Developmental Influences on the Functional Organization of the Vestibular System

描述(申请人提供)：我的长期目标是在一家顶级研究机构运营一个独立的实验室，在那里我们将使用斑马鱼幼体前庭系统作为模型，了解神经元群体如何协调它们的活动来产生行为。我推测，负责将一组神经元组装成一个功能电路的发育过程将限制该电路可以执行的计算。因此，当我们试图了解神经元群体的功能时，我们将从它们的历史和组织它们的分子水平的事件中学到很多东西。斑马鱼幼体的前庭系统是检验这一假设的理想场所。像其他脊椎动物一样，斑马鱼幼体使用一组高度保守的神经元，在面对外部干扰时强有力地稳定凝视和姿势。然而，与其他脊椎动物不同的是，它们的前庭系统由少量可通过遗传途径获得的神经元组成，每个神经元在整个发育过程中都是通过光学方式获得的。在博士培训接近尾声时，我开始对斑马鱼幼体作为一个模型系统感兴趣。我当时正在研究灵长类动物的眼球运动系统，很明显，由于涉及到大量神经元，我为解释重复眼球运动的可变性而产生的群体活动模型最终是无法检验的。我寻求一种更简单的系统，可以控制完整的神经元群体，结果发现它是哈佛大学Schier和Engert实验室的联合博士后。我重新开始，作为一名行为的分子遗传学家接受培训，努力了解一种特殊的多肽--下丘脑泌素--在调节唤醒过程中的作用。我创造了一个转基因的鱼类品系，可以让我标记表达下丘脑泌素受体的神经元，试图识别多肽的下游靶标，可能还有行为调节的位置。我的转基因系中标记的神经元的解剖表明，前庭系统是一种潜在的底物，我们与我的合作者一起开始测试这一假设。我们测量了正常斑马鱼的前庭眼球反射(VOR)，追踪了它的解剖底物，并通过消融和激活表明它们是行为所必需的和充分的。此外，我们通过基因过表达的亚克隆素表明，VOR是敏感的该肽。我们目前正在总结一系列电生理学实验，以测量下丘脑肌醇对中央前庭神经元的影响。在计划过渡到经营自己的实验室时，我试图回到我发现非人类灵长类动物难以解决的电路功能问题上。然而，很明显，作为Schier实验室的一员，我对发育生物学的小小品味深刻地塑造了我接近神经回路的方式：我发现，我经常想知道，不仅神经元的聚集体是如何协同工作的，还有是什么力量决定了它们作为功能回路成员的命运。很明显，斑马鱼的方法优势非常适合回答这样的问题，我试图框定这些优势，使我能够作为博士后继续学习和成长，同时为独立工作设定课程。在这项提议中，我从斑马鱼幼体的中央前庭回路的工作模型开始，该模型提出了两类神经元的功能，与高等脊椎动物中发现的神经元相匹配，足以解释我迄今观察到的所有行为和解剖。第一组实验旨在测试负责VOR的中央前庭神经元的网络级组织和计算模型。我提出了一系列实验，首先从解剖学上识别我的模型中的神经元类别，然后使用单个神经元的消融和激活来探索它们在种群中的功能角色。在这样做的过程中，我将直接测试更高级别的网络交互，到目前为止，这些交互只是理论上的假设。具体地说，我将测试类似的调优 神经元是冗余的(即单独足以产生行为，但不是必需的)，无论它们是协同作用(即每个必要的，但本身都不是充分的)，或者斑马鱼幼体VOR的功能结构是否反映了简单的线性求和。除了对种群水平的计算进行第一次直接测试之外，这些功能架构中的每一个都表明了某种发育安排，这是我第二个目标的主题：在发育过程中测量和操纵中央前庭系统，以揭示它的连接方式。中央前庭系统面临着一个深刻的发育困境：它必须将具有定向调谐信息的外周传入连接到适当的运动神经元池，以产生稳定的代偿反应。由于在活体内研究前庭系统的发育很困难，我们有关于这种双突触协调如何发生的假设，但没有模型系统来测试它们。我和一位合作者一起，创造了第二个斑马鱼转基因品系，使我能够对外周和中央前庭系统(大脑)中的每个神经元进行颜色编码。我建议用这条鱼来监测前庭系统是如何结合在一起的。为了测试跨突触协调的假设，我将有选择地损害神经元群体，以扰乱信息流，监测颜色编码轴突投射的稳定性，并测量我的扰动的行为结果。总之，这些实验将填补我们在理解负责VOR的神经元如何组装和功能方面的深刻空白。此外，他们将填补特定的技术和智力空白，为我成功地从博士后实习生过渡到独立研究员做好准备。

DESCRIPTION (provided by applicant): My long-term goal is to run an independent laboratory at a top-tier research institution, where we will use the larval zebrafish vestibular system as a model to understand how populations of neurons coordinate their activity to produce behavior. I postulate that the developmental processes responsible for assembling an aggregate of neurons into a functional circuit will constrain the computations the circuit can perform. Therefore, as we seek to understand how populations of neurons function, we stand to learn much from their history, and the molecular-level events that organize them. The larval zebrafish vestibular system is an ideal place to test this hypothesis. Like other vertebrates, the larval zebrafish uses a highly conserved set of neurons to robustly stabilize gaze and posture in the face of external perturbations. Unlike other vertebrates, though, their vestibular system is comprised of a small number of genetically accessible neurons, each one optically accessible throughout development. I became interested in the larval zebrafish as a model system towards the end of doctoral training. I was studying the primate oculomotor system, and it had become clear that because of the large number of neurons involved, the models of population activity I had generated to explain variability in repeated eye movements were ultimately untestable. I sought a simpler system that would allow control over complete populations of neurons, and found it as a joint post-doc in the Schier and Engert labs at Harvard. I began anew, training as a molecular geneticist of behavior, working to understand the role of a particular peptide, Hypocretin, in modulating arousal. I generated a transgenic line of fish that would let me label the neurons expressing the Hypocretin receptor, in an attempt to identify the downstream targets of the peptide, and possibly the locus of behavioral modulation. The anatomy of the neurons labeled in my transgenic line implicated the vestibular system as a potential substrate, and together with my