Development and Plasticity of a Retinotectal System

描述（由申请人提供）：非洲爪蟾的视网膜顶盖系统被用作研究神经系统中地形有序投射发育的模型系统。使用在体电生理记录和光学成像技术，连同细胞信号的分子操作，我们建议检查的作用，细胞表面结合的轴突导向分子ephrin-B1和EphB 2在调节视网膜神经节细胞（RGC）的轴突后，视网膜顶盖突触的成熟和可塑性的功能突触接触，其目标顶盖细胞。建议的研究是基于我们的初步发现，在RGC的ephrin-B1反向信号的扰动影响突触功能的成熟，RGC轴突乔木的动力学，和活动诱导的长时程增强（LTP）的视网膜顶盖突触的幅度。在本项目中，我们建议进一步研究ephrin-B1反向信号和EphB 2正向信号在调节视网膜顶盖连接的成熟和可塑性方面的贡献，并确定这种调节如何有助于顶盖细胞感受野（RF）特性的发育和视觉体验驱动的细化。将通过单独或组合使用三种不同的方法进行肝配蛋白-B1和EphB 2信号传导的选择性操作，以剖析肝配蛋白-B1相对于EphB 2信号传导的调节作用：（1）用胞外域融合蛋白EphB 2-Fc或肝配蛋白-B1-Fc急性灌注顶盖，（2）通过电穿孔在少数RGC或顶盖细胞中表达野生型或突变形式的Ephrin-B1或EphB 2，以及（3）在所有突触前RGCs或所有突触后顶盖细胞中这些蛋白质的整体四环素诱导的转基因表达。在目的1中，我们将研究ephrin-B1反向信号和EphB 2正向信号对突触功能成熟的相对贡献，确定ephrin-B1/EphB 2信号调制的突触前和突触后位点，并分别测试动力蛋白依赖性内吞和谷氨酸受体募集参与突触前和突触后调制。在目标2中，我们将研究ephrin-B1/EphB 2信号的调制LTP和长期抑郁症（LTD）的贡献，这种调制的突触后机制，以及ephrin-B1/EphB 2信号在调节RGC轴突和顶盖细胞树突的乔木动力学的作用。在目标3中，我们将检查肝配蛋白-B1/EphB 2信号传导是否有助于RF大小的发育减小、兴奋性和抑制性RF的渐进匹配以及视顶盖细胞对视觉刺激的反应中的视觉体验诱导的方向选择性。总之，这些体内研究提供了独特的机会，以解决一组重要的轴突导向分子在调节突触成熟和可塑性中的功能，并了解跨突触分子信号传导在神经回路的发育和经验驱动的完善中的作用。公共卫生相关性：大脑的发育关键取决于突触连接的及时成熟，这一过程既受到遗传程序编码的分子因子的调控，也受到感官体验触发的神经活动的调控。本项目的重点是一组重要的分子因子，ephrin-B1和EphB 2受体，在调节神经回路的成熟的功能。这些研究结果将有助于阐明正常神经回路发育的机制以及发育过程中神经回路畸形的潜在原因。

DESCRIPTION (provided by applicant): The retinotectal system of Xenopus laevis is used as a model system for studying the development of topographically ordered projections in the nervous system. Using in vivo electrophysiological recording and optical imaging techniques, together with molecular manipulations of cellular signaling, we propose to examine the role of cell surface-bound axon guidance molecules ephrin-B1 and EphB2 in regulating the maturation and plasticity of retinotectal synapses after the axons of retinal ganglion cells (RGCs) have made functional synaptic contacts with their targeted tectal cells. The proposed studies are based on our preliminary findings that perturbation of ephrin-B1 reverse signaling in the RGCs affects the maturation of synaptic functions, the dynamics of RGC axon arbors, and the magnitude of activity-induced long-term potentiation (LTP) of retinotectal synapses. In the present project, we propose to further examine the contribution of both ephrin-B1 reverse signaling and EphB2 forward signaling in regulating the maturation and plasticity of retinotectal connections and to determine how this regulation contributes to developmental and visual experience-driven refinement of the receptive field (RF) properties of tectal cells. Selective manipulations of ephrin-B1 and EphB2 signaling will be performed to dissect the modulatory effects of ephrin-B1 vs. EphB2 signaling, by using three different methods, either alone or in combination: (1) acute perfusion of the tectum with the ectodomain fusion protein EphB2-Fc or ephrin-B1-Fc, (2) expression of wild-type or mutated forms of Ephrin-B1 or EphB2 in a few RGCs or tectal cells by electroporation, and (3) global tetracycline-induced transgenic expression of these proteins in either all presynaptic RGCs or all postsynaptic tectal cells. In Aim 1, we will examine the relative contribution of ephrin-B1 reverse signaling and EphB2 forward signaling to the maturation of synaptic functions, identify the pre- and postsynaptic loci of modulation by ephrin-B1/EphB2 signaling, and test the involvement of dynamin-dependent endocytosis and glutamate receptor recruitment in pre- and postsynaptic modulation, respectively. In Aim 2, we will examine the contribution of ephrin-B1/EphB2 signaling to the modulation of LTP and long-term depression (LTD), the postsynaptic mechanisms underlying such modulation, and the role of ephrin-B1/EphB2 signaling in modulating the arbor dynamics of both RGC axons and tectal cell dendrites. In Aim 3, we will examine whether ephrin-B1/EphB2 signaling contributes to the developmental reduction of the RF size, the progressive matching of excitatory and inhibitory RFs, and visual experience- induced direction selectivity in the tectal cell responses to visual stimuli. Together, these in vivo studies offer unique opportunities to address the function of an important set of axon guidance molecules in regulating synapse maturation and plasticity, and to understand the role of trans-synaptic molecular signaling in developmental and experience-driven refinement of neural circuits. PUBLIC HEALTH RELEVANCE: Brain development depends critically on the timely maturation of synaptic connections, a process known to be regulated by both molecular factors coded by the genetic program and neural activities triggered by sensory experience. This project focuses on the function of an important set of molecular factors, ephrin-B1 and EphB2 receptor, in regulating the maturation of neural circuits. Results from the proposed studies will shed light on the mechanisms underlying normal neural circuit development and the potential causes of malformation of neural circuits during development.