Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头
Summary
此过程使用蓝色光激活藻通道和细胞特异性基因表达的工具，以唤起光脉冲在神经肌肉接头（NMJ）突触潜力<em>果蝇</em>幼虫。这种技术是一种廉价和易于使用替代吸电极刺激突触生理学研究，在研究和教学实验室。
Abstract
“<em>果蝇</em已被证明是一个有用的工具，为研究突触生理学>幼虫神经肌肉准备<sup> 1,2,3</sup>。目前，唯一可利用的手段，以唤起兴奋（EJPs）交界的潜力，这种准备包括吸电极使用。在研究和教学实验室，学生往往难以操纵和操纵这种类型的刺激电极。在目前的工作，我们将展示如何远程使用吸电极刺激幼虫NMJ突触的潜力。表达channelrhodopsin2（ChR2）<sup> 4,5,6</sup>在<em>果蝇</em>使用的运动神经元<em> GAL4 – UAS</em>系统<sup> 7</sup>和一个基本的电钻机小的变化，我们能够可靠地唤起人们的蓝色光脉冲EJPs。这种技术可以在神经生理学教学实验室的学生钻机实践的时间和资源是有限的特定用途。
Protocol
第1部分：动物保健和遗传杂交保持无人机，ChR2和8个单独的瓶含有标准的飞行媒体OK371 GAL4飞线。 收集OK371 – GAL4飞线和UAS – ChR2线的男性处女。 将男性和女性在包含标飞1毫米全反式视网膜（ATR）的混合介质的小瓶。 ATR食品应首先融化在定期飞〜1分钟微波介质。一旦融化，使冷却约30秒到一分钟，然后加入100μL100％乙醇10毫升每飞行媒体的ATR 100毫米?…
Discussion
关键步骤涉及的初步解剖和进入肌肉细胞。如果神经被切断，或在初始剥离肌肉受损，就很难继续实验的休息。在清扫过程中，必须非常谨慎的角度在背的切口，向上为尽可能解剖剪刀。在第二个关键的一步，进入肌肉细胞，一个必须注意的一个极化过去〜30 mV的。 -30毫伏以上的值指示电极是不是肌肉细胞内或在一个不健康的细胞正常。
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生赠款RO1GM – 33205和MH – 067284研究院立法会格里菲斯和一个布兰代斯大学的夏季本科生科研奖学金，新泽西州Hornstein。作为2008年神经系统和行为暑期课程（镍氢补助金：R25 MH059472）的一部分，马在伍兹霍尔海洋生物实验室进行这项技术的初步实验。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
| Sylgard | Ellsworth adhesives | 4019862 | www.ellsworth.com | |
| Minutens Pins | Fine Science Tools | 26002-10 | www.finescience.com | |
| Dissecting Dish | Fisher Scientific | www.fishersci.com | ||
| Neuroprobe Intracellular Amplifier and Head Stage | A-M Systems | 680100 | www.a-msystems.com | |
| Powerlab 4/30 data acquisition system | AD instruments | www.adinstruments.com | ||
| Grass stimulator | Grass instruments | www.grasstechnologies.com | ||
| Desktop Computer | Dell | www.dell.com | ||
| Dissecting Scope | Leica | www.leica-microsystems.com | ||
| Light Source | Dolan-Jenner | 41446-062 | www.dolan-jenner.com | |
| Fly Media | ||||
| All-Trans-Retinal | Sigma-Aldrich | 116-31-4 | www.sigmaaldrich.com | |
| OK-371 Gal4 Flies | Aberle lab, Griffith lab, Bloomington stock center | |||
| UAS-ChR2 Flies | Fiala lab, Griffith lab | |||
| LED controller circuit | Built in Griffith lab | http://www.ledsupply.com http://www.futureelectronics.com Composed of: 1. 200 mA Buck Puck 2. Blue LED 3. Insulated wire 4. Circuit bread board |
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| LED Heat Sink | Thor Labs | http://www.thorlabs.com/ | ||
| Air Table | TMC | http://www.techmfg.com/products/accessories/intro3.html | ||
| Faraday Cage | Built in Griffith lab | |||
| Leica Leitz M Micro-Manipulator | Leica Leitz | ACS01 | www.leica-microsystems.com | |
| Electrode Holder | Axon Instruments | www.axon.com | ||
| Borosilicate Glass | FHC | www.fh-co.com/p14-15.pdf | ||
| Electrode Puller | Sutter Instruments | www.sutter.com | ||
| HL 3.1 Saline with 0.8mM Ca2+ | Contents (mM): NaCl:70 KCl:5 CaCl2: 0.8 MgCl2:4Sucrose:115 NaHCO3: 10 Trehalose: 5 HEPES |
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| Micro-Dissection Tools | Fine Science Tools | www.finescience.com |
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Cite This Article
Hornstein, N. J., Pulver, S. R., Griffith, L. C. Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions. J. Vis. Exp. (25), e1133, doi:10.3791/1133 (2009).