果蠅會感測濕度的差異並且喜歡乾的環境。除了了解完整的觸角第三節對於正常溼度感測行為是必須的之外，神經網路如何影響溼度感測行為所知無幾。結合功能性影像技術 (functional imaging) 與利用神經遺傳工具進行行為操控 (neurogenetic behavior manipulation) 的方法，我們在觸角的第三節找到一群兩極的溼度感測神經元 (bipolar HSNs)。這些神經元分布於球囊的三個空腔 (sacculus chambers，分為chamber I，chamber II 與 chamber III) 中，並與腦中的ventral posterior region (VP region，由VP1，VP2 與VP3組成) 連接。每顆兩極的溼度感測神經元的樹突 (dendrites) 從空腔內的感器 (sensillium) 將訊號送至嗅葉 (antennal lobe) 的VP region。由初步MARCM的分析得到球囊的空腔與VP region相互的連結關係。接著，結合光激發綠螢光蛋白的技術 (PaGFP) 與功能性影像技術 (functional imaging) 的結果，我們將連結VP region與高層腦內處理中心 (higher brain centers) 的投射神經元 (PNs) 分為四類，而這些高層腦內處理中心包括：calyx，lateral horn，superior dorsal frontal protocerebrum，superpeduncular protocerebrum，ventral lateral protocerebrum與 caudo-ventrolateral protocerebrum。藉由研究結果，我們發現這些所有的HSNs與VP PNs都能感受溼度的變化。藉由我們的研究揭示了果蠅的溼度感測系統。

Flies can sense humidity changes and run toward dry environment. Except knowing intact third antenna segment is necessary for normal hygrosensory behavior, neural circuits orchestrating hygrosensory behaviors are largely unknown. Combining functional imaging and neurogenetic behavior manipulation, we found a group of bipolar hygrosensory neurons (HSNs) with cell bodies located at the third antenna segment. These HSNs connect three sacculus chambers (chamber I, chamber II, and chamber III) on the antenna to three ventral posterior regions (VP1, VP2 and VP3) in the brain. Each bipolar HSN gives a single dendrite within a sensillum in the sacculus at one end and gives axonal terminals in the ventral posterior (VP) region of the antennal lobe at the other end. Initial MARCM analysis suggests a stereotyped connection between sacculus chambers and VP region. Next, combining PaGFP tracing and functional imaging, we identified four types of projection neurons (PNs) connecting VP regions to higher brain centers including calyx, lateral horn, superior dorsal frontal protocerebrum, superpeduncular protocerebrum, ventral lateral protocerebrum, and caudo-ventrolateral protocerebrum. All these HSNs and VP PNs were sensitive to humidity changes. Our study revealed a hygrosensory system in Drosophila.

致謝 2
摘要 3
Abstract 4
Introduction 5
Materials and Methods 8
Fly Stocks 8
Sample Preparation and Immunohistochemistry 8
Photoactivation of Pa-GFP 9
Mosaic Analysis with a Repressible Cell Marker (MARCM) Technique 10
Confocal Microscopy 10
Results 11
The Sacculus and Arista are Hygrosensing Related Organs 11
Identification of the Downstream of the Hygrosensory Circuits 13
Discussion 15
References 20
Figures 23
Tables 38
Appendix Figures 40

1. Couto, A., Alenius, M., and Dickson, B.J. (2005). Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr Biol 15, 1535-1547.
2. Feinberg, E.H., Vanhoven, M.K., Bendesky, A., Wang, G., Fetter, R.D., Shen, K., and Bargmann, C.I. (2008). GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron 57, 353-363.
3. Foelix, R.F., Stocker, R.F., and Steinbrecht, R.A. (1989). Fine structure of a sensory organ in the arista of Drosophila melanogaster and some other dipterans. Cell Tissue Res 258, 277-287.
4. Han, R.-D., Parajulee, M., He, Z., and Ge, F. (2008). Effects of environmental humidity on the survival and development of pine caterpillars, Dendrolimus tabulaeformis (Lepidoptera: Lasiocampidae). Insect Science, 6.
5. Iwasak, M., Itoh, T., Yokohari, F., and Taminaga, Y. (1995). Identification of Antennal Hygroreceptive Sensillum and Other Sensilla of the Firefly, Luciola cruciata. Zoological Science, 8.
6. Kamikouchi, A., Inagaki, H.K., Effertz, T., Hendrich, O., Fiala, A., Gopfert, M.C., and Ito, K. (2009). The neural basis of Drosophila gravity-sensing and hearing. Nature 458, 165-171.
7. Lee, T., and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, 451-461.
8. Lee, T., and Luo, L. (2001). Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci 24, 251-254.
9. Liu, L., Li, Y., Wang, R., Yin, C., Dong, Q., Hing, H., Kim, C., and Welsh, M.J. (2007). Drosophila hygrosensation requires the TRP channels water witch and nanchung. Nature 450, 294-298.
10. Nichols, C.D. (2006). Drosophila melanogaster neurobiology, neuropharmacology, and how the fly can inform central nervous system drug discovery. Pharmacol Ther 112, 677-700.
11. Patterson, G.H., and Lippincott-Schwartz, J. (2002). A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873-1877.
12. Rajashekar, K.P., and Shamprasad, V.R. (2004). Maxillary palp glomeruli and ipsilateral projections in the antennal lobe of Drosophila melanogaster. J Biosci 29, 423-429.
13. Sayeed, O., and Benzer, S. (1996). Behavioral genetics of thermosensation and hygrosensation in Drosophila. Proc Natl Acad Sci U S A 93, 6079-6084.
14. Schmidt-Nielsen, K., Schroter, R.C., and Shkolnik, A. (1981). Desaturation of Exhaled Air in Camels Proceedings of the Royal Society of London Series B, Biological Sciences 211, 15.
15. Shanbhag, S.R., Singh, K., and Singh, R.N. (1995). Fine structure and primary sensory projections of sensilla located in the sacculus of the antenna of Drosophila melanogaster. Cell Tissue Res 282, 237-249.
16. Stocker, R.F. (1994). The organization of the chemosensory system in Drosophila melanogaster: a review. Cell Tissue Res 275, 3-26.
17. Stocker, R.F., Singh, R.N., Schorderet, M., and Siddiqi, O. (1983). Projection patterns of different types of antennal sensilla in the antennal glomeruli of Drosophila melanogaster. Cell Tissue Res 232, 237-248.
18. T. Itoh, F., Yokohari, Tanimura, T., and Tominaga, Y. (1991). External morphology of sensilla in the sacculus of an antennal flagellum of the fruit fly Drosophila melanogaster meigen (Diptera.Drosophilidae). Int J Insect Morphol & Embryol 20, 8.
19. Tichy, H. (2007). Humidity-dependent cold cells on the antenna of the stick insect. J Neurophysiol 97, 3851-3858.
20. Tichy, H., and Kallina, W. (2010). Insect hygroreceptor responses to continuous changes in humidity and air pressure. J Neurophysiol 103, 3274-3286.
21. Vosshall, L.B., and Stocker, R.F. (2007). Molecular architecture of smell and taste in Drosophila. Annu Rev Neurosci 30, 505-533.
22. Yorozu, S., Wong, A., Fischer, B.J., Dankert, H., Kernan, M.J., Kamikouchi, A., Ito, K., and Anderson, D.J. (2009). Distinct sensory representations of wind and near-field sound in the Drosophila brain. Nature 458, 201-205.