Optogenetic analysis of the developing spinal circuit in zebrafish

神经回路的形成依赖于发育过程中的活动依赖和独立机制。在感觉系统向神经系统提供输入之前，脊髓、视网膜和海马体等网络显示出自发的、有节奏的动作电位爆发。这种自发活动的时空模式已被证明在视觉系统的发展中发挥着重要作用。然而，关于脊髓中的神经活动模式如何有助于运动神经回路的成熟，人们知之甚少。揭示自发活动对中枢模式生成器(CPG)成熟的影响，将为支持行走和游泳等基本行为的发育机制提供初步见解。这一知识将为再生技术提供信息，这些技术寻求通过识别类似发育过程的活动需求来刺激新神经网络的生长和整合到脊髓中。 在大鼠和小鸡等脊椎动物模型中，脊髓自发活动的整体模式被证明是类似运动的，在脊髓的同侧区域和左右交替同步。我们的研究计划的目标是确定这些自发活动的协调模式是如何获得的，以及它们在形成功能性运动神经回路中所起的作用。我们选择了斑马鱼作为一个模型系统。为了实现我们的目标，我们将：1)描述斑马鱼脊髓自发活动的时空模式，2)确定调节这一活动协调的细胞类型，3)确定这些活动模式在CPG的形成和基本行为的产生中所起的作用。我们将充分利用斑马鱼的透明度和遗传可及性，并将遗传编码的光学工具应用于我们的研究。我们将使用遗传编码的钙指示剂GCaMP来监测特定细胞群体中自发活动的时空模式。为了确定介导协调活动的细胞类型，我们将使用光驱动的氯泵卤视紫质和光敏剂KillerRed来执行特定细胞类型的急性和慢性光学损伤。最后，我们将改变卤视紫质和光门控阳离子通道视紫红质的自发活动模式，并通过分析简单行为来观察对CPG发展的影响。

The formation of neural circuits relies on activity-dependent and independent mechanisms during development. Before sensory systems provide input to the nervous system, networks such as the spinal cord, retina and hippocampus display spontaneous, rhythmic bursts of action potentials. The spatiotemporal patterns of this spontaneous activity have been shown to play a significant role in the development of the visual system. Little is known, however, about how patterns of neural activity in the spinal cord contribute to the maturation of motor circuitry. Uncovering the influence of spontaneous activity on the maturation of the central pattern generator (CPG) would provide elementary insights into developmental mechanisms supporting basic behaviors like walking and swimming. This knowledge would inform regenerative technologies that seek to stimulate the growth and integration of new neural networks into the spinal cord by identifying the activity requirements for analogous developmental processes. Global patterns of spontaneous activity in the spinal cord have been shown to be locomotor-like in vertebrate models such as the rat and chick, with synchronization in ipsilateral regions of the spinal cord and alternation left and right. The goal of our research plan is to establish how these coordinated patterns of spontaneous activity are acquired and what role they play in the formation of a functional motor circuit. We have chosen the zebrafish as a model system. To accomplish our goals, we will: 1) characterize the spatiotemporal patterns of spontaneous activity in the zebrafish spinal cord, 2) identify the cell types that mediate the coordination of this activity, and 3) determine the role that these patterns of activity play in the formation of the CPG and the generation of basic behaviors. We will take full advantage of the transparency and genetic accessibility of the zebrafish and apply genetically-encoded optical tools for our study. We will monitor the spatiotemporal patterns of spontaneous activity in defined cell populations with the genetically-encoded calcium indicator GCaMP. To identify cell types mediating coordinated activity, we will use the light-driven chloride pump Halorhodopsin and the photosensitizer KillerRed to perform acute and chronic optical lesions of defined cell types. Finally, we will alter the patterns of spontaneous activity with Halorhodopsin and the light-gated cation channel Channelrhodopsin and observe consequences on the development of the CPG by assaying simple behaviors.