臺灣博碩士論文加值系統

認出過去遭遇物品的能力對動物的生存至關重要，幫助我們更快的理解所處情境，但在背後支撐該能力的神經機制尚未明朗。對物品的辨識需要形成與回憶物品再認記憶，而新奇事物認知則是在齧齒類動物上研究物品再認記憶時常使用到的的行為典範。科學家利用了齧齒類動物偏愛新奇事物勝過熟悉事物的天性設計該行為典範，以測試動物形成或回憶再認記憶的能力，擁有正常再認記憶的齧齒動物會花上較多時間在新奇物體上，反之則否。不同區域的腦損傷對新奇事物認知的影響揭示該行為典範與海馬迴的可能聯繫，但該腦區的神經元參與建構物品再認記憶的方式尚未解開。我們希望使用活體螢光成像與活體單細胞膜片鉗技術來深入了解海馬迴內神經元在物品再認記憶建構中的角色，然而上述實驗方式需要動物的頭部固定以取得更高質量的數據，為此需要建構一套用於測試頭部固定動物再認記憶的行為典範，我們於此試著滿足此需求。藉由在動物頭顱骨上安裝金屬棒，我們能限制行為實驗中動物頭部的移動。同時結合一個由氣浮支撐的迷宮，動物便能藉由撥動迷宮本身來探索迷宮中的熟悉與新奇物品。依此我們嘗試讓動物在頭部固定狀況下進行新奇事物認知，寄望未來能將其與活體螢光成像或活體單細胞膜片鉗技術結合以研究海馬迴或其他腦區神經元在動物進行新奇事物認知時的活性。

The ability to remember the objects encountered before is crucial to survival, but its underlying neuronal mechanism has not yet been clear. Recognizing objects requires formation and retrieval of recognition memory. Novel object recognition task is a common method to study this kind of memory in rodent models. Scientists exploit rodents’ internal preference of novel objects over familiar ones to test whether subjects form proper recognition memory or not. Rodents without recognition memory deficits would spend more time interacting with novel objects. Lesion studies combined with such behavioral task have revealed important brain regions involved in forming/maintaining the memory. Nevertheless, to study how neurons of these brain regions achieve memory function requires measurements of their activities and/or connectivity using in vivo two-photon imaging or intracellular recordings. Such measurements, however, depend upon animals’ heads to be fixed to harvest high-quality data. There is a need, thus, to develop behavioral tasks to test recognition memory in head-fixed animals. Here we suggest a solution to solve this problem. By implanting a metal strip on mice skulls, we restrict their head movement. During the behavior tests, subjects explore new and familiar objects in an arena lifted by air. With this, we aim to test novel object recognition paradigms in head-fixed mice hoping to combine the new behavioral tasks with in vivo recordings for further investigation of neuronal mechanism underlying novel object recognition.

