Optogenetic analysis of the developing spinal circuit in zebrafish

描述（由申请人提供）：在发育过程中，神经回路的形成依赖于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起着重要作用。然而，关于脊髓中的神经活动模式如何促进运动回路的成熟，我们所知甚少。揭示自发性活动对中枢模式发生器（CPG）成熟的影响，将为研究支持行走和游泳等基本行为的发育机制提供初步的见解。这一知识将为再生技术提供信息，通过识别类似发育过程的活动需求，寻求刺激新神经网络的生长和整合到脊髓中。脊髓自发活动的整体模式在脊椎动物模型（如大鼠和小鸡）中显示为运动样，在脊髓的同侧区域同步，左右交替。我们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，以及它们在功能性运动电路的形成中起什么作用。我们选择斑马鱼作为模型系统。为了实现我们的目标，我们将：1)表征斑马鱼脊髓自发活动的时空模式，2)识别介导这种活动协调的细胞类型，3)确定这些活动模式在CPG形成和基本行为产生中的作用。我们将充分利用斑马鱼的透明性和遗传可及性，应用遗传编码光学工具进行研究。我们将用基因编码的钙指示剂GCaMP监测特定细胞群中自发活动的时空模式。为了鉴定介导协调活动的细胞类型，我们将使用光驱动氯化物泵Halorhodopsin和光敏剂KillerRed对特定细胞类型进行急性和慢性光学损伤。最后，我们将改变光视紫红质和光门控阳离子通道的自发活动模式，并通过分析简单的行为来观察对CPG发展的影响。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Advances in regenerative medicine to treat spinal cord injuries and other CNS diseases depend on the effective integration of new axons and synapses into the existing neural network. This research plan aims to understand the role that neural activity plays in establishing a functional neural circuit during development. Knowledge of the steps taken during circuit formation in development could inform treatments that reiterate these processes for repair purposes.