Development and Plasticity of a Retinotectal System

描述（由申请人提供）：非洲爪蟾的视网膜系统被用作研究神经系统中地形有序投影发展的模型系统。利用体内电生理记录和光学成像技术，结合细胞信号的分子操作，我们提出研究细胞表面结合的轴突引导分子ephrin-B1和EphB2在视网膜神经节细胞（RGCs）轴突与其靶顶细胞进行功能性突触接触后，在调节视网膜顶突突触成熟和可塑性中的作用。我们提出的研究是基于我们的初步发现，即RGC中ephrin-B1反向信号的扰动会影响突触功能的成熟、RGC轴突的动态以及活动诱导的视网膜顶突触的长期增强（LTP）的大小。在目前的项目中，我们建议进一步研究ephrin-B1反向信号和EphB2正向信号在调节视网膜顶端连接的成熟和可塑性中的作用，并确定这种调节如何有助于发育和视觉经验驱动的顶端细胞感受野（RF）特性的完善。通过使用三种不同的方法（单独或联合使用），将对ephrin-B1和EphB2信号传导进行选择性操作，以剖析ephrin-B1对EphB2信号传导的调节作用：(1)用外域融合蛋白EphB2- fc或Ephrin-B1 - fc急性灌注顶盖；(2)通过电穿孔在少数RGCs或顶盖细胞中表达野生型或突变型Ephrin-B1或EphB2；(3)四环素诱导这些蛋白在所有RGCs或所有突触后顶盖细胞中进行转基因表达。在Aim 1中，我们将研究ephrin-B1反向信号和EphB2正向信号对突触功能成熟的相对贡献，确定ephrin-B1/EphB2信号调节的突触前和突触后位点，并分别测试动力蛋白依赖性内吞作用和谷氨酸受体募集在突触前和突触后调节中的作用。在Aim 2中，我们将研究ephrin-B1/EphB2信号在LTP和长期抑制（LTD）的调节中的作用，这种调节的突触后机制，以及ephrin-B1/EphB2信号在调节RGC轴突和顶突细胞树突的树突动力学中的作用。在Aim 3中，我们将研究ephrin-B1/EphB2信号是否有助于RF大小的发育减少，兴奋性和抑制性RF的逐步匹配，以及视觉经验诱导的顶盖细胞对视觉刺激反应的方向选择性。总之，这些体内研究提供了独特的机会来解决一组重要的轴突引导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在发育和经验驱动的神经回路优化中的作用。公共卫生相关性：大脑发育关键取决于突触连接的及时成熟，这一过程已知由遗传程序编码的分子因子和由感觉体验触发的神经活动共同调节。本项目重点研究ephrin-B1和EphB2受体这组重要的分子因子在调节神经回路成熟中的作用。这些研究的结果将揭示神经回路正常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.