目錄
致謝……………………………………………………………………………………………………………………………i
中文摘要 ……………………………………………………………………………………………………………………ii
英文摘要……………………………………………………………………………………………………………………iii
目錄 …………………………………………………………………………………………………………………………iv
圖目錄 ………………………………………………………………………………………………………………………ix
表目錄 ………………………………………………………………………………………………………………………xi
程式編碼目錄………………………………………………………………………………………………………………xii
第一章 引言 …………………………………………………………………………………………………………………1
1.1再認記憶與新奇事物認知 ………………………………………………………………………………………………1
1.2新奇事物認知與海馬迴的聯繫 …………………………………………………………………………………………3
1.3於頭部固定動物測試行為典範的需求 …………………………………………………………………………………5
第二章 實驗材料以及方法 …………………………………………………………………………………………………7
2.1動物與其待遇 ……………………………………………………………………………………………………………7
2.2照明與攝影設備 …………………………………………………………………………………………………………7
2.3動物的上手 ………………………………………………………………………………………………………………7
2.4自由移動新奇事物認知 …………………………………………………………………………………………………7
2.4.1設備 ……………………………………………………………………………………………………………………7
2.4.2物品 ……………………………………………………………………………………………………………………8
2.4.3實驗流程 ………………………………………………………………………………………………………………8
2.4.4設備清潔 ………………………………………………………………………………………………………………8
2.4.5動物探索物品時間的紀錄 ……………………………………………………………………………………………8
2.5 頭棒植入手術……………………………………………………………………………………………………………9
2.5.1手術器具與藥物 ………………………………………………………………………………………………………9
2.5.2實驗流程 ………………………………………………………………………………………………………………9
2.6 頭部固定新奇事物認知 ………………………………………………………………………………………………10
2.6.1 MHC印製………………………………………………………………………………………………………………10
2.6.2物品……………………………………………………………………………………………………………………10
2.6.3 MHC系統架設…………………………………………………………………………………………………………10
2.6.4 MHC使用訓練 ………………………………………………………………………………………………………11
2.6.5實驗流程………………………………………………………………………………………………………………11
2.6.6設備清潔………………………………………………………………………………………………………………11
2.6.7動物探索物品時間的紀錄……………………………………………………………………………………………11
2.7 統計數據分析 …………………………………………………………………………………………………………12
2.7.1量化動物對物品的偏好………………………………………………………………………………………………12
2.7.2量化動物的活動力……………………………………………………………………………………………………12
2.7.3單一樣本T檢定 ………………………………………………………………………………………………………12
2.7.4獨立樣本T檢定 ………………………………………………………………………………………………………13
2.7.5成對樣本T檢定 ………………………………………………………………………………………………………13
2.7.6自由移動新奇事物認知熱圖繪製……………………………………………………………………………………13
2.7.7頭部固定新奇事物認知熱圖繪製……………………………………………………………………………………13
第三章 結果…………………………………………………………………………………………………………………15
3.1嘗試在尺寸與MHC相仿的環境中進行自由移動新奇事物認知 ………………………………………………………15
3.1.1動物在尺寸與MHC相仿的環境中表現出對新奇物品的偏好 ………………………………………………………15
3.1.2確認動物對新出現物品的偏好並非出於物品擺放的位置或是物品的種類………………………………………15
3.1.3不同對探索的定義/不同測試期長度對動物分辨率的影響 ………………………………………………………16
3.2訓練動物使用MHC ………………………………………………………………………………………………………18
3.2.1使用於動物頭部固定的手術與設備…………………………………………………………………………………18
3.2.2 MHC的印製與MHC系統的架設 ………………………………………………………………………………………18
3.2.3使動物習慣實驗者、頭部固定的流程與MHC的使用 ………………………………………………………………19
3.3頭部固定新奇事物認知…………………………………………………………………………………………………21
3.3.1規劃頭部固定新奇事物認知的實驗流程……………………………………………………………………………21
3.3.2修正物品尺寸與頭部固定動物較長休息時間對實驗造成的影響…………………………………………………21
3.3.3頭部固定新奇事物認知無法在不同的實驗室位置重複……………………………………………………………23
3.3.4修改實驗流程與設備以改善動物的探索時間………………………………………………………………………23
3.3.5接受修改過實驗流程訓練的動物由於對特定物品的偏愛，並未展現出對新奇物品的偏好……………………25
3.4結論………………………………………………………………………………………………………………………26
第四章 討論…………………………………………………………………………………………………………………28
4.1即使經過四天的訓練，頭部固定與MHC的使用依然有可能使動物感受到壓力 ……………………………………28
4.2 利用自動追蹤軟體分析動物行為 ……………………………………………………………………………………29
4.3頭部固定新奇事物認知中物品採用的條件……………………………………………………………………………30
4.4 造成頭部固定動物與自由移動動物移動力指數差異的可能原因 …………………………………………………31
4.5 MHC材質的挑選與不同的設計…………………………………………………………………………………………32
4.6基於MHC系統設計頭部固定新奇事物認知的優點與限制 ……………………………………………………………33
4.7未來展望…………………………………………………………………………………………………………………34
參考文獻 ……………………………………………………………………………………………………………………83
圖 目 錄
圖1實驗中採用之物品………………………………………………………………………………………………………36
圖2在尺寸與MHC相似環境中進行自由移動新奇事物認知動物的分辨率 ………………………………………………37
圖3採用不同長度的測試期與採用不同的探索定義對計算分辨率的影響………………………………………………39
圖4頭部固定新奇事物認知中使用到的儀器………………………………………………………………………………41
圖5進行頭部固定新奇事物認知前動物所需經過的訓練…………………………………………………………………42
圖6訓練期中頭部固定動物在MHC上的軌跡與速度紀錄 …………………………………………………………………43
圖7縮短訓練期的時長並不會影響實驗動物的移動力指數………………………………………………………………45
圖8應用於頭部固定新奇事物認知的實驗流程……………………………………………………………………………47
圖9移動力指數較低的實驗動物有著極端的分辨率………………………………………………………………………48
圖10頭部固定新奇事物認知中使用的物品更動 …………………………………………………………………………49
圖11頭部固定動物較長的休息時間與其休息位置的選擇對動物的分辨率有所影響…………………………………………50
圖12在舊日實驗室位置取得的頭部固定新奇事物認知實驗結果無法在新實驗室位置重複 ……………………………52
圖13對頭部固定新奇事物認知實驗細節的修改 …………………………………………………………………………53
圖14接受修改過實驗流程訓練的動物在移動力指數與探索時間上優於接受舊式實驗流程訓練的動物 ……………55
圖15接受修改過實驗流程訓練的動物表現出對特定的物品種類的偏好 ………………………………………………57
圖16除未達探索時間標準後動物在熟悉期與測試期的分辨率 …………………………………………………………59
表 目 錄
表1自由移動新奇事物認知中所使用動物資訊……………………………………………………………………………60
表2訓練期長度為2小時的頭部固定新奇事物認知中所使用動物資訊 …………………………………………………61
表3訓練期長度為1小時的頭部固定新奇事物認知中所使用動物資訊 …………………………………………………62
表4在每次頭部固定前讓動物上手的實驗中所使用動物資訊……………………………………………………………63
表5嘗試重複舊實驗室位置實驗中所使用動物資訊………………………………………………………………………64
表6使用修改頭部固定新奇事物認知實驗流程的實驗中所使用動物資訊………………………………………………65
程 式 編 碼 目 錄
編碼1將自由移動新奇事物認知影片檔案轉為mat檔案並降低影片的幀率 ……………………………………………66
編碼2將半自動自由移動動物鼻尖、身體中心位置紀錄換算為分辨率與移動力指數…………………………………67
編碼2.1 explorationdefine_ver_2 …………………………………………………………………………………… 68
編碼3自由移動新奇事物認知測試期熱圖繪製……………………………………………………………………………69
編碼4 2小時訓練期移動力指數計算………………………………………………………………………………………71
編碼5 2小時訓練期中每15分鐘時間區間的移動力指數計算……………………………………………………………72
編碼6 2小時訓練期中第一小時與第二小時時間區間中移動力指數計算………………………………………………73
編碼7 1小時訓練期移動力指數計算………………………………………………………………………………………74
編碼8頭部固定動物在測試期中的分辨率與移動力指數計算……………………………………………………………75
編碼8.1 TrainingExam ……………………………………………………………………………………………………78
編碼9頭部固定新奇事物認知測試期熱圖繪製……………………………………………………………………………79
編碼10頭部固定新奇事物認知測試期移動狀態下熱圖繪製 ……………………………………………………………81

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