Optogenetic analysis of the developing spinal circuit in zebrafish

斑马鱼发育中的脊髓回路的光遗传学分析

• 批准号: 8085732
• 负责人: Erica Warp
• 金额: $ 3.38万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8085732
• 关键词: Action Potentials; Acute; Animals; Axon; Behavior; Behavioral; Biological Assay; Biological Models; Biological Neural Networks; Calcium; Cations; Cells; Central Nervous System Diseases; Chloride Ion; Chlorides; Chronic; Development; Developmental Process; Electrophysiology (science); Event; Generations; Genetic; Goals; Growth; Halorhodopsins; Hippocampus (Brain); Hour; Image; Individual; Ion Channel; Ion Pumps; Ipsilateral; Knowledge; Left; Lesion; Light; Mediating; Modeling; Monitor; Motor; Motor Neurons; Nervous system structure; Optics; Pattern; Pharmacology; Photosensitizing Agents; Play; Population; Process; Pump; Rattus; Regenerative Medicine; Research; Retina; Retinal; Role; Side; Spinal; Spinal Cord; Spinal cord injury; Swimming; Synapses; System; Technology; Time; Visual system structure; Walking; Zebrafish; calcium indicator; cell type; central pattern generator; insight; kinematics; light gated; neural circuit; neural patterning; regenerative; relating to nervous system; repaired; sensory system; spatiotemporal; tool; vision development

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.

神经回路的形成依赖于发育过程中的活动依赖性和独立机制。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会表现出自发的、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育中发挥着重要作用。然而，人们对脊髓中的神经活动模式如何促进运动回路的成熟知之甚少。揭示自发活动对中枢模式发生器（CPG）成熟的影响将为支持步行和游泳等基本行为的发育机制提供基本见解。这些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已被证明是类似运动的，在脊髓的同侧区域具有同步性和左右交替性。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回路的形成中发挥什么作用。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）识别介导该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。我们将充分利用斑马鱼的透明度和遗传可及性，并应用基因编码光学工具进行我们的研究。我们将使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。为了识别介导协调活动的细胞类型，我们将使用光驱动的氯泵盐视紫红质和光敏剂 KillerRed 对特定细胞类型进行急性和慢性光学损伤。最后，我们将用盐视紫红质和光门控阳离子通道视紫红质改变自发活动的模式，并通过分析简单的行为来观察对 CPG 发育的影响。