Name: optogenetic stimulation of escape behavior in drosophila melanogaster
Uploaded: 2013-01-25T13:00:00
Description: Watch this Scientific Journal Video about Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster at JoVE.com

这项工作是由斯坦福大学的院长奖学金（SE​​JdV），美国国立卫生署署长的先锋奖（TRC DP0035350），一个McKnight基金会学者奖（TRC）和R01 EY022638的（TRC）。

1。生成Channelrhodopsin苍蝇<ol><li>跨UAS-CHR 2的苍蝇与GAL4您所选择的驱动程序，我们使用G105-GAL4，表示这是在福马-1在视叶神经元。 </li><li>为了消除可能的蓝色光刺激的视觉反应，无论是飞线是在AW + norpA背景。 </li><li>最终的结果：W + norpA“G105-Gal4/UAS-ChR2 + </li><li>后成蝇E关闭，把选中的女性在10μM全反式视黄醛（辅助因子所需的CHR 2），避光，新鲜的食物，补充前3天进行行为分析。 <...

<ol>
	<li>Venken, K., Simpson, J., Bellen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulations+of+genes+and+cells+in+the+nervous+system+of+the+fruit+fly.">Genetic manipulations of genes and cells in the nervous system of the fruit fly.</a> Neuron. 72, 202-230 (2011).</li><li>Gordon, M., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+control+in+a+Drosophila+taste+circuit.">Motor control in a Drosophila taste circuit.</a> Neuron. 61, 373-384 (2009).</li><li>Suh, G. S. B., 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+an+innate+olfactory+avoidance+response+in+Drosophila.">Light activation of an innate olfactory avoidance response in Drosophila.</a> Current Biology. 17, 905-908 (2007).</li><li>Zhang, W., Ge, W., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolbox+for+light+control+of+Drosophila+behaviors+through+Channelrhodopsin+2-mediated+photoactivation+of+targeted+neurons.">A toolbox for light control of Drosophila behaviors through Channelrhodopsin 2-mediated photoactivation of targeted neurons.</a> European Journal of Neuroscience. 26, 2405-2416 (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=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proceedings of the National Academy of Science. 100, 13940-13945 (2003).</li><li>de Vries, S., Clandinin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loom-sensitive+neurons+link+computation+to+action+in+the+Drosophila+visual+system.">Loom-sensitive neurons link computation to action in the Drosophila visual system.</a> Current Biology. 22, 353-362 (2012).</li><li>Card, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+behaviors+in+insects.">Escape behaviors in insects.</a> Current Opinion in Neurobiology. 22, 1-7 (2012).</li><li>Card, G., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visually+mediated+motor+planning+in+the+escape+response+of+Drosophila.">Visually mediated motor planning in the escape response of Drosophila.</a> Current Biology. 18, 1300-1307 (2008).</li><li>Fotowat, H., Fayyazuddin, A., Bellen, H. J., Gabbiani, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+neuronal+pathway+for+visually+guided+escape+in+Drosophila+melanogaster.">A novel neuronal pathway for visually guided escape in Drosophila melanogaster.</a> Journal of Neurophysiology. 102, 875-885 (2009).</li><li>Card, G., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+trade-offs+in+the+flight+initiation+of+Drosophila+melanogaster.">Performance trade-offs in the flight initiation of Drosophila melanogaster.</a> The Journal of Experimental Biology. 211, 341-353 (2008).</li><li>Hammond, S., O'Shea, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+flight+initiation+in+the+fly.">Escape flight initiation in the fly.</a> Journal of Comparative Phsyiology A. 193, 471-476 (2007).</li><li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+mutants+of+Drosophila+isolated+by+countercurrent+distribution.">Behavioral mutants of Drosophila isolated by countercurrent distribution.</a> PNAS. 58, 1112-1119 (1967).</li><li>Bloomquist, B., 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=Isolation+of+a+putative+phospholipase+C+gene+of+Drosophila,+norpA,+and+its+role+in+phototransduction.">Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction.</a> Cell. 54, 723-733 (1988).</li><li>Gohl, 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=A+versatile+in+vivo+system+for+directed+dissection+of+gene+expression+patterns.">A versatile in vivo system for directed dissection of gene expression patterns.</a> Nature Methods. 8, 231-237 (2011).</li><li>Zhang, F., 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=Red-shifted+optogenetic+excitation:+a+tool+for+fast+neural+control+derived+from+Volvox+cateri.">Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox cateri.</a> Nature Neuroscience. 11, 631-633 (2008).</li></ol>

