Neural Circuitry in the Developing Zebrafish Spinal Cord

描述(申请人提供)：斑马鱼胚胎在其存在的最初24小时内从单细胞发育到具有正常神经系统的生物体。受精后24小时，胚胎具有机械感觉介导的反射运动行为，96小时时，鱼是一种自由游泳的幼体。这些行为的表现是因为在发育中的鱼的脊髓和后脑中建立了一个简单但有效的神经网络。这个简单的神经系统为生物体成熟过程中的进一步扩张提供了支架。阐明斑马鱼早期神经系统形成的机制可能会对我们理解人类出现多少神经系统疾病产生巨大影响。胚胎/幼体斑马鱼的特性使其非常适合研究神经网络的形成--它在外部受精，在发育过程中是透明的，很容易采用反义治疗和转基因等分子技术。使用染料标记的解剖学研究已经获得了关于这些早期时间点的脊髓神经元的形态信息，表明在24-96HPF之间，脊髓的每半段存在大约11-15个神经元。此外，这些研究还为神经元如何相互连接形成网络提供了一些见解。然而，关于这些神经元之间是如何相互通信的，几乎一无所知。这项建议的目的是创造转基因鱼类品系，使阐明发育中的脊髓的神经化学解剖成为可能。这些鱼不仅将提供关于各种脊髓和脊髓上通路的连通性的信息，而且它们还将提供标记物，用于利用共表达(例如，与钙指示剂)或电生理方法进行功能研究。此外，它们还可以为特定神经元类型提供标记，这些标记可用于正向遗传筛选，目的是识别这些细胞中决定表型或连接所需的基因。

DESCRIPTION (provided by applicant): A zebrafish embryo goes from a single-cell to an organism with a functioning nervous system within its first 24 hours of existence. By 24 hours post-fertilization, the embryo has mechanosensory-mediated reflex motor behaviors and by 96 hr the fish is a free-swimming larva. The manifestation of these behaviors is due to the establishment of a simple but effective neural network in the spinal cord and hindbrain of the developing fish. This simple nervous system provides the scaffolding for further expansion during maturation of the organism. Elucidating the mechanisms underlying early nervous system formation in the zebrafish could have a huge impact on our understanding of how many neurological disorders arise in man. The properties of the embryonic/larval zebrafish has made it uniquely suited to study the formation of a neural network- it's fertilized externally, transparent during its development and lends itself easily to molecular techniques such as anti-sense treatment and transgenesis. Anatomical studies using dye labeling have yielded information regarding the morphologies of spinal neurons during these early time points, suggesting the presence of some 11-15 neurons per hemi-segment of the cord between 24-96 HPF. In addition, these studies have provided some insight into how the neurons connect to one another to form a network. However, almost nothing is known about how these neurons communicate amongst each other. It is the purpose of this proposal to generate transgenic fish lines that will enable the elucidation of the neurochemical anatomy of the developing spinal cord. Not only will these fish provide information as to the connectivity of the various spinal and supraspinal pathways, but they will also provide markers for use in functional studies utilizing either co-expression (e.g., with calcium indicators) or electrophysiological approaches. In addition, they can provide markers for specific neuronal types that can be used in forward genetic screens aimed at identifying genes required for either phenotype or connectivity decisions in these cells