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생물학

수확 및 준비 Drosophila 태아

Published: May 20, 2009
doi:
10.3791/1347
, ,
1Department of Biological Sciences,University of Illinois, 2Department of Biological Sciences,Vanderbilt University

Summary

이 기술은 immunohistochemistry 또는 electrophysiological 레코딩을위한 Drosophila 배아의 neuromusculature를 제공​​합니다. 그것은 신경근육학 개발 초기에 이벤트를 공부하거나 부화 수없는 돌연변이의 전기 생리학을 수행하는 데 유용합니다.

Abstract

Drosophila는 배아 발달과 기능 모두의 신경 과학 연구를위한 최고의 유전자 모델입니다. 전통적으로,이 필드는 주로 독립적인 역사 및 과학 사회와 서로 꽤을 분리하고 있습니다. 그러나 이러한 일반적으로 서로 다른 분야 사이의 인터페이스는 기능 전기적 신호 특성과 신경 회로 형성의 마지막 단계에서 기능성 화학 시냅스의 분화 취득을 기본 발달 프로그램입니다. 이 인터페이스는 조사를위한 중요한 지역입니다. Drosophila에서 기능의 개발이 단계는 embryogenesis의 마지막 세번째 동안 <8시간 기간 (25 ° C에서)하는 동안 발생합니다. 이렇게 늦게 발달 기간 동안 힘든, 스며들지 표피 표피의 증착에 수사 때문에하는 어려운 여겨졌다. 획기적인 사전은 로컬 말기 태아의 제어 절개를 사용하는 표피에 적용할 수있는 물 polymerizing 수술 접착제의 적용되었다. 지느러미 세로 절개로 배아가 실험 조사하기 위해 복부 신경 코드 및 신체 벽면 근육을 노출, 평면 마련하실 수 있습니다. 이 제도는 크게 배아 버렸네 형성에 악영향을하고, 따라서 버렸네 연결과 기능 시냅스 신호 속성의 사양과 차별을 규제하는 분자 메커니즘을 밝혀 유전자 돌연변이를 분리하고 특성화하는 데 사용되었습니다.

Protocol

1 부 : 장비 및 소모품 좋은 해부 현미경은 배아 해부가 필요합니다, 40X 배율은 maximally 배율을 증가 25X 눈길을 조각으로 제안합니다. 좋은 포셉 (숫자 5) 배아와 태아의 devitellinization의 수동 선택이 필요합니다. 미세 유리 바늘을 만들고 수정할 수있는 장비가 필요합니다. 아이고 – 유리 바늘은 절개가 필요합니다. 우리는 해부 (오래)에 대한 단단한 유리를 선호하지만, 표준 ?…

Discussion

배아의 정확한 준비로 인해 단지 몇 시간의 시간 코스 이상의 기능 속성의 급속한 성숙에 중요합니다. 몇 가지 문제는이 준비를 복잡하게. 첫째, 대부분의 연구자들은 초과 계란 단계 배아에 들이죠를 사용하지만, 계란 누워있는 시간이 서로 다른 조건 (Broadie 외. 1992)의 동물에서 동물로 매우 다를 수 있습니다. 특히, 제한된 다이어트 여성 전에 누워로 연장 기간 동안 수정된 달걀을 유지하는 경?…

Acknowledgements

킬로바이트는 NIH 부여 GM54544에 의해 지원됩니다.

Materials

Material Name Type Company Catalogue Number Comment
Embryo Collection Cages   Genesee Scientific (www.flystuff.com) 59-100 (for 60 mm dish; other sizes available) Cages can also be home made using punctured tri-pour beakers, as shown in video
Sylgard 184   Dow Corning Available from various companies Surgical glue adheres better to sylgard-coated coverslips
Fine glass tubing, outer diameter 1.0-1.5 mm   various   For pulling into fine glass needles for dissection and tubes for glue delivery
Plastic tubing, to attach to glass pipette for mouth suction and glue application   Tygon   Tubing inner diameter needs to match glass outer diameter.
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References

