纤毛对小鼠的主要嗅觉系统整装标签
Summary
Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.
Abstract
The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.
Introduction
鼻腔鼠标嗅上皮包括6-10万双极嗅感觉神经元1。每个嗅觉神经元选择的1200气味受体基因的表达之一。检测加臭剂的开始由气味结合到嗅觉受体2，其然后激活腺苷酸环化酶III型（ACIII）3经由嗅觉特异性G蛋白GαOLF 4。由此而造成的环磷酸腺苷（cAMP）打开一个环核苷酸门控（CNG），非选择性阳离子通道，导致钙离子和钠离子，并随后钙涌入2+内流导致开口的Ca 2+活化的Cl –通道5,6。所得向外Cl –的磁通变得容易通过高细胞内氯–吸收，有可能通过的Na + / K + / Cl –的–协同转运NKCC1中，通过稳定氯浓度保持CL – / HCO3 –换热器SLC4A1，也许更多的尚未确定运输6-8。
双极嗅觉神经元具有单一的，无支链的轴突直接投射到嗅球和树枝状延伸到上皮的表面和端部作为专门隔室，树枝状旋钮。从这个旋钮，10-30纤毛，其可以达到50-60微米的长度，发出入粘液覆盖的上皮表面9。规范化的信号转导级联的蛋白质主要定位于这些纤毛的膜。上皮的增加感官表面放大，以检测气味物质的能力。由于感觉神经元的密度，纤毛从相邻的树突旋钮延伸弄乱。这种交织的结果是从不同的神经元，表达不同类型的嗅觉受体，上皮的表面上的随机混合物纤毛。的检测和细胞睫状蛋白，只存在于感觉神经元的一个子集的ular分配，因此在冷冻切片困难。另外，这种蛋白质沿纤毛的精确定位是几乎不可能的，因为冷冻切片通常比纤毛的平均长度薄。
为了使在嗅觉神经元至今未鉴定的膜蛋白的纤毛本土化的调查中，我们优化的恩面对制剂技术，它允许蛋白定位于纤毛的详细分析。简言之，将小鼠处死并在头分割中线附近。鼻甲，鼻，和额叶骨骼被去除以暴露隔膜。用的衬里上皮嗅觉部隔膜通过切割所有连接到鼻腔松开。把隔膜变成培养皿充满林格氏液后，将上皮剥离UND转移到涂覆的玻璃载片。经过简短fixat离子一步，免疫组化方法可以如果操作尽可能轻柔，以免脆弱的组织损伤进行。我们证明在古典冷冻切片和中所描述的连接面制备比较两种不同的膜蛋白的染色在嗅觉纤毛可达到的分辨率。
Protocol
Representative Results
Discussion
The en face preparation technique described in this protocol provides a powerful tool for the detailed analysis of the olfactory system. So far, most studies characterizing the localization of signaling proteins use immunostainings of cryosections. They present a good overview of the olfactory epithelium, and protein expression in distinct cell types or regions can be easily identified. However, expression in olfactory cilia is sometimes hard to detect. Even if ciliary localization is obvious, cryosections offer…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by the Deutsche Forschungsgemeinschaft DFG (Exc257, SFB958).
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Spring scissors | straight tip, multiple suppliers | ||
| Surgical scissors | sharp and blunt end, multiple suppliers | ||
| Fine forceps | curved tips, Dumont #7, multiple suppliers | ||
| Razor blade | extra thin, multiple suppliers | ||
| Binocular with illumination | multiple suppliers, Stemi 2000-C, Zeiss | ||
| Petri dish | multiple suppliers | ||
| Liquid-blocker pen | Science Services | N71310 | |
| Polysine coated slides | Thermo Scientific | J2800AMNZ | |
| Confocal microscope | Leica Microsystems | TCS SPE | |
| primary antibody Goat anti-Kirrel2 | R&D Systems | AF2930 | 1:200 |
| primary antibody Rabbit anti-mOR-EG | Baumgart et al., 2014 | 1:200 | |
| secondary antibodies | Life Technologies | A21206, A11057 | 1:500 |
| Mounting medium, ProLong Gold antifade reagent | Life Technologies | P36930 | |
| Paraformaldehyde | Sigma | 441244 | toxic, work under fume hood |
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