臺灣博碩士論文加值系統

在脊椎動物，N-methyl-D-aspartate subtype of glutamate receptor (NMDA receptor)在神經發育，神經可塑性以及記憶的形成過程中扮演著重要的角色，然而到目前為止，有關於非脊椎動物的NMDA receptor的研究相當有限， NMDA receptor在果蠅腦中的表現位置更是一概不知，此篇論文主要是利用免疫螢光標示的方法觀察drosophila NMDA receptor 1 & 2 (DrNR1 & DrNR2) 在果蠅腦中的分布。首先利用大腸桿菌(E.coli)表現果蠅NMDA receptor 2 cDNA序列2190~2651，可以得到一個分子量大約為20.8kDa的蛋白質片段，接著以 Ni column純化出此蛋白質片段，將此蛋白質片段命名為 : DrNR2-L1，利用此蛋白質片段當抗原，分別免疫老鼠跟兔子生產出果蠅NMDA receptor 2的單株抗體與多株抗體，之後再以免疫沉澱跟西方點墨法證明果蠅腦中確實存在著NMDA receptors。然後利用免疫染色的方式並配合共軛焦雷射顯微鏡的技術，找出NMDA receptors 在果蠅腦中所表現的位置，結果發現 DrNR2 主要表達在果蠅腦中少數幾顆細胞，而且跟DrNR1並非完全表現在相同的細胞。為了了解細胞內有哪一些分子是和DrNR互相調控的，利用一些長期記憶缺損的果蠅突變株進行檢查，結果發現在腦中一對位於dorsal anterior lateral (DAL)的神經細胞不但有表現DrNR1，同時在一些果蠅的突變株 : C133 (phospholipase A2) , krasavietz (eIF-5C) , pastrel (novel)等也有表現。另外Nitric oxide synthase (NOS)也被證明有表現在神經細胞DAL上面。由實驗結果發現許多跟記憶有關的基因都在此細胞有所表達，因此可以推論DAL細胞很可能在記憶形成的過程扮演著重要的角色。

In vertebrates, the N-methyl-D-aspartate subtype of glutamate receptors (NMDA receptor) play an important role in neuronal development, synaptic plasticity and, memory formation. However, there are few researches about NMDAR of invertebrate. So far, The expression patterns and function of NMDA receptors in insect brain are less understood. Here, We labeled drosophila NMDA receptor 1 & 2 (DrNR1 & DrNR2)DrNR1 & DrNR2 in fly brain by immunohistochemical localization. The Drosophila NMDA receptor subunit 2 (DrNR2) 2190~2651 cDNA was constructed in an E. coli expression vector (pET15b). A 20.8KDa fusion protein was purified for generation of polyclonal and monoclonal antibodies. Spatial distribution of DrNR1 and DrNR2 in the brain was identified using immunohistochemical labeling. Surprisingly, DrNR2 expresses in only few cells and are not all colocalized with DrNR1. To identify other proteins working together with DrNR, the expression of several memory mutants were examined. Among brain neurons expressing DrNR1, a pair of dorsal-anterior-lateral (DAL) neuron also expressed in C133 (encode phospholipase A2), krasavietz (encode eIF-5C), pastrel (novel). Nitric oxide synthase (NOS) also expressed in DAL neuron. Co-expression of several candidate memory genes suggests that DAL neuron may implicate an important role in memory formation.

中文摘要 1
英文摘要 2
導論 3
實驗材料及方法 11
結果 18
討論 24
參考資料 29
圖表 37
圖說 51
附錄 56

Barria, A., Derkach, V., & Soderling, T. (1997). Identification of the Ca2+/ calmodulin-dependent protein kinaseII regulatory phosphorylation site in thealpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-type glutamate receptor. Journal of Biological Chemistry. 272, 32727—32730.
Bliss, T.V.P. & Collingridge, G.L. (1993). A synaptic model of memory: long-term potentiation in the hippocampus. Nature. 36, 31-39.
Böhme GA, Bon C, Lemaire M, Reibaud M, Piot O, Stutzmann JM, Doble
A, Blanchard JC. (1993). Altered synaptic plasticity and memory formation in nitric oxide synthase inhibitor-treated rats. Proc Natl Acad Sci U S A. 90, 9191-9194.
Brockie, P.J., Mellem, J.E., Hills, T., Madsen, D.M. & Maricq, A.V. (2001). The C. elegans glutamate receptor subunit NMR-1 is required for slow NMDA -activated currents that regulate reversal frequency during locomotion. Neuron . 31, 617630.
Burnashev, N., Schoepfer, R., Monyer, H., Ruppersberg, J.P., Gunther, W., Seeburg, P.H. & Sakmann, B. (1992). Control by asparagines residues of calcium permeability and magnesium blockade in the NMDA receptor. Science. 257, 14151419.
