摘要

免疫染色是一種廣泛利用在研究神經多胜肽的方法，目前並被視為得知神經多胜肽在生物體中分佈的最好的方法。經由免疫染色，我們可以知道不同的神經多胜肽在果蠅的腦中所表現的位置。通常不同的神經多胜肽在腦中所表現的位置具有高度的相異性，因此我們利用以下幾種抗體多胜肽：Allatostatin-A, Allatostatin-C, Allatotropin, Amnesiac, pyrokinin以及pdf (pigment dispersing factor)針對果蠅大腦進行免疫染色，經由共軛焦顯微鏡掃成圖像，再由Amira的軟體分析，將表達上述幾種神經多胜肽的神經由腦中單獨分離出來，並以細胞本體在腦中的所在位置命名，然後套到標準腦中，便可得到一個初步的各種神經多胜肽在果蠅腦中所表現的地圖。在這個神經多胜肽的表現的地圖裡，有表現Allatastatin-A的DAL3, VAL1以及VAL2神經，表現Allatostatin-C的VPL神經，表現Amnesiac的VPL2神經，表現Pyrokinin的PM神經，表現pdf的LNvs以及同時表現Allatotropin以及Amnesiac的DPM2神經。

此外，免疫染色做為辨認位於不同果蠅株的細胞是否為同一顆的工具；例如我們利用Amnesiac的抗體辨識了位於G0054，G0086，G0338，E0101及E0163等GAL4果蠅株中的一對大細胞都是表現AMN的VPL2神經。我們假設與AMN有colocalize的果蠅株在嗅覺學習上也有缺失，如果經由行為測驗證實了這個推論，我們便可利用更多和學習與記憶有關的蛋白當作抗原，對大量的GAL4株進行免疫染色篩選，由染色和GFP的colocalization快速蒐集到大量的果蠅株當作研究學習與記憶的行為測試的工具，搭起免疫染色和學習與記憶兩個不同領域間的橋樑。

Abstract

Immunohistochemistry is often used to analyze expression patterns of different neuropeptides in our laboratory. Single neuron was segmented from image stacks of immunohistochemical results or Gal4 lines that were crossed to flies carrying reporter genes (Green fluorescent protein) and transformed into the standard brain by using Amira program. DAL3, VAL1 and VAL2 expressing Allatostatin-A, VPL expressing Allatostatin-C, DPM2 expressing both Allatostatin-C and Allatotropin, VPL2 expressing Ammesia, PM expressing Pyrokinin, and LNvs expressing pdf (pigment dispersing element) were analyzed separately and then put together in a standard brain to display as a simple neuropeptide-expression map. Furthermore, we have known that Amnesiac (AMN), a neuropeptide that is associated with learning and memory in Drosophila, expressed in VPL2 neuron and VPL2 is also expressed in several GAL4 lines as G0054, G0086, G0338, E0101 and E0163 by immunohistochemistry. We presume that GAL4 lines colocalized with AMN are also associated with learning and memory as other amnesiac mutant lines. If it is proved by behavior tests, we can use antibodies against other proteins associated with learning and memory to perform a large scale screen of all GAL4 lines. Finally, we can collect lots of GAL4 line colocalized with the protein and use them as tools for behavior tests to know more about learning and memory. Thus, immunohistochemistry can be a simple and fast method for us to collect lots of tools in studying learning and memory in Drosophila.

