嗅觉受体神经元在全山免疫标记<em>果蝇</em>天线
Summary
Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.
Abstract
气味分子结合到他们的目标受体的精确和协调的方式。每个受体识别的特定信号并中继该信息到大脑。因此，确定嗅觉信息是如何传递到大脑，修改这两个认知和行为，值得调查。有趣的是，新出现的证据表明，细胞转导和转录因子参与了嗅觉受体神经元的多样化。在这里，我们提供了一个强大的整体安装免疫标记法来测定体内嗅觉受体神经元组织。使用这种方法，我们发现所有的嗅觉受体神经元具有抗ELAV抗体，已知的泛神经标记物和Or49a-mCD8 :: GFP，用抗GFP抗体是NBA神经元中特异性表达嗅觉受体神经元。
Introduction
嗅觉系统使用一个巨大的各种气味分子之间进行区分，并随后发送所得到的信息到大脑高级中心。这种输入是用来精确地控制动物的基本行为，如喂养和交配1-6。因为每个嗅觉神经元类型与一组特定的气味，嗅觉受体神经元的关联的多样化（ORN），s是正常嗅觉系统功能7是必不可少的。
果蝇遗传学使我们能够执行涉及与ORN发育和生理功能8-16相关的分子机制单细胞水平的调查。 果蝇触角整装免疫染色，使我们更详细地了解所涉及的嗅觉受体神经元的多样化的分子机制（ORN）秒7。本文中，我们提供了一种简单的方法的全面描述，以交流hieve这一点。
Protocol
Representative Results
Discussion
果蝇天线，我们描述了解剖简单，易于在实验室环境中进行。以确保成功剥离，有必要利用细边剪刀。而免疫染色的解剖天线，它孵育他们在一个湿气填充容器内，避免抗体溶液的蒸发是很重要的。解剖天线有一种倾向，在溶液中自由浮动。用0.1％的Triton的PBS中固定期间和阻断步骤，将促进溶液中的天线的浸没并以确保更好地染色。使用“玻璃底培养皿”可以减少免疫染色过程中天线的损…
Declarações
The authors have nothing to disclose.
Acknowledgements
本研究为战略研究基金会在民办高校和学术振兴会青年科学家乙津贴为HT我们要感谢大竹德仁编辑的视频片段支持文部科学省支持的项目。
Materials
| Stemi DV4 dissection microscope | Zeiss | Stemi DV4 | |
| Glass bottom culture dishes | MatTek corporation | P35G-0-10-C | |
| Dissection scissor | Fine Science Tools | 15000-08 | |
| Rat anti-ELAV | Developmental Studies Hybridoma Bank | 7E8A10 | Dilution 1:200 |
| Mouse anti-GFP | Invitrogen | A11122 | Dilution 1:400 |
| Donkey Anti-Rabbit IgG | Jackson ImmunoResearch Laboratories | 711-225-152 | Dilution 1:200 |
| Donkey Anti-Rat IgG | Jackson ImmunoResearch Laboratories | 712-165-150 | Dilution 1:200 |
Referências
- Christensen, T. A., White, J. . Representation of olfactory information in the brain In The Neurobiology of Taste and Smell. , 201-232 (2000).
- Ache, B. W. Towards a common strategy for transducing olfactory information. Sem. Cell Biol. 5, 55-63 (1994).
- Bargmann, C. I., Hartwieg, E., Horvitz, H. R. Odorant-selective genes and neurons mediate olfaction. C. elegans. Cell. 13, 515-527 (1993).
- Barth, A. L., Justice, N. J., Ngai, J. Asynchronous onset of odorant receptor expression in the developing zebrafish olfactory system. Neuron. 16, 23-34 (1996).
- Firestein, S. How the olfactory system makes sense of scents. Nature. 413, 211-218 (2001).
- Stockinger, P., et al. Neural circuitry that governs Drosophila male courtship behavior. Cell. 121, 795-807 (2005).
- Endo, K., et al. Chromatin modification of Notch targets in olfactory receptor neuron diversification. Nat Neurosci. 15, 224-233 (2011).
- Karim, M. R., Moore, A. W. Morphological analysis of Drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics. J Vis Exp. , (2011).
- Suh, G. S., et al. A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila. Nature. 431, 854-859 (2004).
- Sachse, S., Galizia, C. G. Role of inhibition for temporal and spatial odor representation in olfactory output neurons: A calcium imaging study. J Neurophysiol. 87, 1106-1117 (2002).
- Hallem, E. A., Ho, M. G., Carlson, J. R. The molecular basis of odor coding in the Drosophila antenna. Cell. 117, 965-979 (2004).
- Vosshall, L. B., Wong, A. M., Axel, R. An olfactory sensory map in the fly brain. Cell. 102, 147-159 (2000).
- Couto, A., Alenius, M., Dickson, B. J. Molecular, anatomical and functional organization of the Drosophila olfactory system. Curr Biol. 15, 1535-1547 (2005).
- Clyne, P., et al. Odorant response of individual sensilla on the Drosophila antenna. Invert Neurosci. 3, 127-135 (1997).
- Vosshall, L. B., et al. A spatial map of olfactory receptor expression in the Drosophila antenna. Cell. 96, 725-736 (1999).
- Wang, J. W., et al. Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain. Cell. 112, 271-282 (2003).
