Name: channelrhodopsin2 mediated stimulation of synaptic potentials at drosophila neuromuscular junctions
Uploaded: 2009-03-16T00:00:00
Description: Watch this Scientific Journal Video about Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions at JoVE.com

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。

第1部分：动物保健和遗传杂交<ol><li>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。 </li><li>收集OK371 - GAL4飞线和UAS - ChR2线的男性处女。 </li><li>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?...

<ol>
	<li>Keshishian, H., Broadie, K., Chiba, A., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+neuromuscular+junction:+a+model+system+for+studying+synaptic+development+and+function.">The Drosophila neuromuscular junction: a model system for studying synaptic development and function.</a> Annu Rev Neurosci. 19, 545 (1996).</li><li>Collins, C. A., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+development:+insights+from+Drosophila.">Synaptic development: insights from Drosophila.</a> Curr Opin Neurobiol. 17 (1), 35 (2007).</li><li>Lagow, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+a+hydrophobic+layer+by+a+point+mutation+in+syntaxin+1A+regulates+the+rate+of+synaptic+vesicle+fusion.">Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.</a> PLoS Biol. 5 (4), e72 (2007).</li><li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses.">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Curr Biol. 15 (24), 2279 (2005).</li><li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proc Natl Acad Sci U S A. 100 (24), 13940 (2003).</li><li>Schroll, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+activation+of+distinct+modulatory+neurons+triggers+appetitive+or+aversive+learning+in+Drosophila+larvae.">Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae.</a> Curr Biol. 16 (17), 1741 (2006).</li><li>Lin, D. M., Auld, V. J., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+neuronal+cell+ablation+in+the+Drosophila+embryo:+pathfinding+by+follower+growth+cones+in+the+absence+of+pioneers.">Targeted neuronal cell ablation in the Drosophila embryo: pathfinding by follower growth cones in the absence of pioneers.</a> Neuron. 14 (4), 707 (1995).</li><li>Ralph Greenspan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fly Pushing: The Theory and Practice of Drosophila Genetics, 2nd ed. , (2004).</li></ol>

