Developmental Influences on the Functional Organization of the Vestibular System

描述（由申请人提供）：我的长期目标是在一家顶级研究机构建立一个独立的实验室，在那里我们将使用斑马鱼幼体前庭系统作为模型来了解神经元群体如何协调它们的活动来产生行为。我假设，负责将神经元集合成功能电路的发育过程将限制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以及组织它们的分子水平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，斑马鱼幼虫在面对外部扰动时使用一组高度保守的神经元来稳定凝视和姿势。然而，与其他脊椎动物不同的是，它们的前庭系统是由少数基因上可接近的神经元组成的，每个神经元在发育过程中都是光学上可接近的。我开始对幼体斑马鱼产生兴趣，并将其作为一种模型系统。我当时正在研究灵长类动物的动眼系统，很明显，由于涉及到大量的神经元，我用来解释重复眼球运动的可变性的群体活动模型最终是不可检验的。我寻找一种更简单的系统，可以控制整个神经元群，并在哈佛大学Schier和Engert实验室联合做博士后时找到了它。我重新开始，作为一名行为分子遗传学家接受培训，努力了解一种特殊肽下丘脑泌素（Hypocretin）在调节觉醒中的作用。我培育了一种转基因鱼类，让我可以标记表达下丘脑分泌素受体的神经元，试图确定肽的下游目标，以及可能的行为调节位点。在我的转基因系中标记的神经元的解剖结构暗示了前庭系统是一个潜在的基质，我和我的合作者一起，开始测试这个假设。我们测量了正常斑马鱼前庭反射（VOR），在我的线上追踪了它的解剖学基础，并通过消融和激活表明它们对行为是必要和充分的。此外，我们通过下丘脑分泌素的基因过表达表明，VOR对这种肽敏感。我们目前正在完成一系列测量下丘脑泌素对中央前庭神经元影响的电生理实验。在计划过渡到管理自己的实验室时，我试图回到我发现在非人类灵长类动物身上难以解决的电路功能问题上。然而，很明显，作为席尔实验室的一员，我对发育生物学的小小体验深刻地影响了我研究神经回路的方式：我发现，我经常想知道的不仅仅是神经元群是如何一起工作的，还有什么力量决定了它们作为功能回路成员的命运。很明显，斑马鱼在方法上的优势非常适合回答这些问题，我试图构建一些问题，让我在作为博士后继续学习和成长的同时，为独立工作设定路线。在这个提议中，我从斑马鱼幼体的前庭中枢回路的工作模型开始，该模型提出了两个功能级别的神经元，与在高等脊椎动物中发现的神经元相匹配，足以解释我迄今为止观察到的所有行为和解剖结构。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。我提出了一系列的实验，首先从解剖学上确定我的模型中的神经元类别，然后使用消融和激活单个神经元来探测它们在群体中的功能角色。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，这些交互还只是理论命题。具体来说，我将测试是否进行了类似的调整

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.