臺灣博碩士論文加值系統

在現今的感官神經科學研究當中，已經可以利用果蠅嗅覺當作一個模式系統，在腦中描繪出用以理解外部世界的部份圖譜。在腦內的嗅覺訊息處理，現在已知有三個層次的線路分佈，第一層次是在觸角(Ant)和小顎鬚(MP)的感覺神經元偵測到氣味分子，然後藉由它們的軸突傳送氣味訊息到嗅球窩(AL)。第二層是指將第一層送來的訊號在嗅球窩加以計算。最後再由第三層的投射神經元將這些訊號傳送到蕈狀體(MB)和側角區(LH)。 在第三層中，投射神經元從嗅球窩經由三條不同的線路傳送資訊到蕈狀體和側角區等高層腦中心:分別是內嗅腦束、中嗅腦束以及外嗅腦束。為了詳細研究這些線路的連結，我們製造出帶有UAS-PaGFP的基因轉殖果蠅，可以在GAL4蛋白的控制下產生光激發綠色螢光蛋白(PaGFP)。在這次研究當中，第一部分是去確認出光激發綠色螢光蛋白在果蠅腦中的特性，包括光激發和擴散等現象。第二部份是利用波長為820奈米的雙光子雷射激發在果蠅腦中的光激發綠色螢光蛋白，以顯示單一嗅小球和整個嗅球窩的連結。這些影像結果顯示，我們可以追蹤任何有興趣的網路連結。我們還發現，除了目前三條典型的嗅覺網路之外，另有其他區域與嗅球窩連接，分別是中表面區(MS)和腹側邊區(VL)。光激發綠色螢光蛋白提供了一個新穎的工具來追蹤神經網路，使我們得以更完整的繪製出果蠅腦的神經網路連結系統。

Recent advances in sensory neuroscience using Drosophila olfaction as a model system have revealed partial map for the representation of the external world within its brain. Currently, three levels of wiring in antenna lobe (AL) are known. Odorants are detected by the olfactory sensory neurons (OSNs), located on the antenna (Ant) and maxillary palp (MP), sending axons via the antennal nerve (AN) and the labial nerve (LN), respectively, to AL (Level 1); where the projection neurons (PNs) receive and calculate the information (Level 2) and then relay to the mushroom body (MB) and the lateral horn (LH) (Level 3). Projection neurons convey the information via three tracks: the inner antenna-cerebrum track (iACT), the medial ACT (mACT), the outer ACT (oACT). In order to investigate the detailed anatomy along these tracks, we generate a transgenic fly, UAS-Photoactivatable GFP (PaGFP), which produces PaGFP under GAL4 control. In this study, the first part is to characterize PaGFP in fly brain, including photoactivation and diffusion. The second part is to demonstrate the anatomical applications of PaGFP, such as revealing single glomerulus or the whole AL, following its activation by a two-photon laser at 820 nm. These results illustrate that circuits tracing is feasible in any region we are interested in Drosophila brains. In this way, we discover some new circuits in addition to the typical three tracts of olfactory circuits and find two regions, medial superior (MS) and ventral lateral (VL), which may also connect with AL. Therefore, PaGFP provides us a novel tool tracing neuronal circuitry and mapping the complete network in Drosophila brains.

Contents........................1
Acknowledgement.................2
Abstract........................3
Introduction....................5
Materials and methods..........10
Results........................13
Discussion.....................17
References.....................22
Figure legends.................27
Table..........................32
Figures........................33
Appendixes.....................40

Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., and Miyawaki, A. (2002). An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 99, 12651-12656.

Barnea, G., O'Donnell, S., Mancia, F., Sun, X., Nemes, A., Mendelsohn, M., and Axel, R. (2004). Odorant receptors on axon termini in the brain. Science (New York, NY 304, 1468.

Belluscio, L., Gold, G.H., Nemes, A., and Axel, R. (1998). Mice deficient in G(olf) are anosmic. Neuron 20, 69-81.