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。

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comment</td>
</tr>	
<tr>
<td>Sylgard</td>
<td>&nbsp;</td>
<td>Ellsworth adhesives </td>
<td>4019862</td>
<td><a href="http://www.ellsworth.com" target="_blank">www.ellsworth.com</a></td>
</tr>
<tr>
<td>Minutens Pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td><a href="http://www.finescience.com" target="_blank">www.finescience.com</a></td>
</tr>
<tr>
<td>Dissecting Dish</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td><a href="http://www.fishersci.com" target="_blank">www.fishersci.com</a></td>
</tr>
<tr>
<td>Neuroprobe Intracellular Amplifier and Head Stage</td>
<td>&nbsp;</td>
<td>A-M Systems</td>
<td>680100</td>
<td><a href="http://www.a-msystems.com" target="_blank">www.a-msystems.com</a></td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">Powerlab 4/30 data acquisition system</a></td>
<td>&nbsp;</td>
<td><a href="http://www.adinstruments.com" target="_blank">AD instruments</a></td>
<td>&nbsp;</td>
<td><a href="http://www.adinstruments.com" target="_blank">www.adinstruments.com</a></td>
</tr>
<tr>
<td>Grass stimulator</td>
<td>&nbsp;</td>
<td>Grass instruments</td>
<td>&nbsp;</td>
<td><a href="http://www.grasstechnologies.com" target="_blank">www.grasstechnologies.com</a></td>
</tr>
<tr>
<td>Desktop Computer</td>
<td>&nbsp;</td>
<td>Dell</td>
<td>&nbsp;</td>
<td><a href="http://www.dell.com" target="_blank">www.dell.com</a></td>
</tr>
<tr>
<td>Dissecting Scope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td><a href="http://www.leica-microsystems.com" target="_blank">www.leica-microsystems.com</a></td>
</tr>
<tr>
<td>Light Source</td>
<td>&nbsp;</td>
<td>Dolan-Jenner</td>
<td>41446-062</td>
<td><a href="http://www.dolan-jenner.com" target="_blank">www.dolan-jenner.com</a></td>
</tr>
<tr>
<td>Fly Media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>All-Trans-Retinal</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>116-31-4</td>
<td><a href="http://www.sigmaaldrich.com" target="_blank">www.sigmaaldrich.com</a></td>
</tr>
<tr>
<td>OK-371 Gal4 Flies</td>
<td>&nbsp;</td>
<td>Aberle lab, Griffith lab, Bloomington stock center </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>UAS-ChR2 Flies</td>
<td>&nbsp;</td>
<td>Fiala lab, Griffith lab </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>LED controller circuit</td>
<td>&nbsp;</td>
<td>Built in Griffith lab</td>
<td>&nbsp;</td>
<td><a href="http://www.ledsupply.com" target="_blank">http://www.ledsupply.com</a> 
<a href="http://www.futureelectronics.com" target="_blank">http://www.futureelectronics.com</a> 
Composed of: 
1. 200 mA Buck Puck 
2. Blue LED 
3. Insulated wire 
4. Circuit bread board </td>
</tr>
<tr>
<td>LED Heat Sink</td>
<td>&nbsp;</td>
<td>Thor Labs</td>
<td>&nbsp;</td>
<td><a href="http://www.thorlabs.com/" target="_blank">http://www.thorlabs.com/</a></td>
</tr>
<tr>
<td>Air Table</td>
<td>&nbsp;</td>
<td>TMC</td>
<td>&nbsp;</td>
<td><a href="http://www.techmfg.com/products/accessories/intro3.html" target="_blank">http://www.techmfg.com/products/accessories/intro3.html</a></td>
</tr>
<tr>
<td>Faraday Cage</td>
<td>&nbsp;</td>
<td>Built in Griffith lab</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Leica Leitz M Micro-Manipulator</td>
<td>&nbsp;</td>
<td>Leica Leitz</td>
<td>ACS01</td>
<td><a href="http://www.leica-microsystems.com" target="_blank">www.leica-microsystems.com</a></td>
</tr>
<tr>
<td>Electrode Holder</td>
<td>&nbsp;</td>
<td>Axon Instruments</td>
<td>&nbsp;</td>
<td><a href="http://www.axon.com" target="_blank">www.axon.com</a></td>
</tr>
<tr>
<td>Borosilicate Glass</td>
<td>&nbsp;</td>
<td>FHC</td>
<td>&nbsp;</td>
<td><a href="http://www.fh-co.com/p14-15.pdf" target="_blank">www.fh-co.com/p14-15.pdf</a></td>
</tr>
<tr>
<td>Electrode Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td><a href="http://www.sutter.com" target="_blank">www.sutter.com</a></td>
</tr>
<tr>
<td>HL 3.1 Saline with 0.8mM Ca2+</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Contents (mM): 
NaCl:70 KCl:5 CaCl2: 0.8 MgCl2:4Sucrose:115 
NaHCO3: 10 Trehalose: 5 HEPES
</td>
</tr>
<tr>
<td>Micro-Dissection Tools</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td><a href="http://www.finescience.com" target="_blank">www.finescience.com</a></td>
</tr>		
</tbody>
</table>

channelrhodopsin2 mediated stimulation of synaptic potentials at drosophila neuromuscular junctions

“<em&gt;果蝇</em已被证明是一个有用的工具，为研究突触生理学&gt;幼虫神经肌肉准备<sup&gt; 1,2,3</sup&gt;。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&gt; 4,5,6</sup&gt;在<em&gt;果蝇</em&gt;使用的运动神经元<em&gt; GAL4 - UAS</em&gt;系统<sup&gt; 7</sup&gt;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。

Watch this Scientific Journal Video about Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions at JoVE.com

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

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&gt;果蝇</em&gt;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。

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Research

JoVE Journal

Biology

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

Loading Drosophila Nerve Terminals with Calcium Indicators

载入果蝇与神经末梢钙指标

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

科研

生物学

Operant Learning of Drosophila at the Torque Meter

果蝇在操作性学习扭力计

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

一个磁性的系绳系统，以调查在果蝇的视觉和嗅觉介导的飞行控制

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

电生理方法记录从果蝇幼虫NMJ突触潜力

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Visually Mediated Odor Tracking During Flight in Drosophila

在果蝇的飞行过程中可视介导的气味追踪

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

线虫神经肌肉交界处的解剖和录制

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

定量比较顺调节元件（CRE）的活动，在转基因果蝇</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Recombineering Homologous Recombination Constructs in Drosophila

Recombineering Homologous Recombination Constructs in Drosophila

重组工程同源重组构建<em&gt;果蝇</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...