光轉換性突觸探針之研發與應用-偵測果蠅大腦中功能性神經傳導

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研究生：	蔡居霖 Tsai, Chu-Lin
論文名稱：	光轉換性突觸探針之研發與應用-偵測果蠅大腦中功能性神經傳導 The Development and Application of Photoconvertible Synapse Sensor for Detecting Functional Connectivity in Drosophila Brain
指導教授：	桑自剛 Sang, Tzu-Kang
口試委員:	羅中泉 Lo, Chung-Chuan 張慧雲 Chang, Hui-Yun
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	48
中文關鍵詞：	探針、PSmOrange2 、光轉換、功能性、果蠅、連接
外文關鍵詞：	probe, PSmOrange2, photoconverting, functional, Drosophila, connectivity
相關次數：	點閱：3 下載：0
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摘要
神經系統中的傳遞信息可以藉由突觸間的傳遞來標示。為了看到突觸間的傳遞，過去我們的實驗室已設計了具有活性的綠色螢光蛋白探針稱作 adGRASP的系統。adGRASP系統藉由綠色螢光蛋白重組的概念設計探針並偵測功能性突觸，並且在過去我們已經確定探針具有該功能。儘管我們已經確立其功能，但我們發現 adGRASP系統中已經重組的綠色螢光蛋白訊號消失的速率很慢，這使得我們無法區分突觸過去與新產生的神經傳遞。我們嘗試在 adGRASP系統中利用光轉換蛋白 PSmOrange2替換掉綠色螢光蛋白，以此來偵測更具有即時性與動態的突觸傳遞。我們認為透過光轉換的特性，可以重置先前突觸傳遞產生的螢光並記錄即時產生的反應，進而不會受到”過去”產生的螢光訊號干擾。然而，我們並不清楚 PSmOrange2被拆解成兩個部份是否還能保持其光轉換的特性。近期我建立了帶有 PSmOrange2的基因轉殖果蠅並用實驗去證明此探針仍保有特性。在果蠅嗅覺跟視覺的實驗中，我們證明了此探針可以進行重組且仍然保有光轉換的特性。然而，我們發現 PSmOrange2在果蠅的嗅覺系統中產生了細胞的毒性或其他的影響導致果蠅的死亡。儘管如此，我們希望 PSmOrange2探針的特性可以幫助我們研究神經的功能性連接以及成為一個理想的光學工具去研究即時性的神經動態。近期內，我們利用傳統 adGRASP探針在果蠅的交配行為中發現了有趣的結果，因此我們將利用 PSmOrange2的探針利用其光轉換的特性去探討在交配後的雌果蠅是否會改變其神經中突觸的連結。

Abstract
Synaptic transmission marks the information relay in the nervous system. To visualize such incidence, our lab has developed an activity-dependent GFP probe called adGRASP. This set of probes adopts split-GFP concept to interrogate functional synapses, and we have demonstrated its capability in reporting functional synapses. Despite its utility, the turnover rate of GFP signal from adGRASP appears slow, which hinders its capacity of discriminating the newly-triggered neurotransmission from the previous incidences. To differentially detect the synaptic transmission in a dynamic and activity-dependent manner, we attempt to modify adGRASP by swapping the split GFP by a photoconvertible protein PSmOrange2. We reason that through the photoconversion, we could reset the pre-existed synaptic fluorescence and record the immediate response of the synapse without the interference of those "old" incidences. However, it is unclear whether PSmOrange2 can be separated into two portions and remains photoconvertible. Here I show the recently established transgenic flies bearing PSmOrange2 and provide experimental evidence to validate this set of probes in vivo. I present data from the olfactory and visual systems of fruit fly to demonstrate that PSmOrange2 probes can be reconstituted and still preserve the photoconvertible feature. However, I find that flies expressing PSmOrange2 probes in Drosophila olfactory system shows a potentially cytotoxicity or some unknown effects which led to the premature death. Despite that, we expect the utility of PSmOrange2 probes could benefit the study of functional connectome and offer an adequate optical tool for visualizing an acute neuronal response. Using these fluorescent tools, I have observed neuronal circuits that control the Drosophila mating behavior. To test these connections, I have applied PSmOragne2 probes in the SAG-SPSNs circuit to ask whether the synaptic communications of female flies might be altered after the courtship.

