- 學位論文
斑馬魚行為分析系統的發展與應用
Development and application of Zebrafish Behavior Tracking System
摘要
斑馬魚的行為可定義為對外在刺激的內部協調反應。目前有數種範例已被開發用以評估斑馬魚行為，包括三維空間游泳行為測試，新魚缸潛水測試，鏡像啄食測試，社群互動測試，群游測試，和掠食者脫逃分析。通常測量斑馬魚行為需要不同的裝置和設備，評估法通常很複雜又昂貴。此外，斑馬魚三維空間游泳量測通常需要倚賴商業軟體，較複雜的程序編寫或依賴兩台以上的相機來同步擷取影像。此外，斑馬魚行為分析資料相當多變，資料特性往往不符合常態分佈，因此為了同步得到較高的樣品數據，利用開源軟體建立高通量斑馬魚行為測試是必須的。在此，我們建立了一簡單且經濟的設施來偵測成年斑馬魚的三維泳動。使用成年魚做測試乃因成魚有展現複雜行為的能力，譬如具有較強的社群互動與學習能力，對不同的外來刺激顯示明顯的反應。這套三維泳動追蹤設備涉及單一相機擷取系統，可記錄多尾斑馬魚在一特殊設計的魚缸(具有一面傾斜45度的鏡子)中的移動影像，被錄下的影片可用開源軟體idTracker分析，idTracker軟體是藉著組合物體影像橫切面以收集物體移動軌跡，而空間位置是用ImageJ軟體來校正，三維空間移動軌跡是用Origin9.1軟體繪製。這套簡易設備可讓科學家以低費用、高空間精準度追蹤多尾斑馬魚三維游泳軌跡。此外在本論文第二部分，我們將呈現一個嶄新且多樣的裝置，運用5層2平行欄的魚架裝置10個壓克力魚缸，可以進行如新魚缸潛水測試，鏡像啄食測試，社群互動測試，群游測試，和掠食者脫逃等多種行為分析。在一次的錄像中可同時量測多達10尾斑馬魚的多種行為，而各物體的X軸及Y軸座標位移可用idTracker及ImageJ等開源軟體計算求得，這個簡易的裝置的建立，可以協助科學家利用斑馬魚行為學來進行藥理與毒理的高通量評估。
並列摘要
Zebrafish behavior can be defined as the coordination of the internal responses to a stimulus. There are several simple paradigms that have been developed to assess the zebrafish behavior including three-dimensional (3D) swimming behavior test, novel tank diving test, mirror biting test, social interaction test, shoaling test and predator escape assay. Generally, the measurement of behaviors in zebrafish requires different equipment and settings, and assessments are usually complicated and costly. In addition, the measurement of 3D swimming behavior in zebrafish relies on commercial software or requires sophisticated scripts, and depends on more than two cameras to capture the video. Moreover, zebrafish behavioral data is highly variable, even when collected within the same conditions. Thus, a high-throughput screening setup for zebrafish behavior tests by using open source software is needed in order to obtain high sample numbers. Here, we establish a simple and economic apparatus to detect 3D locomotion in adult zebrafish. Adult zebrafish were used since its capability to exhibit complex behaviors such as having strong social interaction with others, effective learning abilities, and portraying distinctive reactions to different environmental stimuli. This setup involves a single camera capture system that records zebrafish movement in a specially designed water tank with a mirror tilted at 45 degrees. The recorded videos are analyzed using idTracker, an open source program that is used for collecting motion trajectories by matching the feature on all crossing frames of objects based on appearance analysis, while spatial positions are calibrated by ImageJ software and 3D trajectories are plotted by Origin 9.1 software. This easy setting allowed scientists to track 3D swimming behavior of multiple zebrafish with low cost and precise spatial position, showing great potential for fish behavioral research in the future. In this report, we also presented a versatile setup that applied acrylic tanks into 5 vertical layers and 2 horizontal columns to perform multiple behavior assays such as novel tank diving test, mirror biting test, social interaction, shoaling and predator escape assay. In total, 10 different fish behaviors can be recorded simultaneously in a single video file, and the XY coordination of locomotion can be calculated by open source software of idTracker and ImageJ. This simple setting will allow scientists to assess behavioral endpoints of multiple zebrafish individuals, even other animals with high reliability and reproducibility.