功能。我们将检验我们的假设 目的如下：#1)确定GABA能神经元间回路的功能特异性；#2) 研究肌源性NT-3对GABA-Pre突触形成的影响；以及#3)评估 GABA前感觉突触形成中的Contactin-5。在第一个目标中，我们结合了定时他莫昔芬注射， 用小鼠遗传学和CTB标记分析个体GABApre的特异性连接性 中间神经元可能在功能上相关。在第二个目标中，我们检测了Gabrg1在 不同功能的本体感觉神经元并评估改变NT-3水平对两者的影响 Gabrg1的表达和GABApre的终端数。在第三个目标中，我们使用 小鼠遗传学和使用分子、显微解剖和功能分析进行表型分析， 包括突触前抑制的电生理测量。我们还使用了体外结合试验来 筛选与体内GABApre突触特异性相关的新的黏附分子候选。这项研究 在本申请中提出的是创新的，因为它结合了分子定义的神经元间电路和 独一无二的方法学组合，将功能特异性与目标信号相结合。这项研究 将提供对不同功能的GABApre电路的理解，并促进我们对 基本电路范例如何适应不同的运动功能。拟议的工作意义重大 因为它将有助于形成电路级机制和神经的基础知识 在中枢神经系统中使用的策略；这反过来将推动我们开发有效的治疗方法来重建电路 以及脊髓损伤或其他神经疾病后的肌肉功能。

Establishing specific neuronal circuits is fundamental for the generation of coordinated muscle function. However, the signals underlying the specificity of connection between interneurons and their targets in the mammalian central nervous system (CNS) remain largely unknown to date. Our long-term research goal is to understand the rules of interneuron circuit wiring and the molecular mechanisms that control it. The objective of the proposed research is to describe how a combination of adhesive and neurotrophic signals determine GABAergic interneuron circuit connectivity. A class of GABAergic interneurons, termed GABApre, forms synaptic contacts with the terminals of proprioceptive sensory afferents, and thus directly controls proprioceptive sensory input through an inhibitory strategy known as presynaptic inhibition. We will test the hypothesis that the connectivity of a class of spinal GABAergic neurons varies between functionally-distinct sensory neurons and that this connectivity is mediated via (ii) differential expression of muscle-derived neurotrophin (NT)-3 and (ii) a matrix of IgSF adhesion proteins. The rationale underlying this proposal is that through understanding the mechanisms underlying GABAergic interneuron circuit formation, we will enhance our understanding of — and ultimately control over — inhibitory neuronal circuit development and function in vivo. We will test our hypothesis with the following three aims: #1) Determine the functional specificity of GABAergic interneuron circuitry; #2) Investigate the influence of muscle-derived NT-3 on GABApre synapse formation; and #3) Assess the role of Contactin-5 in GABApre-sensory synapse formation. In the first aim, we combine timed tamoxifen injections, mouse genetics and CTb labeling to analyze whether the specific connectivity of individual GABApre interneurons may be functionally relevant. In the second aim, we examine the expression of Gabrg1 in functionally distinct proprioceptive sensory neurons and assess consequences of changing NT-3 levels on both Gabrg1 expression and GABApre terminal number. In the third aim, we perturb cell adhesion signaling using mouse genetics and perform phenotypic analysis using molecular, micro-anatomic and functional assays, including an electrophysiological measure of presynaptic inhibition. We also use an in vitro binding assay to screen for new adhesion molecule candidates relevant for GABApre synaptic specificity in vivo. The research proposed in this application is innovative because it combines a molecularly-defined interneuronal circuit with a unique constellation of methodologies to integrate functional specificity with target-derived signals. The research will provide an understanding of functional diverse GABApre circuitry and also advance our understanding of how basic circuit paradigms may be adapted for diverse motor functions. The proposed work is significant because it will contribute to fundamental knowledge of the formation of circuit-level mechanisms and neural strategies used in the CNS; this in turn will advance our efforts to develop effective therapies to rebuild circuitry and muscle function after spinal cord injury or other neurological diseases.