Functional Development of Premotor Neurons That Mediate the Vestibulo-Ocular Reflex

摘要 大脑的前庭神经元将外周平衡感觉转化为反射性命令，稳定姿势、步态和凝视。在这里，我们提出了一系列实验来测试流行的模型，即发育中的前庭神经元如何将感觉信息适当地传递到特定的运动神经元，从而实现凝视稳定。我们的建议有三个目标，每个目标都针对一个关于中枢神经元发育的特定假说： 目前该领域的模型提出，负责垂直/扭转前庭眼反射的中央前庭神经元通过对连接到目标肌肉的眼外运动神经元分泌的提示做出反应，采用两种命运中的一种。相反，我们的初步数据表明，前庭神经元的命运可能不依赖于这些信号。我们建议利用斑马鱼制剂的光学可及性、快速的外部开发和分子可处理性。为了在活体内直接量化中央前庭神经元的时空发育，我们将使用我们之前优化过的运动神经元出生测定技术。我们将在正常情况下，以及随后的动眼神经和滑车神经的光学损伤下这样做。 其次，目前的前庭发育模型表明，半规管在决定中央前庭神经元的感觉选择性方面起着至关重要的作用。然而，我们在斑马鱼幼体上的工作支持了这样的观点，即对于正常的垂直/扭转VOR来说，管道输入是必不可少的，这表明感觉选择性的发展与管道输入无关。为了确定前庭神经元的调节，我们将使用我们开发的一种新的电生理制剂直接测量发育中的前庭神经元的感觉选择性。我们将为完整的斑马鱼提供前庭刺激(平移)，同时从中央前庭神经元细胞内记录。同样，我们将测量兴奋性和抑制性突触输入对前庭神经元的反应，以确定上游前庭信号如何塑造其目标的反应。 最后，大多数脊椎动物前庭神经元接受来自两个末端器官的输入，即耳石和半规管。斑马鱼幼体的功能性半规管输入的延迟出现提供了一个独特的机会来确定是否需要感觉活动来建立适当的连接。我们将首先测量斑马鱼发育过程中神经管传入神经元的电生理特性，以确定电活动的出现模式。下一步，我们将确定在发育过程中，耳管和耳石传入神经何时适当会合。我们将通过首先在单通道传入中表达一个光敏通道来实现这一点。然后我们将从中央前庭神经元进行记录，定义它们的感觉调谐。然后我们将定义特定的通道传入之间的连通性概率，每个通道 调谐到特定的旋转轴，具有类似和垂直调谐的中央前庭神经元。 总而言之，这些实验的影响将是确定负责凝视稳定的前庭神经元何时以及如何出现解剖特化和感觉选择性。这些数据是评估和治疗发育异常以及改善急性中枢紊乱(如中风后)的先决条件。

Abstract The brain's vestibular neurons transform peripheral balance sensations into reflexive commands, stabilizing posture, gait, and gaze. Here we propose a series of experiments to test prevailing models of how developing central vestibular neurons come to properly relay sensory information to particular motoneurons, enabling gaze stabilization. Our proposal has three aims, each addressing a specific hypothesis about central neuron development: The current model in the field proposes that central vestibular neurons responsible for the vertical/torsional vestibuloocular reflex adopt one of two fates by responding to cues secreted from extraocular motoneurons that have wired to their target muscles. Our preliminary data suggests instead that vestibular neuron fate may instead proceed independently of such signals. We propose to leverage the optical accessibility, rapid external development, and molecular tractability of the zebrafish preparation. To directly quantify, in vivo, the spatiotemporal development of central vestibular neurons, we will use a birthdating technique we have previously optimized for motoneurons. We will do so both under normal conditions, as well as following optical lesions of oculomotor and trochlear nerves. Next, the current model of vestibular development proposes a vital role for the semicircular canals in determining the sensory selectivity of central vestibular neurons. However, our work in the larval zebrafish supports the idea that canal input is dispensable for a normal vertical/torsional VOR, suggesting the development of sensory selectivity is independent of canal in put. To define vestibular neuron tuning, we will directly measure the sensory selectivity of developing vestibular neurons using a novel electrophysiological preparation we have developed. We will provide vestibular stimuli (translation) to intact zebrafish while recording intracellularly from central vestibular neurons. Similarly, we will measure the response of the excitatory and inhibitory synaptic inputs to vestibular neurons to determine how the upstream vestibular signals shape the response of their target. Finally, most vertebrate vestibular neurons receive input from two end organs, the otoliths and the semicircular canals. The delayed emergence of functional semicircular canal input in the larval zebrafish provides a unique opportunity to determine whether or not sensory activity is required to establish proper connectivity. We will first measure the electrophysiological properties of canal afferent neurons as zebrafish develop to define emergent patterns of electrical activity. Next, we will determine when during development the canal and otolith afferents properly converge. We will do so by first expressing a light-sensitive channel in single canal afferents. We will then record from central vestibular neurons, defining their sensory tuning. We will then define the probability of connectivity between specific canal afferents, each tuned to a particular axis of rotation, with similarly and orthogonally tuned central vestibular neurons. Together, the impact of these experiments will be to define when and how anatomical specialization and sensory selectivity emerge in the vestibular neurons responsible for gaze stabilization. Such data are a prerequisite to evaluate and treat abnormal development, and to ameliorate acute central perturbations, such as follow stroke.