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研究生: 許竣博
Hsu Chun-Po
論文名稱: 探討斑馬魚恐懼記憶之神經機制
To elucidate the neural mechanism of fear memory in zebrafish
指導教授: 呂國棟
Lu, Kwok-Tung
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2009
畢業學年度: 97
語文別: 中文
論文頁數: 62
中文關鍵詞: 斑馬魚恐懼記憶神經機制
英文關鍵詞: Zebrafish, Fear memory, Neural mechanism
論文種類: 學術論文
相關次數: 點閱:192下載:12
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  • 斑馬魚在脊椎動物學習和記憶能力之基因體研究方面是一種功能強大的模式動物。科學家利用斑馬魚的基因轉殖技術,發展出多樣不同基因變異的品系。而在各種突變斑馬魚被大量建立的同時,更迫切需要進行專門針對斑馬魚學習與記憶功能的行為研究。在許多研究抑制性逃避行為實驗,常以其他物種─例如大白鼠當動物模式。然而,目前對硬骨魚類情緒性記憶相關的研究依然相當缺乏,有礙於斑馬魚於神經科學相關研究的推廣。本研究採用改良型之抑制性逃避行為箱來研究斑馬魚之抑制性逃避行為。
    本研究之實驗結果顯示:(1)於訓練後24小時,所有斑馬魚將會被再放回淺水區進行測試。此時,斑馬魚會對深水區產生抑制性逃避行為,且停留在淺水區的時間較訓練前有明顯的延長。(2)在消減階段,所有重新暴露於深水區的斑馬魚,會改變其對深水區已建立之抑制性逃避行為,而停留在淺水區的時間較訓練前有明顯的縮短。(3)在訓練後7天,所有斑馬魚放回淺水區進行測試,斑馬魚仍對深水區產生抑制性逃避行為,顯示此恐懼記憶被保存下來的時間至少可達到7天。(4)經過(+)MK-801(一種非競爭性麩胺酸NMDA受體拮抗劑)處理的斑馬魚,其抑制性逃避行為將會被阻斷。 (+) MK-801處理組和對照組之間,斑馬魚對深水區產生抑制性逃避行為而停留在淺水區的時間有顯著差異。(5)在經過訓練後的斑馬魚,端腦內的MAPK的磷酸化程度,會隨著時間而增加,在訓練後1.5小時到達高峰,同時與naïve組比較也有明顯增加。而端腦內的MAPK的表現量並沒有明顯變化。
    綜合上述各點,本實驗不只建立一套操作簡單的行為儀器來研究斑馬魚的恐懼記憶,且實驗結果推論斑馬魚恐懼記憶相關的神經機轉與陸生的脊椎動物相似。因此,也許可以增進我們使用斑馬魚來研究神經科學的可行性,並且拓展對精神疾病藥物的開發領域,而在未來配合多樣不同突變的斑馬魚,將有助釐清脊椎動物基因與學習以及記憶功能之間的關係。

    The zebrafish (Danio rerio) represents a powerful model for genetic studies involved in learning and memory function in vertebrates. Scientist developed a variety of genetic mutants by using genetic techniques in zebrafish. While zebrafish mutants are being developed, behavioral researches on learning and memory function in zebrafish are in urgent need. Numerous studies investigated inhibitory avoidance conditioning in other animal model such as rat. However, the study of emotional behavior in teleost fish was still lacking. We used a modified behavioral chamber in our study to study the inhibitory avoidance learning in zebrafish.
    Results showed: (1) Twenty-four hour after the training day, all fishes were returned to the shallow compartment again. The escape latency to enter the deeply compartment was significantly longer than pre-training. (2) After exposing to the deeply compartment (extinction training), all fishes were decline in escape latency when compared with the previously test. (3) Seven day after the training day, all fishes were returned to the shallow compartment again. The escape latency to enter the deeply compartment was significantly longer than pre-training. It suggested that this memory can be stored at least seven days. (4) The administration of (+)MK-801(a noncompetitive NMDA receptor antagonist) blocked the expression of inhibitory avoidance behavior in zebrafish. There was a significant difference in escape latency between (+)MK-801-treated group and control group. (5) The phospho-MAPK expression of trained zebrafish’s telencephalon was gradually increasing after training. One and half hour after training, the phospho-MAPK expression of trained zebrafish’s telencephalon reached peak, and there was a significant increase compared with naïve group. The MAPK expression of trained zebrafish’s telencephalon was no significant change.
    The present study not only establishes a simple behavior model to study the fear memory in zebrafish, but also suggest the similar involvements of neurochemicals in learning and memory among vertebrates. Thus, it may improve our usage of zebrafish model in neuroscience researches, which may extend to the pace of psychiatric drug discovery, and future studies with zebrafish mutants may be helpful for identify genes involved in learning and memory in vertebrates.

    壹、中文摘要(Abstract in Chinese)…………………………………2-3 貳、英文摘要(Abstract in English)…………………………………4-5 參、緒論(Introduction) 一、研究背景(Research Background)……………………………6-16 二、研究目的(Research Aim)……………………………………16 肆、研究材料與方法(Materials & Methods) A. 實驗動物(Animals)…………………………………………17 B. 實驗藥物(Drug)………………………………………………17 C. 抑制性趨避行為(Inhibitory Avoidance Apparatus)………18-20 D. 運動行為監測(Locomotor Activity Monitoring)……………20 E. 西方轉漬法(Western Blotting Assay)……………………20-27 F. 統計分析(Statistics)…………………………………………27 伍、實驗結果(Results)……………………………………………28-38 陸、實驗討論(Discussion)…………………………………………39-44 柒、參考文獻(References)…………………………………………45-52 捌、附圖/表(Figures & Tables)……………………………………53-62

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