Chazot, P. L., & Stephenson, F. A. (1997). Molecular dissection of native mammalian forebrain NMDA receptors containing the NR1 C2 exon: direct demonstration of NMDA receptors comprising NR1, NR2A, and NR2B subunits within the same complex. J Neurochem. 69, 2138— 2144.
Chiang, A.-S., Lin, W.-Y., Liu, H.-P., Pszczolkowski, M.A., Fu, T.-F., Chiu, S.-L. & Holbrook, G.L. (2002). Insect NMDA receptors mediate juvenile hormone biosynthesis. Proc. Natl. Acad. Sci. USA. 99, 3742.
Collingridge, G.L. & Singer, W. (1990). Excitatory amino acid receptors and synaptic plasticity. Trends Pharmacol. Sci. 11, 379-387
Das, S., Sasaki, Y. F., Rothe, T., Premkumar, L. S., Takasu, M., Crandall,
J. E., Dikkes, P., Conner, D. A., Rayudu, P. V., Cheung,W., Chen, H. S.,
Lipton, S. A., & Nakanishi, N. (1998). Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A. Nature. 393, 377—381.
Davies S. (2000). Nitric oxide signalling in insects. Insect Biochem. Mol. Biol.
30, 1123-1138.
Dale, N. & Kandel, E.R. (1993). L-glutamate may be the fast excitatory transmitter of Aplysia sensory neurons. Proc. Natl. Acad. Sci. USA. 90, 71637167.
Daniel C, Serge Birman. (2001). Blockade of the Central Generator of locomotor rhythm by noncompetitive NMDA receptor antagonists in drosophila larvae. J. Neurobiology. 48, 58-73.
Davis, R. L. (1993). Mushroom bodies and Drosophila learning. Neuron. 11, 1—14.
Dubnau J, Chiang AS, Grady L, Barditch J, Gossweiler S, McNeil J, Smith P, Buldoc, Scott R, Certa U, Broger C, Tully T. (2003). The staufen/pumilio pathway is involved in Drosophila long-term memory. Curr. Biol. 13, 286-296.
Dubnau J, Grady L, Kitamoto T, Tully T. (2001). Disruption of neurotransmission in Drosophila mushroom body blocks retrieval but not acquisition of memory. Nature. 411, 476-480.
Dubnau, J. & Tully, T. (1998). Gene discovery in Drosophila: new insights for learning and memory. Ann. Rev. Neurosc i. 21, 407-444.
Eriksson, M., Nilsson, A., Froelich-Fabre, S., Akesson, E., Dunker, J.,
Seiger, A., Folkesson, R., Benedikz, E., & Sundstrom, E. (2002). Cloning
and expression of the human N-methyl-D-aspartate receptor subunit
NR3A. Neurosci Lett. 321, 177— 181.
Ferrer-Montiel, A.V., Sun, W. & Montal, M. (1995). Molecular design of the N-methyl- D-aspartate receptor binding site for phencyclidine and dizolcipine. Proc. Natl. Acad. Sci. USA. 92, 80218025.
Giese, K. P., Fedorov, N. B., Filipkowski, R. K., & Silva, A. J. (1998). Autophosphorylation at Thr286 of the alpha calcium—calmodulin kinase II in LTP and learning. Science. 279, 870—873.
Goebel, D. J., & Poosch, M. S. (1999). NMDA receptor subunit gene expression in the rat brain: a quantitative analysis of endogenous mRNA levels of NR1Com, NR2A, NR2B, NR2C, NR2D and NR3A. Brain Res Mol Brain Res. 69, 164— 170.
Granger, N. A., Sturgis, S. L., Ebersohl, R., Geng, C. & Sparks, T. C. (1996) Arch. Insect Biochem. Physiol. 32, 449—466.
Hollmann, M., Boulter, J., Maron, C., Beasley, L., Sullivan, J., Pecht, G., &
Heinemann, S. (1993). Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor. Neuron. 10, 943— 954.
Hollmann, M., & Heinemann, S. (1994). Cloned glutamate receptors. Annu Rev Neurosci. 17, 31— 108.
Ishii, T., Moriyoshi, K., Sugihara, H., Sakurada, K., Kadotani, H., Yokoi, M., Akazawa, C., Shigemoto, R., Mizuno, N., & Masu, M. (1993). Molecular characterization of the family of the N-methyl-D-aspartate receptor subunits. J Biol Chem. 268, 2836—2843.
Jennifer M. Loftis, Aaron Janowsky. (2003). The N-methyl-D-aspartate receptor subunit NR2B: localization,functional properties, regulation, and clinical implications . Pharmacology & Therapeutics. 97, 55— 85.
Johnston, M. V., Trescher, W. H., Ishida, A., & Nakajima, W. (2000). Novel
treatments after experimental brain injury. Semin Neonatol. 5, 75— 86.
Kaczmarek, L., Kossut, M. & Skangiel-Kramska, J. (1997). Glutamate receptors in cortical plasticity: molecular and cellular biology. Physiol. Re. 77, 217-255.
