Monitoring Neural Activity in Freely Behaving Zebrafish Larvae with Bioluminescen

用生物发光监测自由行为的斑马鱼幼虫的神经活动

• 批准号: 8145447
• 负责人: Florian Engert
• 金额: $ 32.42万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8145447
• 关键词: Aequorin; Animals; Area; Behavior; Behavior monitoring; Behavioral Paradigm; Binding; Biological Assay; Biological Models; Bioluminescence; Calcium; Calcium Signaling; Cells; Detection; Dopamine; Electrophysiology (science); Electroporation; Environment; Fishes; Fluorescence; Genes; Genetic; Hypothalamic structure; Image; Individual; Laboratories; Larva; Light; Lighting; Luminescent Proteins; Methods; Monitor; Neurons; Neurosciences; Noise; Optics; Photons; Pilot Projects; Plasmids; Population; Property; Proteins; Proxy; Reporting; Research; Resolution; Serotonin; Signal Transduction; Specific qualifier value; Specificity; Structure; System; Systems Development; Techniques; Technology; Testing; Time; Transgenic Organisms; Vertebrates; Visual; Zebrafish; apoaequorin; awake; base; cell type; coelenterazine; design; detector; dopaminergic neuron; genetic manipulation; hypocretin; in vivo; interest; luminescence; patch clamp; promoter; raphe nuclei; relating to nervous system; research study; response; sensor; tool

DESCRIPTION (provided by applicant): Existing techniques for monitoring neural activity in awake, behaving vertebrates are invasive and often require restraining the animal. Here we propose the use of bioluminescence to non- invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. The photoprotein GFP-apoAequorin (Ga) will be expressed in neurons of larval zebrafish and constituted in vivo with its substrate coelenterazine (CLZN) to form the Ca2+-sensitive bioluminescent sensor GFP-Aequorin (GA). Flashes of luminescence will then report spontaneous and evoked Ca2+ signals in the targeted neurons. These 'neuroluminescence' responses can be recorded with a large-area photon-counting detector while simultaneously monitoring behavior with an infrared-sensitive camera. Pilot studies have shown that transgenic, pan-neuronal GA fish produced large and fast neuroluminescent signals that could be recorded continuously for many days. The relationship of these light signals with the neurons underlying electrical activity will be explored by targeting patch-clamp recordings to individual neurons expressing the protein under pan-neuronal promoters. To explore the limits and the sensitivity of this technique, GA will specifically be targeted to the hypocretin-positive neurons of the hypothalamus, the serotonergic neurons of the raphe nuclei and the dopaminergic neurons of the ventral hypothalamus. To overcome a major limitation of existing bioluminescence monitoring strategies, which require a completely dark environment, we propose an extension of this method for fast temporal gating that is able to count single photons during normal lighting conditions. Thus, this assay will allow us to monitor, with high temporal resolution and stability, the activity of small subsets of neurons during unrestrained, visual behavior over a time period of many days. We believe that the fast, stable properties of GA's report of neural activity along with non- imaging detection strategies can provide a useful, easily implemented tool for monitoring the activity of genetically specified cell types during natural behavior; an attractive alternative to other more technically challenging imaging approaches currently being pursued. PUBLIC HEALTH RELEVANCE: The ability to record from genetically identified neurons in freely behaving animals is highly desirable in modern neuroscience. We propose here a technology based on bioluminescence that exploits the translucence of the larval zebrafish combined with its availability for genetic manipulation. Bioluminescent and calcium sensitive proteins can be targeted to specific neurons of interest and will report neural activity with high temporal resolution and stability, during unrestrained, visual behavior over a time period of many days.

描述(由申请人提供)：现有的监测清醒、行为脊椎动物神经活动的技术是侵入性的，通常需要约束动物。在这里，我们建议使用生物发光来非侵入性地监测自由行为斑马鱼中遗传特定神经元的活动。将光蛋白GFP-Aequorin(GA)在斑马鱼幼体神经元中表达，并在体内与底物腔肠净(CLZN)组成钙离子敏感的生物发光传感器GFP-Aequorin(GA)。然后，发光的闪光将报告目标神经元中自发的和诱发的钙信号。这些“神经发光”反应可以用大面积的光子计数探测器记录下来，同时用红外线敏感的相机监测行为。初步研究表明，转基因泛神经元GA FISH产生了大量快速的神经发光信号，可以连续记录很多天。通过将膜片钳记录定位于泛神经元启动子下表达该蛋白的单个神经元，将探索这些光信号与潜在电活动的神经元的关系。为了探索这项技术的局限性和敏感性，GA将专门针对下丘脑的下丘脑下丘脑的下丘脑阳性神经元、中缝核群的5-羟色胺能神经元和下丘脑腹侧的多巴胺能神经元。为了克服现有生物发光监测策略的一个主要局限性，即需要完全黑暗的环境，我们提出了一种扩展的快速时间门控方法，能够在正常照明条件下计数单光子。因此，这项测试将使我们能够以高时间分辨率和稳定性监测在许多天的时间段内不受限制的视觉行为中的小神经元亚群的活动。我们相信，GA的神经活动报告的快速、稳定的特性以及非成像检测策略可以提供一个有用的、易于实施的工具来监控自然行为期间遗传特定类型的细胞的活动；对于目前正在追求的其他更具技术挑战性的成像方法来说，这是一个有吸引力的替代方案。 与公共卫生相关：在现代神经科学中，从行为自由的动物身上通过遗传识别的神经元进行记录的能力是非常可取的。在这里，我们提出了一种基于生物发光的技术，该技术利用斑马鱼幼体的半透明特性，并结合其可用于遗传操作。生物发光和钙敏感蛋白可以针对感兴趣的特定神经元，并将在许多天的时间段内报告不受限制的视觉行为中具有高时间分辨率和稳定性的神经活动。