科學家已研究果蠅的各種行為數十年，但是產生行為的神經迴路仍未被完整地了解。透過光遺傳學的方法，利用自動雷射追蹤及光遺傳調控系統(ALTOMS)，使我們可以在短時間內篩選出與特定行為相關的神經元。經由ALTOMS激發特定被挑選的GAL4啟動子標定的神經，我們發現果蠅會分別做出公母特有的生殖行為，例如：公果蠅求偶行為中的振翅、交配動作，或是母果蠅將產卵器伸出並將腹部往下彎的產卵動作。為了確認上述的神經與交配或產卵行為相關，我們以遺傳學的技術去抑制這些神經後觀察果蠅行為是否變化，實驗結果顯示交配率及下蛋數量皆明顯下降。另一方面，我們也想知道上述神經是否與fruitless有關，出乎意料的是，儘管下蛋與求偶行為屬於雌性及雄性分別專屬的動作，結果卻與調控果蠅雌雄相異性的轉錄因子fruitless無關，證明這些神經並不隸屬fruitless的神經迴路。最後，我們以共軛焦顯微鏡觀察神經在果蠅腹部神經管束以及大腦的分布，並利用不同GAL80 lines去找出與求偶及下蛋行為相關的神經傳導物。未來我們可以運用這些GAL4 line果蠅進一步研究求偶與下蛋行為的神經迴路，讓我們更深度地了解果蠅神經網路連結體的功能。

Diverse behaviors in Drosophila Melanogaster have been studied for many years, the neural circuits behind these behaviors, however, are still not fully understood. New developed automated laser tracking and optogenetic manipulation system (ALTOMS) provides us a platform to high-throughput screen multiple lines with different behaviors. By activating neurons labeled by different GAL4 driver lines, we verified a couple of lines with specific reproductive behaviors, including abdomen bending and wing extension in males and ovipositor extension in females, implying their functions in courtship and egg laying behaviors respectively. We further modulated the activities of target neurons by expressing temperature-sensitive shibire gene, and the results indicated that thermogenetic inhibition of these specific neurons decreased the copulation rate as well as the number of laying eggs in corresponding lines. Surprisingly, while both behaviors are sexually dimorphic, the intersection study suggested that none of these neurons is fruitless positive. The neural anatomies of these targeting GAL4 lines were also examined under confocal microscopy. Finally, in order to narrow down the target neurons, the molecular identities of the labeled neurons were characterized by multiple GAL80 driver lines for different neurotransmitters. Identifying these candidate GAL4 lines led us to reveal new circuits involved in courtship and egg laying behaviors, which would eventually enhance our understanding of functional connectome in fruit fly.

Abstract 2
摘要 3
致謝 4
Table of content 5
1. Introduction 7
1.1. Drosophila is a suitable model animal for studying different behaviors 7
1.2. Courtship behavior 7
1.3. Ovipositor extension 10
1.4. Behavioral screening and research aim 12
2. Materials and methods 14
2.1. Fly stocks 14
2.2. Immunohistochemistry 14
2.3. Optogenetic manipulation 15
2.4. Courtship assay 15
2.5. Climbing assay 16
2.6. Egg laying 17
2.7. Statistical analysis 17
3. Results 18
3.1. Courtship candidate GAL4 lines 18
3.1.1. Activating different GAL4 driver lines neurons by ALTOMS drives different behaviors 18
3.1.2. Inhibiting the activities of targeting neurons by temperature-sensitive Shibire decrease courtship drive 19
3.1.3. Intersection experiments showed the courtship-related neurons are not fruitless-positive neurons 21
3.1.4. Fluorescence images show GAL4 labeling neurons innervate into different regions in the brain and ventral nerve cord (VNC) 21
3.1.5. Narrowing down the GAL4 lines by different GAL80-labeled neurotransmitters 22
3.2. Egg laying candidate GAL4 lines 23
3.2.1. Activating different Gal4 driver lines neurons by ALTOMS elicits ovipositor extension 23
3.2.2. Inhibiting the activities of targeting neurons by temperature-sensitive shibire decrease the amount of laying eggs 24
3.2.3. Fluorescence images show GAL4 labeling neurons innervate into different regions in the brain and ventral nerve cord (VNC) 24
3.2.4. Narrowing down the GAL4 lines by different GAL80-labeled neurotransmitters 25
4. Discussion 27
4.1. Courtship candidate GAL4 lines 27
4.1.1. The neurons labeled by VT28160 and VT9573 are less likely responsible for courtship circuits. 27
4.1.2. The neurons labeled by VT0252 and VT20037 are candidate courtship neurons. 29
4.1.3. The neurons labeled by VT20037 GAL4 line are sexually dimorphic. 29
4.1.4. Identify the neurons by multiple neurotransmitter GAL80 driver lines. 30
4.1.5. Conclusion 31
4.2. Egg laying candidate GAL4 lines 31
4.2.1. The neurons labeled by VT17180 are not involved in egg laying circuitry. 31
4.2.2. VT58561 GAL4 line may involve in both egg laying and courtship behavior circuits. 32
4.2.3. Activating the neurons labeled by GR46428 and GR48003 may lead to post-mating behaviors 32
4.2.4. No female-specific behavior was detected by activating the neurons labeled by GR46428 and GR48003 in male flies. 33
4.2.5. Narrowing down the neurons labeled by GR46428 and GR48003 33
4.2.6. Conclusion 34
5. Reference 36
6. Figures 42
7. Appendix 65

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