Department of Biology, Institute of Zoology, University of Fribourg, Fribourg, Switzerland.
Department of Biology, Brandeis University, Waltham, Massachusetts, USA.
Nat Protoc. 2016 Dec;11(12):2389-2400. doi: 10.1038/nprot.2016.144. Epub 2016 Nov 3.
Monitoring neuronal responses to defined sensory stimuli is a powerful and widely used approach for understanding sensory coding in the nervous system. However, providing precise, stereotypic and reproducible cues while concomitantly recording neuronal activity remains technically challenging. Here we describe the fabrication and use of a microfluidics system that allows precise temporally restricted stimulation of Drosophila chemosensory neurons with an array of different chemical cues. The system can easily be combined with genetically encoded calcium sensors, and it can measure neuronal activity at single-cell resolution in larval sense organs and in the proboscis or leg of the adult fly. We describe the design of the master mold, the production of the microfluidic chip and live imaging using the calcium sensor GCaMP, expressed in distinct types of Drosophila chemosensory neurons. Fabrication of the master mold and microfluidic chips requires basic skills in photolithography and takes ∼2 weeks; the same devices can be used repeatedly over several months. Flies can be prepared for measurements in minutes and imaged for up to 1 h.
监测神经元对特定感觉刺激的反应是一种强大且广泛应用的方法,可用于了解神经系统中的感觉编码。然而,在同时记录神经元活动的情况下,提供精确、刻板和可重复的线索仍然具有技术挑战性。本文介绍了一种微流控系统的制作和使用方法,该系统可使用一系列不同的化学线索精确地对果蝇化学感觉神经元进行时间受限刺激。该系统可以很容易地与遗传编码的钙传感器结合使用,并可以在幼虫感觉器官以及成年果蝇的喙或腿中以单细胞分辨率测量神经元活动。我们描述了主模具的设计、微流控芯片的制作以及使用钙传感器 GCaMP 的活体成像,该传感器在不同类型的果蝇化学感觉神经元中表达。主模具和微流控芯片的制作需要具备基本的光刻技能,大约需要 2 周的时间;相同的设备可以在几个月内重复使用多次。准备好的果蝇可以在几分钟内进行测量,并可以进行长达 1 小时的成像。