  1. Aravamudan, B., Fergestad, T., Davis, W. S., Rodesch, C. K., Broadie, K. Drosophila UNC-13 is essential for synaptic transmission. Nat. Neurosci. 2, 965-971 (1999).
  2. Auld, V. J., Fetter, R. D., Broadie, K., Goodman, C. S. Gliotactin a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila. Cell. 81, 757-767 (1995).
  3. Baines, R. A., Bate, M. Electrophysiological development of central neurons in the Drosophila embryo. J. Neurosci. 18, 4673-4683 (1998).
  4. Baines, R. A., Robinson, S. G., Fujioka, M., Jaynes, J. B., Bate, M. Postsynaptic expression of tetanus toxin light chain blocks synaptogenesis in Drosophila. Curr. Biol. 9, 1267-1270 (1999).
  5. Baines, R. A., Uhler, J. P., Thompson, A., Sweeney, S. T., Bate, M. Altered electrical properties in Drosophila neurons developing without synaptic transmission. J. Neurosci. 21, 1523-1531 (2001).
  6. Bate, M. The embryonic development of the larval muscles in Drosophila. Development. 110, 791-804 (1990).
  7. Bate, M., Martinez Arias, A., Bate, M., Martinez Arias, A. . The Development of Drosophila melanogaster. , (1993).
  8. Baumgartner, S., JT, L. i. t. t. l. e. t. o. n., Broadie, K., MA, B. h. a. t., Harbecke, R., JA, L. e. n. g. y. e. l., Chiquet-Ehrismann, R., Prokop, A., Bellen, H. J. A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function. Cell. 87, 1059-1068 (1996).
  9. AH, B. r. a. n. d. Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 118, 401-415 (1993).
  10. Brand, A. GFP as a cell and developmental marker in the Drosophila nervous system. Methods Cell Biol. 58, 165-181 (1999).
  11. Broadie, K., Sullivan, W., Ashburner, M., Hawley, R. S. Electrophysiological Approaches to the Neuromusculature. Drosophila Protocols. , 273-296 (2000).
  12. Broadie, K., Bate, M. Development of the embryonic neuromuscular synapse of Drosophila melanogaster. J. Neurosci. 13, 144-166 (1993a).
  13. Broadie, K., Bate, M. Development of larval muscle properties in the embryonic myotubes of Drosophila melanogaster. J. Neurosci. 13, 167-180 (1993b).
  14. Broadie, K., Bate, M. Activity-dependent development of the neuromuscular synapse during Drosophila embryogenesis. Neuron. 11, 607-619 (1993c).
  15. Broadie, K., Bate, M. Synaptogenesis in the Drosophila embryo: innervation directs receptor synthesis and localization. Nature. 361, 350-353 (1993d).
  16. Broadie, K., Bellen, H. J., DiAntonio, A., Littleton, J. T., Schwarz, T. L. The absence of Synaptotagmin disrupts excitation-secretion coupling during synaptic transmission. Proc. Natl. Acad. Sci. USA. 91, 10727-10731 (1994).
  17. Broadie, K., Prokop, A., Bellen, H. J., O’Kane, C. J., Schulze, K. L., Sweeney, S. T. Syntaxin and Synaptobrevin function downstream of vesicle docking in Drosophila. Neuron. 15, 663-673 (1995).
  18. Broadie, K., Rushton, E., Skoulakis, E. C. M., Davis, R. L. e. o. n. a. r. d. o. a 14-3-3 protein involved in learning, regulates presynaptic function. Neuron. 19, 391-402 (1997).
  19. Broadie, K., Skaer, H., Bate, M. Whole-embryo culture of Drosophila: development of embryonic tissues in vitro. Roux’s Arch. Develop. Biol. 201, 364-375 (1992).
  20. Campos-Ortega, J., Hartenstein, V. . The embryonic development of Drosophila melanogaster. , (1985).
  21. Deitcher, D. L., Ueda, A., Stewart, B. A., Burgess, R. W., Kidokoro, Y., Schwartz, T. L. Distinct requirements for evoked and spontaneous release of neurotransmitter are revealed by mutations in the Drosophila gene neuronal-synaptobrevin. J. Neurosci. 18, 2028-2039 (1998).
  22. Featherstone, D. E., Broadie, K. Surprises from Drosophila: genetic mechanisms of synaptic development and plasticity. Brain Res. Bull. 53, 501-511 (2000).
  23. Featherstone, D. E., Rushton, E. M., Hilderbrand-Chae, M., Phillips, A. M., Jackson, F. R., Broadie, K. Presynaptic glutamic acid decarboxylase is required for induction of the postsynaptic receptor field at a glutamatergic synapse. Neuron. 27, 71-84 (2000).
  24. Featherstone, D. E., Davis, W. S., Dubreuil, R. R., Broadie, K. Drosophila alpha- and beta-spectrin mutations disrupt presynaptic neurotransmitter release. J Neurosci. 21, 4215-4224 (2001).
  25. Featherstone, D. E., Rushton, E., Broadie, K. Developmental regulation of glutamate receptor field size by nonvesicular glutamate release. Nat Neurosci. 5, 141-146 (2002).
  26. Featherstone, D. E., Rushton, E., Rohrbough, J., Liebl, F., Karr, J., Sheng, Q., Rodesch, C. K., Broadie, K. An essential Drosophila glutamate receptor subunit that functions in both central neuropil and neuromuscular junction. J. Neurosci. 25, 3199-3208 (2005).
  27. Fergestad, T., Davis, W. S., Broadie, K. The stoned proteins regulate synaptic vesicle recycling in the presynaptic terminal. J Neurosci. 19, 5847-5860 (1999).
