收获和准备果蝇胚胎的电生理记录和其他程序

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Summary

这种技术公开的果蝇胚胎neuromusculature免疫组织化学或电生理记录。它是有用的,为研究神经肌肉发展的早期事件,或执行中的突变体,不能孵化电。

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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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Abstract

果蝇胚胎发育和功能的神经科学的研究是首屈一指的遗传模型。传统上,这些领域都相当彼此孤立的,很大程度上是独立的历史和科学界。然而,这些通常是不同的领域之间的接口是发展计划的基本功能的电子信号的属性和功能的化学突触在神经回路形成的最后阶段分化的收购。该接口是一个极为重要的领域进行调查。在果蝇中,这些功能开发阶段发生在胚胎发育的最后三分之一的<8小时期间(在25 ° C) 。后期发展时期,长期被视为棘手的调查,由于一个艰难的,不透水的表皮角质层的沉积。一个突破性的进展是,可以在本地应用的角质层,使后期胚胎的控制清扫手术,聚合水胶中的应用。胚胎背侧纵切口,可以平放,露出腹侧神经线和体壁肌肉的实验研究。该系统已大量用于分离和鉴定基因突变体,损害胚胎的突触的形成,从而揭示了突触连接和功能的突触信号特性的规范和分化的分子机制。

Protocol

第1部分:设备和用品

  1. 一个良好的解剖镜下胚胎解剖;建议40X放大倍率25X眼睛件,最大限度地增加放大。
  2. 胚胎及胚胎devitellinization手动选择需要精细镊子(5号)。
  3. 制定和修改细玻璃针的设备是必需的。拉玻璃针都需要剥离。我们喜欢实心玻璃夹层(持续更长的时间),但工程,以及标准的厚壁玻璃管(外径1-1.5毫米)。有些人用细的钨针,电解所需的清晰度在1M的NaOH使用自耦变压器电池10 + AMP浴。锐利的钨清扫针,他们是比较有弹性和长期的利益。中空玻璃针(形成类似膜片钳电极)连接到塑料管中,并在清扫吸力和生理盐水驱逐,以及胶水的控制下交付使用。各种玻璃车夫可用于制造玻璃针。电脑编程车夫可预置拉各种形状和大小。布朗阻燃模型(萨特仪器有限公司)的广泛青睐。一个复合式显微镜(20 - 40X的目标)应可用来检查和修改电极在使用前。
  4. 两种类型的解决方案通常用于沐浴在剥离过程中胚胎组织:1)(月和1月,1976a)“标准”或“修订标准”(Broadie 2000年)salines,根据记录在其他无脊椎动物系统常用的解决方案,2)“血淋巴样”(HL)的salines(Stewart等,1994),标准生理盐水和果蝇血淋巴测量离子浓度之间的妥协。应该指出,这些salines都没有准确地模仿在体内的血淋巴中的化学成分(Broadie 2000年)。标准生理盐水(毫米):135氯化钠,氯化钾,4 2 1.8氯化镁2,氯化钙,72蔗糖,5工商业污水附加费,pH值7.2。实验者也可能要考虑商业化准备的昆虫生理盐水(如施耐德公司的昆虫媒体),这是不合适的电生理记录,但最有可能的,以保护暴露组织的健康。

