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研究生:溫榮崑
研究生(外文):Rong-Kun Wen
論文名稱:增加雄果蠅大腦內特定多巴胺神經元之多巴胺合成可提高老齡化雄果蠅之性活動力與性衝動
論文名稱(外文):Increased Dopamine Synthesis In Specific Dopaminergic Neurons Reverse Age-related Declines In Sexual Activity And Sex Drive In Male Drosophila
指導教授:傅在峰
指導教授(外文):Tsai-Feng Fu
口試委員:吳嘉霖汪宏達
口試委員(外文):Chia-Lin WuHorng-Dar Wang
口試日期:2012-07-16
學位類別:碩士
校院名稱:國立暨南國際大學
系所名稱:應用化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:74
中文關鍵詞:果蠅求偶行為多巴胺老化性活動力性衝動
外文關鍵詞:Drosophilacourtshipdopamineagingsexual activitysex drive
相關次數:
  • 被引用被引用:2
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  • 下載下載:22
  • 收藏至我的研究室書目清單書目收藏:0
在各項的臨床醫學研究中證實,動物體會隨著老化(aging)現象改變其行為反應與認知能力;其中,性活動力(sexual activity)與性衝動(sex drive)隨著老化而降低的現象,也在各項研究中被發現。但是,對於此現象所涉及的分子機制至今尚未被釐清。而運用果蠅進行求偶行為的分析及定量的方法已被明確的證實。並且果蠅在遺傳分析工具上具有絕佳的研究優勢。然而,多巴胺(dopamine)已證實參與調控哺乳類動物的性行為與性慾,本論文發現,隨著雄性果蠅的老齡化,性活動力與性衝動皆顯著下降,有趣的是,透過多巴胺的補充可回復此一現象。透過分析果蠅大腦中不同的多巴胺神經元,我們證實一群將神經纖維伸入蕈狀體calyx與lateral horn的PPL2ab神經元,當其多巴胺含量增加時,即可逆轉老齡化果蠅之性活動力與性衝動低下現象。對於調控雄果蠅大腦中少數多巴胺神經元之多巴胺含量,得以回復因老化所造成的性活動低下現象,預期此結果可為針對調節性活動力與性衝動相關分子機制研究,提供一個嶄新的研究分析平台。
The declination of sexual activity and sexual drive along with age has been proven in various clinical studies. The involved cellular and molecular mechanisms have not been clarified. Drosophila provides an appropriate genetic analytical tool and the research advantage of large-scale analyses that the analyses and quantification of courtship behavior have been clearly confirmed. Dopamine has been proven to participate in control of the sexual desire in mammals. In this study, increasing dopamine amount in dopaminergic neurons, the declination of courting activity and sexual drive of Drosophila along with ageing could be slowed down. Interestingly, we demonstrate the existence of critical circuit in PPL2ab neurons which innervate the calyx of mushroom bodies (MBs) and lateral horn through DA enhance the male sexual activity and sex drive. As such a complicated behavior could be simply controlled by this neural circuitry, the outcome may become an effective platform for relevant research on sexual activity.
目錄 I
圖目錄 IV
附錄 V
第1章 研究目的(Specific Aim) 1
第2章 研究背景(Background) 1
第1節 老化(Aging) 1
第2節 神經傳導物質(Neurotransmitter)---多巴胺(Dopamine) 2
第2.1節 多巴胺(Dopamine)調節行為 2
第2.2節 多巴胺(Dopamine)與果蠅的求偶行為(courtship behavior) 3
第3節 果蠅模式動物 3
第3.1節果蠅基因表現系統(Gene Expression Systems in Drosophila) 4
第4節 果蠅的求偶行為(Courtship Behavior) 4
第4.1節 果蠅求偶行為步驟(courtship steps) 4
第4.2節 果蠅求偶行為的影響因子 5
第5節 果蠅求偶行為相關之神經網路(neural circuitry) 6
第5.1節 果蠅大腦蕈狀體(Mushroom bodies; MBs)影響求偶行為 6
第5.2節 果蠅週邊感覺神經元受器影響求偶行為 7
第5.3節 果蠅大腦中的多巴胺神經元 8
第3章 材料與方法(Materials and methods) 9
第1節 果蠅品系及培養條件 9
第2節 求偶觀測盤的設計 9
第3節 果蠅求偶行為紀錄 9
第4節 果蠅求偶行為分析 9
第5節 餵食多巴胺前驅物與抑制劑之藥理測試 10
第6節 運用LexPR藥物誘導式基因表現系統,條件式改變雄性果蠅體內多巴胺含量 11
第7節 即時定量聚合酶鏈鎖反應(Real-Time Polymerase Chain Reaction) 11
第7.1節 RNA萃取 12
第7.2節 反轉錄-聚合酶鏈反應 (RT-PCR) 12
第7.3節 即時定量聚合酶鏈鎖反應 13
第8節 高效能液相層析(Hgih-Performance Liquid Chromatograpy) 13
第8.1節 果蠅真實樣品的萃取 14
第9節 果蠅活動力分析 15
第10節 PER (Proboscis extension reflex) assay 16
第11節 果蠅生存曲線測試 16
第4章 結果(Results) 17
第1節 檢測野生種(Canton-S)雄性果蠅不同年齡之性活動力及性衝動 17
第1.1節 年齡與雄果蠅性活動力之相關性 17
第1.2節 年齡與雄果蠅性衝動(sex drive)之相關性 17
第2節 增加雄性果蠅大腦內多巴胺神經元多巴胺含量,檢測其不同年齡之性活動力及性衝動 17
第3節 利用RNAi方式降低雄性果蠅大腦內多巴胺神經元多巴胺含量,檢測其性活動力(sexual activity)及性衝動(sex drive) 18
第4節 運用藥理方式改變雄性果蠅體內多巴胺含量檢測其性活動力變化 19
第5節 條件式誘導LexPR活性,增加或降低雄果蠅多巴胺含量,並檢測其不同年齡之雄性果蠅性活動力及性衝動 20
第5.1節以TH-Gal4表現LexPR,並以綠色螢光蛋白確認其藥物誘導後之
轉錄活性 20
第5.2節 利用基因調控方式,改變雄果蠅體內多巴胺含量,並檢測其不同年
齡之雄性果蠅性活動力 21
第6節 利用即時定量聚合酶鏈鎖反應技術,分析各UAS-effector果蠅株對酪胺酸氫氧化酶mRNA表現量之影響 21
第7節 利用高效能液相層析結合電化學偵測器分析定量果蠅腦內之多巴胺含量 22
第8節 利用檢測求偶活動力方式,篩選影響果蠅求偶活動力與MBs相關之多巴胺神經元 23
第9節 PPL2ab多巴胺神經元集群之求偶活動力表現 24
第10節 利用基因調控方式,條件式改變老齡化雄果蠅大腦內PPL2ab神經元多巴胺含量,檢測其性活動力之表現 24
第5章 討論(Discussion) 26
第1節 老化對於雄性果蠅行為反應與求偶行為之影響 26
第2節 PPL2ab神經元之多巴胺分泌量改變促進雄性果蠅性活動與性慾之假說 26
第2.1節 增加PPL2ab神經元多巴胺含量造成視覺誘發之雄性間求偶行為 27
第2.2節 PPL2ab多巴胺神經元影響雄果蠅的性活動力或性慾 28
第3節 影響果蠅求偶行為之多巴胺神經元網路 28
第3.1節 PPL2ab多巴胺神經元 29
第3.2節 多巴胺受體(dopamine receptor)影響雄性果蠅求偶行為 29
第4節 多巴胺影響雌果蠅求偶行為 30
第5節 影響果蠅求偶行為之外在因素 31
第6章 參考文獻(References) 33
第7章 附錄 66