Berdnik, D., Chihara, T., Couto, A., and Luo, L. (2006). Wiring stability of the adult Drosophila olfactory circuit after lesion. J Neurosci 26, 3367-3376.

Bozza, T., Feinstein, P., Zheng, C., and Mombaerts, P. (2002). Odorant receptor expression defines functional units in the mouse olfactory system. J Neurosci 22, 3033-3043.

Brand, A.H., and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development (Cambridge, England) 118, 401-415.

Calvert, P.D., Peet, J.A., Bragin, A., Schiesser, W.E., and Pugh, E.N., Jr. (2007). Fluorescence relaxation in 3D from diffraction-limited sources of PAGFP or sinks of EGFP created by multiphoton photoconversion. Journal of microscopy 225, 49-71.

Chen, Y., MacDonald, P.J., Skinner, J.P., Patterson, G.H., and Muller, J.D. (2006). Probing nucleocytoplasmic transport by two-photon activation of PA-GFP. Microscopy research and technique 69, 220-226.

Chudakov, D.M., Belousov, V.V., Zaraisky, A.G., Novoselov, V.V., Staroverov, D.B., Zorov, D.B., Lukyanov, S., and Lukyanov, K.A. (2003). Kindling fluorescent proteins for precise in vivo photolabeling. Nature biotechnology 21, 191-194.

Chudakov, D.M., Verkhusha, V.V., Staroverov, D.B., Souslova, E.A., Lukyanov, S., and Lukyanov, K.A. (2004). Photoswitchable cyan fluorescent protein for protein tracking. Nature biotechnology 22, 1435-1439.

Davis, R.L. (2004). Olfactory learning. Neuron 44, 31-48.

Dixit, R., and Cyr, R. (2003). Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy. Plant J 36, 280-290.

Dobritsa, A.A., van der Goes van Naters, W., Warr, C.G., Steinbrecht, R.A., and Carlson, J.R. (2003). Integrating the molecular and cellular basis of odor coding in the Drosophila antenna. Neuron 37, 827-841.

Elmore, T., and Smith, D.P. (2001). Putative Drosophila odor receptor OR43b localizes to dendrites of olfactory neurons. Insect biochemistry and molecular biology 31, 791-798.

Gurskaya, N.G., Verkhusha, V.V., Shcheglov, A.S., Staroverov, D.B., Chepurnykh, T.V., Fradkov, A.F., Lukyanov, S., and Lukyanov, K.A. (2006). Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nature biotechnology 24, 461-465.

Habuchi, S., Ando, R., Dedecker, P., Verheijen, W., Mizuno, H., Miyawaki, A., and Hofkens, J. (2005). Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proceedings of the National Academy of Sciences of the United States of America 102, 9511-9516.

Jefferis, G.S., and Hummel, T. (2006). Wiring specificity in the olfactory system. Seminars in cell & developmental biology 17, 50-65.

Jones, W.D., Cayirlioglu, P., Kadow, I.G., and Vosshall, L.B. (2007). Two chemosensory receptors together mediate carbon dioxide detection in Drosophila. Nature 445, 86-90.

Komiyama, T., and Luo, L. (2006). Development of wiring specificity in the olfactory system. Current opinion in neurobiology 16, 67-73.

Kuppers-Munther, B., Letzkus, J.J., Luer, K., Technau, G., Schmidt, H., and Prokop, A. (2004). A new culturing strategy optimises Drosophila primary cell cultures for structural and functional analyses. Developmental biology 269, 459-478.

Kwon, J.Y., Dahanukar, A., Weiss, L.A., and Carlson, J.R. (2007). The molecular basis of CO2 reception in Drosophila. Proceedings of the National Academy of Sciences of the United States of America 104, 3574-3578.

Lin, H.H., Lin, C.Y., and Chiang, A.S. (2007). Internal representations of smell in the Drosophila brain. J Biomed Sci.