  28. Fergestad, T., Wu, M. N., Schulze, K. L., Lloyd, T. E., Bellen, H. J., Broadie, K. Targeted mutations in the syntaxin H3 domain specifically disrupt SNARE complex function in synaptic transmission. J Neurosci. 21, 9142-9150 (2001).
  29. Fergestad, T., Broadie, K. Interaction of stoned and synaptotagmin in synaptic vesicle endocytosis. J Neurosci. 21, 1218-1227 (2001).
  30. Goodman, C. S., Doe, C. Q., Bate, M., Martinez Arias, A. Embryonic Development of the Drosophila Central Nervous System. In The Development of Drosophila melanogaster. , 1131-1206 (1993).
  31. Harrison, S. D., Broadie, K., Goor, J. v. a. n. d. e., Rubin, G. M. Mutations in the Drosophila Rop gene suggest a function in general secretion and synaptic transmission. Neuron. 13, 555-566 (1994).
  32. Huang, F. D., Woodruff, E., Mohrmann, R., Broadie, K. Rolling blackout is required for synaptic vesicle exocytosis. J. Neurosci. 26, 2369-2379 (2006).
  33. Jan, L. Y., Jan, Y. N. Properties of the larval neuromuscular junction in Drosophila melanogaster. J. Physiol. 262, 189-214 (1976).
  34. Jan, L. Y., Jan, Y. N. L-glutamate as an excitatory transmitter at the Drosophila larval neuromuscular junction. J. Physiol. 262, 215-236 (1976b).
  35. Kidokoro, Y., Nishikawa, K. I. Miniature endplate currents at the newly formed neuromuscular junction in Drosophila embryos and larvae. Neuroscience Research. 19, 143-154 (1994).
  36. Landgraf, M., Bossing, T., Technau, G. M., Bate, M. The origin, location, and projections of the embryonic abdominal motorneurons of Drosophila. J. Neurosci. 17, 9642-9655 (1997).
  37. Mohrmann, R., Matthies, H. J., Woodruff III, E., Broadie, K. Stoned B mediates sorting of integral synaptic vesicle proteins. 신경과학. 153, 1048-1063 (2008).
  38. Nishikawa, K. I., Kidokoro, Y. Junctional and extrajunctional glutamate receptor channels in Drosophila embryos and larvae. J. Neurosci. 15, 7905-7915 (1995).
  39. Renden, R., Berwin, B., Davis, W., Ann, K., Chin, C. T., Kreber, R., Ganetzky, B., Martin, T. F., Broadie, K. Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion. Neuron. 31, 421-437 (2001).
  40. Rohrbough, J., Broadie, K. Electrophysiological Analysis of Synaptic Transmission in Central Neurons of Drosophila Larvae. J. Neurophysiol. 88, 847-860 (2002).
  41. Rohrbough, J., Rushton, E., Palanker, L., Woodruff, E., Matthies, H. J., Acharya, U., Acharya, J. K., Broadie, K. Ceramidase regulates synaptic vesicle exocytosis and trafficking. J. Neurosci. 24, 7789-7803 (2004).
  42. Rohrbough, J., Rushton, E., Woodruff, E. 3. r. d., Fergestad, T., Vigneswaran, K., Broadie, K. Presynaptic establishment of the synaptic cleft extracellular matrix is required for postsynaptic differentiation. Genes Dev. 21, 2607-2628 (2007).
  43. Stewart, B. A., Atwood, H. L., Renger, J. J., Wang, J., Wu, C. F. Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions. J. Comp. Physiol.. A175, 179-191 (1994).
  44. Sweeney, S. T., Broadie, K., Keane, J., Niemann, H., O’Kane, C. J. Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron. 14, 341-351 (1995).
  45. Tsunoda, S., Salkoff, L. Genetic analysis of Drosophila neurons: Shal, Shaw, and Shab encode most embryonic potassium currents. J. Neurosci. 15, 1741-1754 (1995).
  46. Ueda, A., Kidokoro, Y. Longitudinal body wall muscles are electrically coupled across the segmental boundary in the third instar larva of Drosophila melanogaster. Invertebrate Neuroscience. 1, 315-322 (1996).
  47. Wu, C. F., Haugland, F. N. Voltage clamp analysis of membrane currents in larval muscle fibers of Drosophila. J. Neurosci. 5, 2626-2640 (1985).
  48. Yan, Y., Broadie, K. In vivo assay of presynaptic microtubule cytoskeleton dynamics in Drosophila. J Neurosci Methods. 162, 198-205 (2007).
  49. Yoshikami, D., Okun, L. Staining of living presynaptic nerve terminals with selective fluorescent dyes. Nature. 310, 53-56 (1984).
  50. Zagotta, W. N., Brainard, M. S., Aldrich, R. W. Single-channel analysis of four distinct classes of potassium channels in Drosophila muscle. J. Neurosci. 8, 4765-4779 (1988).
  51. Zhang, Y. Q., Rodesch, C. K., Broadie, K. A living synaptic vesicle marker: synaptotagmin-GFP.. Genesis. 34, 142-145 (2002).

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DrosophilaEmbryosElectrophysiological RecordingProceduresGenetic ModelEmbryonic DevelopmentFunctional NeuroscienceDevelopmental ProgramsElectrical Signaling PropertiesChemical SynapsesNeural Circuit FormationInvestigationLate Developmental PeriodEpidermal CuticleSurgical GlueControlled DissectionDorsal Longitudinal IncisionVentral Nerve CordBody Wall MusculatureGenetic MutantsSynapse FormationMolecular Mechanisms
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Featherstone, D. E., Chen, K., Broadie, K. Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures. J. Vis. Exp. (27), e1347, doi:10.3791/1347 (2009).
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