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: 8293110
• 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/8293110
• 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.

描述（由申请人提供）：现有的用于监测清醒的行为脊椎动物的神经活动的技术是侵入性的，并且通常需要限制动物。在这里，我们提出了使用生物发光非侵入性监测活动的遗传指定的神经元在自由行为的斑马鱼。将发光蛋白GFP-apoAequorin（Ga）在斑马鱼幼鱼神经元中表达，并与其底物腔肠素（CLZN）在体内构建成钙敏感的生物发光传感器GFP-Aequorin（GA）。然后，发光闪烁将报告靶向神经元中的自发和诱发的Ca 2+信号。这些“神经发光”反应可以用大面积光子计数探测器记录，同时用红外敏感相机监测行为。初步研究表明，转基因，泛神经GA鱼产生大而快的神经发光信号，可以连续记录许多天。这些光信号与神经元潜在的电活动的关系将通过靶向膜片钳记录到在泛神经元启动子下表达蛋白质的单个神经元来探索。为了探索这种技术的局限性和敏感性，GA将专门针对下丘脑的下丘脑泌素阳性神经元、中缝核的多巴胺能神经元和腹侧下丘脑的多巴胺能神经元。为了克服现有的生物发光监测策略，这需要一个完全黑暗的环境中的一个主要限制，我们提出了一个扩展这种方法的快速时间门控，能够计数单光子在正常的照明条件下。因此，该测定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期间神经元的小子集的活动。我们相信，GA报告神经活动的快速、稳定的特性沿着非成像检测策略可以提供有用的、容易实现的工具，用于监测在自然行为期间遗传指定的细胞类型的活动;是目前正在追求的其他更具技术挑战性的成像方法的有吸引力的替代方案。