<html>我们已经证明的逃逸行为optogenetic刺激，洗澡自由地走在明亮的蓝色光线的苍蝇。这种方法可以很容易地适应自由行走苍蝇检查的其他行为，并且可以扩展到更大的平台，通过简单地平铺在一个较大的区域中使用的LED阵列，我们。使用廉价的摄像机，我们描述，或其他可用的相机系统中，用户可以调整图像的帧速率和空间分辨率获得的适应行为兴趣。此外，我们的成像后的时间是有限的LED被?...

越来越基因编码的工具库已经发展到操纵的神经活动在特定的细胞在果蝇 1。这些工具使非侵入性的完整和自由移动的动物在特定的神经元的激活或沉默。的光激活的阳离子通道，Channelrhodopsin2（CHR 2），其中，提供了重要的优势，因为它可以在时间控制，并迅速诱导。当神经元表达CHR 2暴露在明亮的蓝色（470 nm）的光，他们迅速去极化，表现出升高的发射率3-5。这种有针对性的激活特定的神经元在自由活动动物，揭示了充足的特定神经元的行为，如长鼻延长2,4，3，CO 2避免和巨纤维介导的惊吓反应。然而，由于激烈的光源要刺激CHR 2也刺激光感受器，施加运算togenetic视觉系统的技术，已经被限制。通过相结合的optogenetic的方法的突变，会影响光传导，我们已经表明，在果蝇视叶神经元激活一个特定的集群可以开车逃逸行为，以避免碰撞6。 大多数情况下，如果不是全部的，视觉的动物表现出逃避行为，以避免与迎面而来的物体发生碰撞。步行或固定荏苒，当一个迫在眉睫的冲突，进入飞行，起飞距离迎面而来的碰撞7-9。这些起飞的特点是起飞和飞行轨迹不稳定，10,11之前提出的翅膀。这种反应是与巨纤维介导的惊吓反应，跳之前没有提出翅膀不同，通常会导致在自由下落的滚筒4,9。在确定了具体的织机敏感的神经元群在视叶，福马-1神经元，这是尹居良伊利调整到接近对象进行编码，我们试图探讨其参与果蝇的织机逃逸行为。在这里，我们演示了如何使用光遗传学选择性地激活这些神经元，引起果蝇的逃逸行为。 我们使用驱动C​​HR 2的FOMA-1神经元表达的GAL4-UAS转录的激活系统。 CHR 2需要的辅助因子全反式视黄醛，因为这是发现在果蝇中枢神经系统在较低水平，必须要补充在果蝇的饮食。3,4明亮的光线来激活CHR 2和果蝇表现出强烈的趋光行为12，我们试图以消除可能的刺激的视觉反应。要做到这一点，我们使用的动物，它编码的光传导级联反应的重要组成部分，磷脂酶C-β的norpA基因为无效等位基因的纯合突变。在这样的突变果蝇的感光器无法respond来点亮13。的逃避反应要测试optogenetic的刺激，我们需要隔离一只苍蝇，沐浴在明亮的蓝色光线。要做到这一点，我们把个人的苍蝇在枪头。一个枪头放置在一个自定义的夹持器，使得飞将geotactically步行尖端和流出到一个矩形平台。苍蝇是能够自由地走动这个平台的顶部。该平台所包围四个蓝色LED阵列，各含3个LED，重点的顶部上的平台。苍蝇在该平台后，指示灯亮起，并使用高速摄像机6苍蝇的反应记录。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td>全反式视网膜</td><td>促进科学和化学公司</td><td> R3041 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>散热片9.2 C / W </td><td> Luxeonstar </td><td> LPD30-30B </td><td> 30平方毫米X 30毫米高</td></tr><tr><td> CARCLO 18°三镜头</td><td> Luxeonstar </td><td> 10507 </td><td></td></tr><tr><td>蓝色的反叛LED上三星基地</td><td> Luxeonstar </td><td> MR-B0030-20T </td><td> 470纳米，174流明@ 700毫安。 </td></tr><tr><td> 700毫安BuckPuck DC驱动器</td><td> Luxeonstar </td><td> 3021-DE-700 </td><td></td></tr><tr><td>线束BuckPuck驱动</td><td> Luxeonstar </td><td> 3021-HE </td><td></td></tr><tr><td>预切的热胶带</td><td> Luxeonstar </td><td> LXT-S-12 </td><td> 20毫米内六角基地</td></tr><tr><td>扣-LOC冷却液软管，6.3“ID </td><td>麦克马斯特 - 卡尔</td><td> 5307K49 </td><td></td></tr><tr><td>扣-LOC冷却液软管接头</td><td>麦克马斯特 - 卡尔</td><td> 5307K39 </td><td> ¼“不扩散核武器条约”男</td></tr><tr><td>实验室等级的开关模式可编程直流电源供应器</td><td> BK精密</td><td> 1698 </td><td></td></tr><tr><td> EXILIM相机</td><td>卡西欧</td><td> EX-FH20 </td><td></td></tr></tbody></table>