圖目錄
圖1. 年齡與雄性果蠅性活動力之關係 41
圖2. 年齡老化所造成多巴胺含量變化影響雄果蠅性衝動 43
圖3. 增加雄果蠅腦內多巴胺含量影響雄果蠅求偶活動力 44
圖4. 降低雄果蠅腦內多巴胺含量影響雄果蠅求偶活動力 46
圖5. 利用藥理與基因方式,條件式性增加雄性果蠅多巴胺神經元多巴胺含量之
研究方法 48
圖6. 運用藥理方式改變雄性果蠅體內多巴胺含量檢測其性活動力 50
圖7. 以UAS-LexPR;TH-Gal4>LexAop-AI-mCD8::GFP-attp2果蠅株檢測LexPR
經不同RU486濃度誘導後之轉錄活性 52
圖8. 利用LexPR基因調控系統使用藥物調控方式改變雄性果蠅體內多巴胺含量
檢測雄性果蠅性活動力之變化 54
圖9. 利用即時定量聚合酶鏈鎖反應技術,檢測不同基因操作後酪胺酸氫氧化酶
之改變 55
圖10. 利用高效能液相層析結合電化學偵測器分析定量野生種(Canton-S)雄性果
蠅之年齡與腦內多巴胺含量變化 57
圖 11.與MBs連結之多巴胺神經元之多巴胺含量對求偶活動力的影響 59
圖 12. 增加PPL2ab神經元之多巴胺含量對求偶活動力之影響 60
圖 13. 利用LexPR基因調控系統使用藥物調控方式改變老年化雄性果蠅大腦內
PPL2ab神經元多巴胺含量檢測雄果蠅性活動力(sexual activity)之變化 62
圖14. 分析PPL2ab神經元之多巴胺含量對果蠅活動力之影響 64
圖15.果蠅活動力分析、生存曲線測試及PER (Proboscis extension reflex) test 65


附錄
附圖1. 多巴胺生合成路徑 66
附圖2. LexPR基因表現系統 67
附圖3. 果蠅的求偶行為 68
附圖4. th-Gal4 driver所表現在果蠅腦中之神經元分布 69
附圖5. 分析改變PPL2ab神經元之多巴胺含量後果蠅對嗅覺之感受性 70
附圖6. 果蠅大腦蕈狀體中多巴胺神經元的分布 71
附圖7. 與果蠅大腦蕈狀體相關之多巴胺神經元 72
附圖8. 以免疫螢光染色證實Murashka-1中之PPL2ab為多巴胺神經元。 73
附圖9. PPL2ab多巴胺神經元表現在果蠅腦中之神經元分布 74

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