Contents
Chapter 1. Introduction    1
Chapter 2. Material and Methods    5
DNA cloning and constructs    5
Fly stocks    5
Odorant releasing    6
Light exposure    7
Lifespan assays    7
Mating behavior assays    7
Immunohistochemistry of brain and whole nervous system    8
Confocal microscopic imaging    8
Chapter 3. Results    9
Testing functional connectivity of adGRASP in the Drosophila visual system    9
Generating the split-PSmOrange2 pre- and postsynaptic probes in the fly strains    11
Validation of functional split-PSmOrange2-based probes in Drosophila olfactory      system.    12
Validation of functional split-PSmOrange2-based probes in Drosophila visual system    14
The reconstituted PSmOrange2 still preserve the property of photoconversion    14
To detect functional connectivity of adGRASP in Drosophila courtship behavior    16
Chapter 4. Discussion    18
Photoconvertable fluorescent proteins, PSmOrange2.    18
Reconstituted split-PSmOrange2 led flies die in the Drosophila olfactory system?    20
Appling the split PSmOrange2-based probes in Drosophila mating behavior.    21
Figure 1. Illustration of adGRASP for detecting functional connectivity of neurons.    23
Figure 2. Functional GRASP in Drosophila R8-Tm5c circuit (Shi-Yu Chen).    24
Figure 3. Functional GRASP in Drosophila R8-Tm5c circuit after light deprivation   (Shi- Yu Chen).    25
Figure 4. Depicted constructs of the split-PSmOrange2 pre- and postsynaptic probe.    26
Figure 5. A point mutation on PSmOrange2-based trans-synaptic probes.    27
Figure 6. Illustration of the split PSmOrange2-based probes for detecting functional   connectivity of neurons.    28
Figure 7. Validation of the split PSmOrange2-based probes in the brain.    29
Figure 8. Functional PsmOrange2 probes in Drosophila ORCO-GH146 by 1/25 geranyl acetate exposure for 3 hours.    30
Figure 9. Lifespan assays of flies expressing the split PSmOrange2-based probes in    Drosophila ORCO-GH146.    31
Figure 10. Functional PsmOrange2 probes in Drosophila PanR8-Tm5c circuit.    32
Figure 11. Blue/Green LED light include 489 nm (Photoconverting).    33
Figure 12. Validation of photoconversion in the reconstituted PSmOrange 2.    35
Figure 13. Functional split PSmOrange2-based probes in the PanR8-Tm5c circuit after UVA (320-400 nm) light exposure.    37
Figure 14. Validation of photoconversion in the reconstituted PSmOrange 2 by        photoconverting light (480-490 nm)    38
Figure 15. Anatomy of the whole nervous system in Drosophila females.    39
Figure 16. Drosophila female expressing adGRASP pre- and postsynaptic probes in    VT50405-3280 and VT00454-3280 circuit.    40
Figure 17. Drosophila female expressing adGRASP pre- and postsynaptic probes in the VT7068-3280 circuit.    42
Figure 18. Sequence alignment of the split-GFP and the two versions of split-        PSmOrange2.    43
Figure 19. Lifespan assays of flies expressing the full-length PSmOrange in    44
ORCO-Gal4 and GH146-LexA.    44
Table 1. Transgenic lines of PSmOrange2 probe constructs.    45
References    46


                                

References

Evan H. Feinberg, M. K. V., 2 Andres Bendesky, 1 George Wang, 2 Richard D. Fetter, 3 Kang Shen, 2, *, & 1, a. C. I. B. (2007). GFP Reconstitution Across Synaptic Partners (GRASP) Defines Cell Contacts and Synapses in Living Nervous Systems. Neuron.

Fan, J.-Y, Z.-Q. C, H.-P. W. (2007). Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein–protein interactions in living cells. Biochemical and Biophysical Research Communications.

Lee, C.-H. (2014). Development of trans-synaptic probes-A potential approach for detecting both synaptic proximity and functional connectivity (Master), NTHU.

Pletnev, S, D. M. S, O. M. S. (2014). Orange Fluorescent Proteins: Structural Studies of LSSmOrange, PSmOrange and PSmOrange2. PLOS ONE.

Subach, O. M., Entenberg, D., Condeelis, J. S., & Verkhusha, V. V. (2012). A FRET-facilitated photoswitching using an orange fluorescent protein with the fast photoconversion kinetics. J Am Chem Soc, 134(36), 14789-14799. doi: 10.1021/ja3034137

Thangavel Karuppudurai, Tzu-Yang Lin, Chun-Yuan Ting, Randall Pursley, Krishna V. Melnattur, Fengqiu Diao & Benjamin H. White, L. J. M., 4 Marco Gallio, Thomas Pohida, and Chi-Hon Lee. (2013). A Hard-Wired Glutamatergic Circuit Pools and Relays UV Signals to Mediate Spectral Preference in Drosophila. Neuron.

Oksana M. Subach, David Entenberg, John S. Condeelis, and Vladislav V. Verkhusha. (2012). A FRET-Facilitated Photoswitching Using an Orange Fluorescent Protein with the Fast Photoconversion Kinetics. Journal of the American Chemical Society.

Subach, O. M., Entenberg, D., Condeelis, J. S., & Verkhusha, V. V. (2012). A FRET-facilitated photoswitching using an orange fluorescent protein with the fast photoconversion kinetics. J Am Chem Soc.

Kai Feng,Mark T. Palfreyman, Martin Ha¨ semeyer, Aaron Talsma. (2014). Ascending SAG Neurons Control Sexual Receptivity of Drosophila Females. Neuron.

Elizabeth J Rideout, Anthony J Dornan, Megan C Neville, Suzanne Eadie & Stephen F Goodwin. (2010). Control of sexual differentiation and behavior by the doublesex gene in Drosophila melanogaster NATURE NEUROSCIENCE.

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