在果蠅嗅覺系統中，環境中的味道分子經由嗅葉（antennallobe）整合，產生的訊號將由嗅覺投射細胞（projection neurons）傳遞到更高層的腦區-蕈狀體（mushroom body）和側號角（lateral horn）。許多證據顯示，在嗅覺學習和記憶行為中主要是藉由蕈狀體調控，但對側號角機制與功能的研究是相當少的，因此我們以側號角作為研究對象，蒐集了108 個表現神經在側號角的果蠅，藉由影像分析方法探討神經細胞的形態及特性並且預測嗅覺資訊的流向，將結果分成兩大類，再細分成22種類型，建構以側號角為主的嗅覺神經網路圖譜，並且藉由免疫染色法標定GABAergic 神經，以此分辨表現於側號角的抑制型細胞，在結果中發現LHD/V-LN,LHD-PN4,LHD-PN6和MB-C5 屬於抑制型細胞。而在我所蒐集的這些表現神經在側號角的果蠅，發現了一群令人感到興奮的類型(LHPNCalyx:MB-C4 到MB-C7)，連接了蕈狀體和側號角，為了釐清此神經的功能，我將這群連接蕈狀體和側號角的神經，用人工的方法使它們表現，在光感應的行為實驗中，發現MB-C4 神經參與吸引的行為，此外，藉由GRASP 實驗，證實MB-C4 和蕈狀體神經以及側號角神經在蕈狀體和側號角這兩個腦區有連接。總和上述實驗，我建構了側號角的神經網路圖譜，並且發現MB-C4 神經會調控嗅覺的先天性吸引行為。

In Drosophila melanogaster, olfactory projection neurons (PNs) deliver odor information from antennal lobe (AL) to two higher brain centers: mushroom body(MB) and lateral horn (LH). Numerous evidences implicate key roles of the MB and
its downstream circuits in olfactory learning and memory,but little is known about the roles of the LH. Here, I characterized more than 100 GAL4 lines containing LH related neurons and constructed a comprehensive connectivity map for predicting odor information flow. Based on morphology and polarity of individual neurons, I classified LH neurons into 2 classes, 7 families and 22 types. Immunohistochemical
labeling of 22 types of LH neurons showed that only 5 types were GABAergic: LHD/V-LN1, LHD/V-LN2, LHD-PN4, LHD-PN5 and MB-C5 neurons. I focused on studying LH-PNCalyx neurons (four types: MB-C4 to -C7) connecting between LH and MB calyx. To understand innate function, I activated each neuron with optogenetics.
and found that activation of MB-C4 and MB-C7, but not MB-C5 and MB-C6,
triggered attraction behavior. Furthermore, using GFP reconstitution across synaptic
partners (GRASP) labeling, I showed that MB-C4 neurons connected with AL-PN
and LH local neurons at the LH and with AL-PN and MB neurons at the calyx. In
conclusion, I constructed a map of LH circuits and showed that innate olfactory
attraction may involve MB-C4 neurons.

致謝 1
摘要 3
Abstract 4
1. Introduction 5
1.1 Taking Drosophila as an Animal Model to Study the Olfactory System……5
1.2 The First Level of Olfactory System-Olfactory Sensor Neuron and Antennal
Lobe………………...……………………….……………………..……………..6
1.3 The Higher Level of Olfactory System-Mushroom Body and Lateral Horn…6
1.4 Summary……………………………………………………………………….9
2. Materials and Methods 10
2.1 Fly Strains……………………………………………………………..……10
2.2. Sample Preparation and Immunohistochemistry 10
2.3. Confocal Imaging and Post-recording Image Processing 11
2.4 Polarity Imaging………….…………………………………………….12
2.5. Single Neuron Imaging 12
2.6. GFP Reconstitution Across Synaptic Partners (GRASP) 12
2.7. Optogenetic Assay 13
2.8. Odor Acuity and Learning Behavior Testing 13
3. Results 15
3.1. Strategy Analysis 15
3.2. Lateral Horn Neurons Classification 15
3.3. Identification of the Class of Lateral Horn-Local Neuron 16
3.4. Identification of the Class of Lateral Horn-Projection Neuron 17
3.5. Identification of the Class of Lateral Horn-Projection NeuronCalyx 18
3.6. Information Flow with Lateral Horn-Projection Neuron 19
3.7 GABAergic staining of Lateral Horn Neurons…………………………...19
3.8 The Behavior Result of Lateral Horn-Projection NeuronCalyx…………19

4. Discussion 21
4.1 Investigation of Lateral Horn Neurons Morphology ………………………….…21
4.2 Information Flow within Lateral Horn Neurons…………………………..….22
4.3 Identification of Lateral Horn Inhibitory Local Neuron and Projection Neuron by
Immunolabling…………………………..……………………………………….22
4.4 Investigate the Character of Lateral Horn-Projection Neuroncalyx in Innate and
Learning Behavior …………………………………………….…………...……23
5. References……………………………...………………………………………25
6. Figures and Figure legends………………………………………………31
7. Appendix Figures and Figure legends 57

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