第2部分:胚胎分期和解剖

  1. 最佳鸡蛋收集,保持年轻(<7天),健康雄性和雌性果蝇的新鲜奠定盆2-3天(20-40)。铺设盆通常由100毫升塑料烧杯(三倒)穿孔,以提供足够的空气流通,涵盖了60毫米琼脂平板。琼脂平板上包含苹果或葡萄汁与琼脂硬化。多个配方存在,但其中一个例子如下:700毫升的水,琼脂25-30克,300毫升的浓缩果汁(葡萄或苹果),0.5克甲基苯甲酸酯(P - hydroxymethylbenzoate),和30克糖。高压灭菌的水和琼脂,并分别熬糖和果汁的P - hydroxymethylbenzoate。混合的琼脂和果汁解决方案,并迅速倒入60毫米的钢板,消除泡沫。
  2. 鸡蛋收集之前,苍蝇喂酵母膏(9毫升的水储存于4 ° C,面包酵母7克)新鲜的板块,每天至少两次,至少两天改变。第3天,好锅应每小时100-200鸡蛋。更好的产蛋观察划伤板琼脂在其一侧保持了锅。许多胚胎将放置在旁边的划痕。
  3. 为了收集大量的胚胎,轻轻地从琼脂平板上转移到一个篮子用画笔胚胎。一篮子可与15或50 mL离心管。切断底部留出足够的表面积陷阱之间的盖子和管顶部的屏幕(70微米网状)。与卫生署2 0冲洗胚胎放入一个培养皿新鲜的50%至100%的漂白粉去除外层的绒毛膜。此外,鸡蛋可以收集单独使用罚款钳,并对其进行手动放入漂白水下降dechorionated。 chorionation可以从30秒到2分钟(新鲜漂白快得多),并应解剖显微镜下监测。去除绒毛膜公开透明的光泽,卵黄膜。 Dechorionated鸡蛋应简单地冲洗,或置于生理盐水中,作为漂白剂迅速破坏devitellinized组织。
  4. 仔细到所需的发育年龄阶段的胚胎,这一点至关重要。定义的果蝇胚胎阶段(1-17;坎波斯奥尔特加和Hartenstein,1985年)是没有用的,所有功能的神经肌肉发展发生在后期的16或阶段17的。因此,胚胎发育小时上演25 °在培养箱中小时后受精或,更普遍的,产蛋后(AEL)。在这些条件下,胚胎持续21 + / - 1小时25 ° C。
  5. 鸡蛋从一个定时蛋打下(1-2小时S),是适当的年龄在旁边观看的形态学标准。 DH 2 O的广泛的洗涤后,dechorionated鸡蛋都放在一个塑料培养皿(卫生署辖下 2)查看。可以确认正确的临时使用反射光的形态与解剖显微镜(坎波斯奥尔特加和Hartenstein,1985年)的标准。成熟后期胚胎孵化的边缘(22 - 24小时机场快线)是普遍认可的角质层和虚增气管分割。胚胎也可以使用GFP平衡器和一个适当的过滤器的荧光解剖镜下基因型。
  6. 清扫,dechorionated和发育上演的胚胎附着在盖玻片下的生理盐水下降(见第1.5步)(背侧)。胚胎从卵黄膜与玻璃微吸管或钨针刺破膜附近的前或后结束,然后轻轻地从膜释放的胚胎。在准备解剖,胚胎应定位于背水面达(背侧了清扫将公开腹侧神经系统和实验neuromusculature盖玻片,但是,胚胎显然是定位在其他方面,以方便访问其他组织。)
  7. 早期胚胎(<16小时机场快线)直接连接到干净的玻璃或镀膜玻璃与多聚赖氨酸(聚- L -赖氨酸氢溴酸; Broadie和巴特,1993,B)。胚胎(机场快线> 16小时)后,角质层的形成,必须粘,最好Sylgard涂层(道康宁)盖玻片。使用一个聚合水的氰基丙烯酸酯胶外科或兽医(Broadie和巴特,1993c,D)。胶是通过一个小玻璃电极吸管口压力控制的胶水流连接橡胶管(10-20微米内径TIP)交付。必须小心胶交付期间,胶水联系生理盐水后迅速聚合。请注意,也不能执行,粘合没有或低的二价离子浓度的盐水,胶水,需要二价离子聚合。首次使用少量的胶水牢固地附着在头部和尾部。
  8. 一旦胚胎的两端粘盖玻片,切口是沿背中线与玻璃电极或削尖的钨针。这是轻轻射孔切口线,晋级决赛之前变得更加容易。切口的两侧,然后连接到盖玻片更多的胶水,轻轻蔓延胚胎单位。内部器官,包括肠,脂肪体,可选,气管,然后由使用橡胶管(Broadie和贝特1993 - C)连接到一个玻璃吸管吸删除。现在应该附加胚胎持平盖玻片,表皮腹中枢神经系统,周围神经和躯体肌肉暴露实验。

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Discussion

胚胎的精确分期是由于功能特性的只是几个小时的时间当然的迅速成熟的关键。几个问题复杂化分期。首先,大多数研究人员使用定时鸡蛋奠定阶段的胚胎,但产蛋时间可以千差万别,在不同的条件(Broadie等,1992)在动物。特别是,在有限的食物的女性倾向于保留奠定前长时间的受精卵。因此,至关重要的丰富的酵母饮食喂养前至少2天收集蛋(Broadie等,1992)“明确的”女性。此外,老年女性也保持一个较长时期奠定的鸡蛋。一个年长的女性可能经常下蛋的只有几个小时之前,他们孵化。因此重要的是使用最一致的铺设时间(Broadie等,1992)的年轻女性(<7天)。其次,在后期的胚胎阶段,它是困难的阶段完全由胚胎形态学标准。最清晰的分期功能完整的<16小时的机场快线(坎波斯,奥尔特加和Hartenstein,1985年),但功能最全的发展出现> 16小时的机场快线。晚期(如气管空气填充,晒黑角质层)发展特点很少,而且往往是少时间限制。基于这些原因,我们建议组合方法:收集鸡蛋从1-2小时定时的规定,现阶段以定义良好的早期形态持续时间<30分钟的事件(如原肠,背封,3部分肠道坎波斯,奥尔特加和Hartenstein,1985年),然后孵化在一个良好的控制,25℃孵化器所需的年龄。

本手册清扫和胶水的应用,需要在显微镜下,很少有人初步具备精细动作技能。随着时间的推移,这些技能必须制定。实验者应愿意承诺在期待一个成功的高频前至少几个星期每天专门的实践。适合镜检的中枢神经系统和一些腹侧神经肌肉接头的筹备工作应该是可以实现比较快,适合用于电健康的准备,而通常会需要更多的努力。

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Acknowledgments

KB是由美国国立卫生研究院授予GM54544支持。

Materials

Name Company Catalog Number Comments
Embryo Collection Cages Genesee Scientific 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. The Development of Drosophila melanogaster. Bate, M., Martinez Arias, A. Cold Spring Harbor Laboratory Press. New York. Vol. I, II (1993).
  8. Baumgartner, S., JT, L. ittleton, Broadie, K., MA, B. hat, Harbecke, R., JA, L. engyel, 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. rand 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. Electrophysiological Approaches to the Neuromusculature. Drosophila Protocols. Sullivan, W., Ashburner, M., Hawley, R. S. Cold Spring Harbor Laboratory Press. New York. 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. eonardo 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. Springer. Berlin. (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. Embryonic Development of the Drosophila Central Nervous System. In The Development of Drosophila melanogaster. Bate, M., Martinez Arias, A. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York. 1131-1206 (1993).
  31. Harrison, S. D., Broadie, K., Goor, J. vande, 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. Neuroscience. 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. 3rd, 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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