collaborators, we set out to test this hypothesis. We measured the normal zebrafish vestibuloocular reflex (VOR), traced its anatomical substrates in my line, and showed by ablation and activation that they were necessary and sufficient for behavior. Further, we showed by genetic overexpression of Hypocretin that the VOR is sensitive to this peptide. We are currently concluding a series of electrophysiologcial experiments measuring the effects of Hypocretin on central vestibular neurons. In planning a transition to run a laboratory of my own, I sought to return to the questions of circuit function that I found intractable in non-human primates. However, it became clear that my small taste of developmental biology as a member of the Schier lab had profoundly shaped the way I approached neural circuits: I found that I often wondered not just how aggregates of neurons worked together, but about what forces shaped their fate as members of a functional circuit. It became apparent that the methodological advantages of the zebrafish were uniquely suited to answer such questions, and I sought to frame ones that would allow me to continue to learn and grow as a postdoc, while setting a course for independent work. In this proposal, I begin with a working model of the central vestibular circuit in the larval zebrafish, which proposes that two functional classes of neuron, matching those found in higher vertebrates, are sufficient to explain all of the behaviors and anatomy I had observed to date. The first set of experiments aims to test models of network-level organization and computations in the central vestibular neurons responsible for the VOR. I propose a series of experiments to first anatomically identify the classes of neurons within my model, and then to use ablations and activation of individual neurons to probe their functional roles within the population. In doing so, I will directly test for higher-level network interaction that are, to date, only theoretical propositions. Specifically, I will test whether similarly tuned neurons are redundant (i.e. individually sufficient to produce behavior, but not necessary), whether they act synergistically (i.e. each necessary, but none sufficient by itself), or whether the functional architecture of the larval zebrafish VOR reflects simple linear summation. Beyond making the first direct tests of population-level computations, each of these functional architectures suggests a certain developmental arrangement, which is the subject of my second Aim: To measure and manipulate the central vestibular system during development, to uncover the way it is wired. The central vestibular system faces a deep developmental quandary: it must link peripheral afferents with directionally tuned information to the appropriate pools of motoneurons that produce stabilizing compensatory responses. Because of the difficulty in studying development of the vestibular system in vivo, we have hypotheses of how this di-synaptic coordination might occur, but no model system in which to test them. Along with a collaborator, I have generated a second transgenic line of zebrafish that allows me to color-code each neuron in the peripheral and central vestibular system (Brainbow). I propose to use this line of fish to monitor how the vestibular system comes together. To test hypotheses of trans-synaptic coordination, I will selectively lesion populations of neurons to disrupt information flow monitor the stability of color-coded axonal projections, and measure the behavioral results of my perturbations. Together, these experiments will fill deep gaps in our understanding of how the neurons responsible for the VOR assemble and function. Further, they will fill specific technical and intellectual gaps, preparing me to successfully transition from a postdoctoral trainee to an independent investigator.