參考文獻

Bendena,W.G., B.C.Donly, and S.S.Tobe. 1999. Allatostatins: a growing family of neuropeptides with structural and functional diversity. Ann. N. Y. Acad. Sci. 897:311-329.
Byers,D., R.L.Davis, and J.A.Kiger, Jr. 1981. Defect in cyclic AMP phosphodiesterase due to the dunce mutation of learning in Drosophila melanogaster. Nature 289:79-81.
Davis,N.T., U.Homberg, P.E.Teal, M.Altstein, H.J.Agricola, and J.G.Hildebrand. 1996. Neuroanatomy and immunocytochemistry of the median neuroendocrine cells of the subesophageal ganglion of the tobacco hawkmoth, Manduca sexta: immunoreactivities to PBAN and other neuropeptides. Microsc. Res. Tech. 35:201-229.
Davis,R.L. 1996. Physiology and biochemistry of Drosophila learning mutants. Physiol Rev. 76:299-317.
Davis,R.L. 1993. Mushroom bodies and Drosophila learning. Neuron 11:1-14.
Feany,M.B. and W.G.Quinn. 1995. A neuropeptide gene defined by the Drosophila memory mutant amnesiac. Science 268:869-873.
Hewes,R.S. and P.H.Taghert. 2001. Neuropeptides and neuropeptide receptors in the Drosophila melanogaster genome. Genome Res. 11:1126-1142.
Holman,G.M., B.J.Cook, and R.J.Nachman. 1986. Primary structure and synthesis of a blocked myotropic neuropeptide isolated from the cockroach, Leucophaea maderae. Comp Biochem. Physiol C. 85:219-224.
Lenz,C., M.Williamson, and C.J.Grimmelikhuijzen. 2000. Molecular cloning and genomic organization of an allatostatin preprohormone from Drosophila melanogaster. Biochem. Biophys. Res. Commun. 273:1126-1131.
Lorenz,M.W., R.Kellner, and K.H.Hoffmann. 1995. Identification of two allatostatins from the cricket, Gryllus bimaculatus de Geer (Ensifera, Gryllidae): additional members of a family of neuropeptides inhibiting juvenile hormone biosynthesis. Regul. Pept. 57:227-236.
Nassel,D.R. 2002. Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones. Prog. Neurobiol. 68:1-84.
Rao,K.R., C.J.Mohrherr, J.P.Riehm, C.A.Zahnow, S.Norton, L.Johnson, and G.E.Tarr. 1987. Primary structure of an analog of crustacean pigment-dispersing hormone from the lubber grasshopper Romalea microptera. J. Biol. Chem. 262:2672-2675.
Renn,S.C., J.H.Park, M.Rosbash, J.C.Hall, and P.H.Taghert. 1999. A pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila. Cell 99:791-802.
Zars,T. 2000. Behavioral functions of the insect mushroom bodies. Curr. Opin. Neurobiol. 10:790-795.
Zupanc,G.K. 1996. Peptidergic transmission: from morphological correlates to functional implications. Micron. 27:35-91.
de Belle,J.S. and M.Heisenberg. 1994. Associative odor learning in Drosophila abolished by chemical ablation of mushroom bodies. Science 263:692-695
Edwards,J.P., N.Audsley, G.C.Marris, M.Cusson, and R.J.Weaver. 2001. The role of allatostatic and allatotropic neuropeptides in the regulation of juvenile hormone biosynthesis in Lacanobia oleracea (Lepidoptera: Noctuidae). Peptides 22:255-261.
Moore,M.S., J.DeZazzo, A.Y.Luk, T.Tully, C.M.Singh, and U.Heberlein. 1998. Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway. Cell 93:997-1007.
Qiu,Y. and R.L.Davis. 1993. Genetic dissection of the learning/memory gene dunce of Drosophila melanogaster. Genes Dev. 7:1447-1458.
Choi,M.Y., A.Rafaeli, and R.A.Jurenka. 2001. Pyrokinin/PBAN-like peptides in the central nervous system of Drosophila melanogaster. Cell Tissue Res. 306:459-465
Kataoka,H., R.G.Troetschler, S.J.Kramer, B.J.Cesarin, and D.A.Schooley. 1987. Isolation and primary structure of the eclosion hormone of the tobacco hornworm, Manduca sexta. Biochem. Biophys. Res. Commun. 146:746-750.
Strand,F.L. 1999. New vistas for melanocortins. Finally, an explanation for their pleiotropic functions. Ann. N. Y. Acad. Sci. 897:1-16.
Strausfeld,N.J., L.Hansen, Y.Li, R.S.Gomez, and K.Ito. 1998. Evolution, discovery, and interpretations of arthropod mushroom bodies. Learn. Mem. 5:11-37.
Sato,Y., M.Ikeda, and O.Yamashita. 1994. Neurosecretory cells expressing the gene for common precursor for diapause hormone and pheromone biosynthesis-activating neuropeptide in the suboesophageal ganglion of the silkworm, Bombyx mori. Gen. Comp Endocrinol. 96:27-36.
Waddell,S., J.D.Armstrong, T.Kitamoto, K.Kaiser, and W.G.Quinn. 2000. The amnesiac gene product is expressed in two neurons in the Drosophila brain that are critical for memory. Cell 103:805-813.
Waddell,S. and W.G.Quinn. 2001. What can we teach Drosophila? What can they teach us? Trends Genet. 17:719-726.
Waddell,S. and W.G.Quinn. 2001. Flies, genes, and learning. Annu. Rev. Neurosci. 24:1283-1309.
Williamson,M., C.Lenz, A.M.Winther, D.R.Nassel, and C.J.Grimmelikhuijzen. 2001. Molecular cloning, genomic organization, and expression of a C-type (Manduca sexta-type) allatostatin preprohormone from Drosophila melanogaster. Biochem. Biophys. Res. Commun. 282:124-130.