Lukyanov, K.A., Chudakov, D.M., Lukyanov, S., and Verkhusha, V.V. (2005). Innovation: Photoactivatable fluorescent proteins. Nature reviews 6, 885-891.

Luo, H., Nakatsu, F., Furuno, A., Kato, H., Yamamoto, A., and Ohno, H. (2006). Visualization of the post-Golgi trafficking of multiphoton photoactivated transferrin receptors. Cell structure and function 31, 63-75.

Marin, E.C., Watts, R.J., Tanaka, N.K., Ito, K., and Luo, L. (2005). Developmentally programmed remodeling of the Drosophila olfactory circuit. Development (Cambridge, England) 132, 725-737.

Miyawaki, A., Sawano, A., and Kogure, T. (2003). Lighting up cells: labelling proteins with fluorophores. Nature cell biology Suppl, S1-7.

Mombaerts, P. (1999). Seven-transmembrane proteins as odorant and chemosensory receptors. Science (New York, NY 286, 707-711.

Patterson, G.H., and Lippincott-Schwartz, J. (2002). A photoactivatable GFP for selective photolabeling of proteins and cells. Science (New York, NY 297, 1873-1877.

Post, J.N., Lidke, K.A., Rieger, B., and Arndt-Jovin, D.J. (2005). One- and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos. FEBS letters 579, 325-330.

Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G., and Cormier, M.J. (1992). Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229-233.

Ramaekers, A., Magnenat, E., Marin, E.C., Gendre, N., Jefferis, G.S., Luo, L., and Stocker, R.F. (2005). Glomerular maps without cellular redundancy at successive levels of the Drosophila larval olfactory circuit. Curr Biol 15, 982-992.

Schermelleh, L., Spada, F., Easwaran, H.P., Zolghadr, K., Margot, J.B., Cardoso, M.C., and Leonhardt, H. (2005). Trapped in action: direct visualization of DNA methyltransferase activity in living cells. Nature methods 2, 751-756.

Schneider, M., Barozzi, S., Testa, I., Faretta, M., and Diaspro, A. (2005). Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region. Biophysical journal 89, 1346-1352.

Shang, Y., Claridge-Chang, A., Sjulson, L., Pypaert, M., and Miesenbock, G. (2007). Excitatory local circuits and their implications for olfactory processing in the fly antennal lobe. Cell 128, 601-612.

Stocker, R.F. (1994). The organization of the chemosensory system in Drosophila melanogaster: a review. Cell and tissue research 275, 3-26.

Stocker, R.F., Lienhard, M.C., Borst, A., and Fischbach, K.F. (1990). Neuronal architecture of the antennal lobe in Drosophila melanogaster. Cell and tissue research 262, 9-34.

Suh, G.S., Wong, A.M., Hergarden, A.C., Wang, J.W., Simon, A.F., Benzer, S., Axel, R., and Anderson, D.J. (2004). A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila. Nature 431, 854-859.

Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N., and Miyawaki, A. (2005). Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO reports 6, 233-238.

Verkhusha, V.V., Chudakov, D.M., Gurskaya, N.G., Lukyanov, S., and Lukyanov, K.A. (2004). Common pathway for the red chromophore formation in fluorescent proteins and chromoproteins. Chemistry & biology 11, 845-854.

Vosshall, L.B., Amrein, H., Morozov, P.S., Rzhetsky, A., and Axel, R. (1999). A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96, 725-736.

Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Rocker, C., Salih, A., Spindler, K.D., and Nienhaus, G.U. (2004). EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proceedings of the National Academy of Sciences of the United States of America 101, 15905-15910.

Wilson, R.I., and Mainen, Z.F. (2006). Early events in olfactory processing. Annual review of neuroscience 29, 163-201.

Wong, A.M., Wang, J.W., and Axel, R. (2002). Spatial representation of the glomerular map in the Drosophila protocerebrum. Cell 109, 229-241.