利用行為組學技術對斑馬魚與稻田魚進行種內與種間差異與環境污染之評價

Behavior Phenomic Approach in Assessing the Effects of Mutations, Pollutants, and Intraspecies and Interspecies Differences in Zebrafish and Medaka

陳良賢(Gilbert Audira)

指導教授 ： 蕭崇德

中原大學/理學院/化學研究所/博士(2021年)

https://doi.org/10.6840/cycu202100693

摘要

近年來，斑馬魚和稻田魚已被廣泛應用在行為神經科學研究。除了低成本，在毒理學和藥理學研究的有效性外，神經行為研究還提供了對人腦疾病複雜性的全面理解。行為 組學是研究行為表型，內源性或外源性因素的領域。實際上，學術界已經提出了表觀基因組學的概念，隊小鼠和斑馬魚在內的幾種動物進行神經行為學研究。但是，仍然沒有透過行為組學方法系統性的去量測斑馬魚或稻田魚的行為。主要困難包括每個實驗中各種不同參數的測量收集、以及實驗動物間較大的個體差異。本論文包括五個章節。第一章節是利用行為組學驗證瘦素基因的在斑馬魚的行為控制功能。通過多種新穎的水箱，鏡子啄食，天敵躲避，社交，群游，晝夜節律運動和顏色偏好分析，發現了瘦素基因剃除斑馬魚表現出過度活躍行為，這是一種焦慮發生表型，與快速游泳，減少凍結時間，減少對天敵動物的恐懼，放鬆群游圈以及晝夜節律的運動活動和顏色偏好調節異常。總之，通過執行行為組學和生化分析，我們首次證明了瘦素基因在調節斑馬魚行為中擔任了關鍵作用。接下來，第二和第三章節中，我分析了幾種廣泛使用的溶劑和奈米材料對斑馬魚行為的影響。在所有測試過的常用溶劑中，丙酮和PEG-400表現出更好的生物相容性和較低的毒性，因為它們引發了較少的行為和生化改變，而甲醇和DMSO在長期暴露於這些溶劑後可以引起斑馬魚的顯著的行為改變。同時，透過行為組學加上生物標誌物的定量量測，我也比較了石墨烯和氧化石墨烯兩種的奈米材料對斑馬魚行為毒理。在第四章節中，我比較了幾種不同斑馬魚品系的行為表現，包括AB，absolute，TL，golden，WIK與寵物商店購買（PET）野生種的種內行為表現差異。第五章節旨在調查四種同屬不同種稻田魚的行為和生物標誌物表達的差異，這四種稻田魚分別是Oryzias latipes，O. dancena，O. woworae和O. sinensis。結果表明每個測試稻田魚彼此之間都表現出明顯不同的行為，這可能與應激反映激素，神經傳遞質和氧化應激標記物的不同基礎水平有關。

關鍵字

斑馬魚 ； 稻田魚 ； 行為組學 ； 瘦素 ； 有機溶劑 ； 石墨烯 ； 氧化石墨烯 ； 種內差異 ； 種間差異

並列摘要

In recent years, a significant escalation in the use of adult zebrafish (Danio rerio) and medaka has been seen in behavioral neuroscience research. Besides its low cost, robustness, and effectiveness for toxicology and pharmacology researches, neurobehavioral study offers a comprehensive understanding of the complexity of human brain disorders. Behavioral phenomics is a field that studying behavioral phenotypes and their regulation by various endogenous, or exogenous factors. Actually, the idea of phenomic has been proposed to facilitate neurobehavioral study in several animals, including mice and zebrafish larvae. However, there was still no study that focused on adult zebrafish behavior with the phenomics approach. This problem might be caused by some common difficulties in this study, including the measurements that were various and different in each experiment. Therefore, these reasons became the main concerns to develop a robust and reliable system to examine zebrafish behavior in a previous study and propose that the phenomic approach conducted in these studies can be used for future experimental standardization. The works included in this dissertation cover five studies. The first study was focused on validating the potential function of leptin gene especially on adult zebrafish’s behavior. By multiple behavior endpoint analyses, including novel tank, mirror biting, predator avoidance, social interaction, shoaling, circadian rhythm locomotor activity, and color preference assay, the lepa KO fish was found to display a hyperactivity behavior, one of the anxiogenic phenotypes, with rapid swimming, less freezing time, less fear to predator, loosen shoal, and circadian rhythm locomotor activity and color preference dysregulations. Taken together, by performing loss-of-function multiple behavior endpoint testing and biochemical analysis, we provide strong evidence for a critical role of lepa gene in modulating behaviors of zebrafish for the first time. Next, the second and third studies were focused on the effect of several extensively used solvents and materials on adult zebrafish’s behavior. Among all tested common solvents, acetone and PEG-400 displayed better biocompatibility and less toxicity since they triggered less behavioral and biochemical alterations while methanol and DMSO caused severe behavior alterations in zebrafish after chronic exposure to these solvents. Meanwhile, the effects of graphene and graphene oxide in zebrafish behavioral dysregulation were also demonstrated, followed by the changes in their biomarker’s level. In the following study, the behaviors of several zebrafish strains, which are AB, absolute, Tübingen long fin (TL), golden, pet store-purchased (PET), and WIK (Wild Indian Karyotype), were further characterized to find out the intraspecies behavior variabilities, and thus, strain-specific zebrafish behavior should be considered when designing experiments using zebrafish behavior. The final study was to investigate the differences in behavioral performance and biomarker expression in the brain of four medaka fishes, which were Oryzias latipes (Japanese Ricefish), O. dancena (Indian Ricefish), O. woworae (Daisy's Ricefish), and O. sinensis (Chinese Ricefish). Later, we found that each tested medaka species explicitly exhibited different behaviors to each other, which might be related to the different basal levels of a stress hormone, several neurotransmitters, and oxidative stress markers measured by ELISA.

並列關鍵字

Zebrafish ； Medaka ； Fish Behaviors ； lepa KO ； Common Solvents ； Graphene ； Graphene Oxide ； Intraspecies ； Interspecies

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