Kutsuwada, T., Kashiwabuchi, N., Mori, H., Sakimura, K., Kushiya, E., Araki, K., Meguro, H., Masaki, H., Kumanishi, T., & Arakawa, M. (1992). Molecular diversity of the NMDA receptor channel. Nature. 358 , 36— 41.
Laube, B., Kuhse, J., & Betz, H. (1998). Evidence for a tetrameric structure of recombinant NMDA receptors. J Neurosci. 18, 2954—2961.
Laurie, D. J., Bartke, I., Schoepfer, R., Naujoks, K., & Seeburg, P. H.Regional. (1997). developmental and interspecies expression of the four NMDAR2 subunits, examined using monoclonal antibodies. Brain Res Mol Brain Res. 51, 23— 32.
Mammen, A. L., Kameyama, K., Roche, K. W., & Huganir, R. L. (1997). Phosphorylation of the alpha-amino- 3-hydroxy -5-methylisoxazole -4- propionic acid receptor GluR1 subunit by calcium/calmodulin-dependent kinase II. Journal of Biological Chemistry. 272, 32528—32533.
McBain, C. J., & Mayer, M. L. (1994). N-methyl-D-aspartic acid receptor structure and function. Physiol Rev. 74 , 723— 760.
Moriyoshi, K., Masu, M.,Ishii, T., Shigemoto, R.,Mizuno, N., and Nakanishi, N. (1991). Molecular cloning and characterization of the rat NMDA receptor. Nature. 354, 31-37.
Nakazawa, T., Komai, S., Tezuka, T., Hisatsune, C., Umemori, H., Semba, K., Mishina, M., Manabe, T., & Yamamoto, T. (2001). Characterization of Fyn-mediated tyrosine phosphorylation sites on GluRe2 (NR2B) subunit of the N-methyl-D-aspartate receptor. J Biol Chem. 276, 693—699.
Nishi, M., Hinds, H., Lu, H. P., Kawata, M., & Hayashi, Y.. Motor. (2001).
neuron-specific expression of NR3B, a novel NMDA-type glutamate
receptor subunit that works in a dominant-negative manner. J Neurosci.
21 , RC185.
Pfeiffer-Linn, C. & Glantz, R.M. (1991). An arthropod NMDA receptor. Synapse. 9, 3542.
Premkumar, L. S., & Auerbach, A. (1997). Stoichiometry of recombinant N-methyl-D-aspartate receptor channels inferred from single-channel current patterns. J Gen Physiol. 110, 485— 502.
Roche, K. W., O’Brien, R. J., Mammen, A. L., Bernhardt, J., & Huganir, R. L. (1996). Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit. Neuron. 16, 1179—1188.
Sans, N., Petralia, R. S., Wang, Y. X., Blahos, J., Hell, J. W., & Wenthold,R. J. (2000). A developmental change in NMDA receptor -associated proteins at hippocampal synapses. J Neurosci. 20, 1260— 1271.
Sattler, R., Xiong, Z., Lu, W. Y., Hafner, M., MacDonald, J. F., & Tymianski,
M. (1999). Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein. Science. 284, 1845— 1848.
Sattler, R., & Tymianski, M. (2000). Molecular mechanisms of calcium dependent excitotoxicity. J Mol Med. 78 , 3 —13.
Takasu, M.A., Dalva, M.B., Zigmond, R.E. & Greenberg, ME. (2002). Modulation of NMDA receptor-dependent calcium influx and gene expression through EphB eceptors. Science. 295, 491—95.
Tang, Y.P. ., Shimizu., E., Dube, G. R., Rampon, C., Kerchner, G.A., Zhuo, M., Liu, G. &. Tsien J. Z. (1999). Genetic enhancement of learning and memory in mice. Nature. 401, 63-69.
Thompson, C. S., Yagi, K. J., Chen, Z. F. & Tobe, S. S. (1990) J. Comp. Physiol.B. 160, 241—249.
Siegmund, T. & Korge, G. (2001). Innervation of the ring gland of drosophila melanogaster. J Comp Neurol. 431, 481-491.
Yin J.C. & Tully, T. (1996). CREB and the formation of long-term memory. Curr Opin Neurobiol. 6(2), 264-268.
Ultsch, A., Schuster, C. M., Laube, B., Betz, H. & Schmitt, B. (1993). Glutamate receptors of Drosophila melanogaster: primary structure of a putative NMDA receptor protein expressed in the head of the adult fly. FEBS Lett. 324, 171—177.
Volkner, M., Lenz-Bohme, B., Betz, H. & Schmitt, B. (2000). Novel CNS glutamate receptor subunit genes of Drosophila melanogaster. J. Neuronchem. 75, 17911799.
Yu Y, Armstrong JD, Clark J, Kaiser K. (1997). Molecular cloning of genes related to Drosophila olfactory function. J Neurogenet. 11, 212.