The olfactory system as a model to study axonal growth patterns and morphology in vivo.
The olfactory system as a model to study axonal growth patterns and morphology in vivo.
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DOI：
10.3791/52143
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发表时间：
2014-10-30
期刊：
影响因子：
--
通讯作者：
Manzini I
中科院分区：
文献类型：
--
作者：
Hassenklöver T;Manzini I
嗅觉系统具有在生物体的整个生命周期中产生新神经元的不寻常的能力。嗅上皮基底部的嗅觉干细胞不断产生新的感觉神经元，这些神经元将其轴突延伸到嗅球中，在那里它们面临着整合到现有电路中的挑战。由于这个特殊的功能，嗅觉系统代表了一个独特的机会，监测轴突布线和指导，并研究突触的形成。在这里，我们描述了一个程序，在体内标记的感觉神经元和随后的可视化轴突在嗅觉系统的两栖动物非洲爪蟾的幼虫。我们采用电穿孔技术对嗅觉器官的感觉神经元进行染色。体内电穿孔是用于将荧光团偶联的葡聚糖或其他大分子递送到活细胞中的已建立的技术。染色的感觉神经元和它们的轴突过程，然后可以在活体动物中使用共聚焦激光扫描或多光子显微镜进行监测。通过将标记细胞的数量减少到每只动物很少或单个细胞，可以将单个轴突追踪到嗅球中，并且可以通过进行一系列体内时间推移成像实验来监测其形态变化。虽然所描述的方案阐明了嗅觉感觉神经元的标记和监测，但它也可以适用于嗅觉和其他系统内的其他细胞类型。
The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.
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