In vivo Neuronal Calcium Imaging in C. elegans
In vivo Neuronal Calcium Imaging in C. elegans
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DOI：
10.3791/50357
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发表时间：
2013-04-01
影响因子：
1.2
通讯作者：
Gabel, Christopher V.
中科院分区：
文献类型：
--
作者：
Chung, Samuel H.;Sun, Lin;Gabel, Christopher V.
线虫C.秀丽线虫是用于相对简单、低成本的体内神经元成像的理想模式生物。它的小透明的身体和简单，良好的神经系统特征允许识别和荧光成像的任何神经元在完整的动物。对动物生理影响最小的简单固定技术允许延长延时成像。遗传编码的钙敏感荧光团（如cameleon（1）和GCaMP（2））的开发允许对与细胞生理学和神经元活性相关的神经元钙进行体内成像。在特定神经元中表达这些荧光团的许多转基因菌株是容易获得的，或者可以使用成熟的技术构建。在这里，我们描述了详细的程序，测量钙动力学在一个单一的神经元在体内使用GCaMP和cameleon。我们讨论的优点和缺点，以及各种方法的样品制备（动物固定）和图像分析。最后，我们给出了两个实验的结果：1）使用GCaMP测量特定神经元对外部电场的感觉反应和2）使用cameleon测量神经元对创伤性激光损伤的生理钙反应。钙成像技术，如这些被广泛用于C。elegans的研究，并已扩展到测量自由移动的动物，多个神经元同时和遗传背景的比较。C. Elegans提出了一种用于体内神经元成像的鲁棒且灵活的系统，其在技术简单性和成本方面优于其它模型系统。
The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon(1) and GCaMP(2) allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.