Activity-dependent modeling of developing dendrites in a retinal neuron

格兰特：视网膜神经元树突发育的活动依赖性建模 神经元通常以其树突状的形状为特征。了解它们在体内发育过程中如何获得形态是神经生物学的一个关键目标。位置和精确的分支模式不仅控制了它们通过电路布线接收的突触输入的类型，而且还决定了如何处理信号并将其转化为尖峰活动。编码视网膜中向上运动的方向选择性神经节细胞（DSGCs）提供了形式和功能之间的这种关系的一个引人注目的例子，因为它们的整个树突树指向它们编码的方向（Trenholm等人，2011年）。这个项目的总体目标是了解如何在发展过程中建立不对称的树枝状乔木。 在中枢神经系统的不同部位，神经元以不同的方式建立它们的不对称树突。或者，它们可以通过向突触前伙伴定向生长而变得不对称。或者，神经元可能首先建立无定形的树枝状分支，树枝状分支指向随机方向，随后成熟为高度定向的乔木。区分这些可能性是具有挑战性的，因为给定大脑区域中的神经元由各种亚型组成，当它们的树突尚未获得其特征形态时，这些亚型在不成熟状态下特别难以区分。为此，我们最近确定了一种转基因小鼠系，其中GFP选择性地在不对称DSGCs中表达，使我们能够通过早期发育跟踪这些神经元。使用传统的解剖学方法，我们将首先表征与突触前伙伴（双极和星爆无长突细胞）的树突状细胞生长的发展，以确定DSGCs获得其不对称树突状乔木的步骤。 虽然已知树突结构的适当发育和建立由遗传程序指定（Jan和Jan，2003），但其可以在很大程度上由活性依赖性过程修饰（麦卡利斯特et al.，1997年，Nedivi等人，1998，Rajan和Cline，1998，Wu等人，1999年，Polleux等人，2000年，Sin等人，2002年，Grueber等人，2003年，哈斯等人，2006年，Jinushi-Nakao等人，2007，Jan和Jan，2010）。在发育中的视网膜中，自发活动（不依赖于光）由NMDA和乙酰胆碱受体介导，已被假定为细化树突动脉化。在第二个目标中，我们将使用Cre-lox策略来特异性地干扰向上编码的DSGCs中的活性。我们将研究通过NMDA和乙酰胆碱受体介导的Ca信号的作用，使用遗传策略敲除NMDA受体或沉默胆碱能神经元来抑制乙酰胆碱的释放。相反，我们将使用DSGCs的光遗传学刺激（使用通道视紫红质的选择性表达）来增加活性水平。这样的操作将使我们能够评估活动对DSGCs在其天然环境中树突状树发育的影响。 最近在透明的斑马鱼和蝌蚪的个别神经元的体内时间推移成像揭示了一个特殊的运动和营业额的树突状分支。在本补助金的第三个目标中，我们将在不同的发展阶段对DSGC树突的动态行为进行成像，并研究活动依赖性过程（在目标2中建立）如何影响哺乳动物神经系统树突的动态重建。

Grant: Activity-dependent modeling of developing dendrites of a retinal neuron Neurons are often characterized by the shape of their dendritic arbours. Understanding how they acquire their morphology during development in vivo is a key goal in neurobiology. The location and precise branching patterns not only controls the type of synaptic input they receive by virtue of the circuit wiring, but also critically determines how the signals are processed and translated into spike activity. Directionally selective ganglion cells (DSGCs)that code upward motion in the retina, offer a striking example of this relationship between form and function, as their entire dendritic tree points in the direction they code (Trenholm, et al., 2011). The overall aim of this project is to understand how asymmetric dendritic arbors are established during development. In different parts of the CNS, neurons establish their asymmetric dendritic arbors in distinct ways. Either, they can become asymmetric by directed outgrowth towards their presynaptic partners. Alternatively, neurons may first establish amorphous arborisations with dendritic branches pointing in random directions and subsequently mature into a highly oriented arbor. To distinguish these possibilities is challenging because neurons in a given brain area consist of a variety of subtypes which are especially hard to distinguish in the immature state when their dendrites have not acquired their characteristic morphologies. To this end, we recently identified a transgenic mouse line in which GFP is expressed selectively in asymmetric DSGCs allowing us to follow these neurons through early development. Using a conventional anatomical approach we will first characterize the development of dendritic growth in relation with presynaptic partners (bipolar and starburst amacrine cells), to determine the steps by which DSGCs acquire their asymmetric dendritic arbors. While the proper development and establishment of dendritic structure is known to specified by genetic programs (Jan and Jan, 2003), it can modified to a large extent by activity-dependent processes (McAllister et al.,1997, Nedivi et al., 1998, Rajan and Cline, 1998, Wu et al., 1999, Polleux et al., 2000, Sin et al., 2002,Grueber et al., 2003, Haas et al., 2006, Jinushi-Nakao et al., 2007, Jan and Jan, 2010)mediated by Ca signaling. In the developing retina, spontaneous activity (independent of light) is mediated by NMDA and acetylcholine receptors, has been postulated to refine dendritic arboursizations. In the second aim, we will use Cre-lox strategies to specifically perturb activity in upward coding DSGCs. We will examine the role of Ca signals mediated through NMDA and acetylcholine receptors using genetic strategies to either knock out NMDA receptors or silence cholinergic neurons to inhibit release of acetlycholine. Conversely, we will use optogenetic stimulation of DSGCs (using selective expression of channelrhodopsin) to increase activity levels. Such manipulations will allow us to assess the impact of activity on the development of the dendritic trees of DSGCs in their native environment. Recent in vivo time lapse imaging of individual neurons in transparent zebra fish and tadpoles have revealed an exceptional amount of motility and turnover of dendritic branches. In the third aim of this grant we will image the dynamic behavior of DSGC dendrites during different stages of development and examine how an activity-dependent process (established in Aim 2) affects dynamic re-modeling of dendrites in the mammalian nervous system.