optogenetic stimulation of escape behavior in drosophila melanogaster

越来越多的基因编码的工具，正成为可用，允许非侵入性操作，在果蝇的特定神经元的神经活动。其中最主要的是optogenetic工具，激活或沉默的完整和自由移动的动物在特定的神经元，使用明亮的光线，使。 channelrhodopsin（CHR 2）是激活时，由蓝色光，使反映它的神经元的去极化的光激活的阳离子通道。 CHR 2已经有效地确定神经元的特定行为，如CO 2避税，长鼻延伸和巨纤维介导的惊吓反应2-4的关键。然而，作为用来刺激CHR 2也刺激感光体的强光源中，这些optogenetic技术已经以前没有被使用的视觉系统。在这里，我们结合的optogenetic的方法的突变，会影响演示的光传导nstrate集群的织机敏感的神经元在果蝇视叶，福马-1神经元的激活，可以开车逃逸行为，以避免碰撞。我们使用了无效等位基因的光传导级联的重要组成部分，磷脂酶C-β，编码基因的norpA，导致苍蝇盲目的，也可以使用CHR 2的福马-1表达的GAL4-UAS转录的激活系统神经元。个人的苍蝇被放置在一个小平台，周围环绕着蓝色的LED。当LED被点亮，过得很快起飞，飞行，视觉驱动的织机逃逸行为方式类似。我们相信，这种技术可以很容易地适应检查的其他行为，在自由移动的苍蝇。

盲飞表达无论是CHR 2或G105驱动程序独秀率较低起飞后带有明亮的蓝色光照明。盲飞，无论照明（ 图2）表现出同样的速度起飞，起飞，这是自发的，而不是由于蓝色光的照明。当CHR 2 Foma1神经元中的表达，然而，与蓝色光的照射引发逃避反应。超过50％的测试苍蝇起飞，在1秒内的照明，75％的5秒内（ 图2）。相比之下，只有10％的控制苍蝇起飞，在1秒内，20％的5秒内。表示...

Watch this Scientific Journal Video about Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster at JoVE.com

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

基因编码的optogenetic的工具，使非侵入性的操作，在特定的神经元<em&gt;果蝇</em&gt;脑。这些工具可以识别神经元的激活是足以引起或抑制特定的行为。在这里，我们提出了有针对性的自由行走苍蝇的神经元激活方法Channelrhodopsin2表示。

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Research

JoVE Journal

Neuroscience

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

在昆虫单感器记录果蝇按蚊</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

科研

神经科学

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

在自发活动的测定<em&gt;果蝇</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

简单的检测方法的社交回避的量化<em&gt;果蝇</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Optogenetic Stimulation of the Auditory Nerve

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Conditions Affecting Social Space in Drosophila melanogaster

Conditions Affecting Social Space in Drosophila melanogaster

在条件影响社会空间<em&gt;果蝇</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

在气味引导行为的高分辨率定量<em&gt;果蝇</em&gt;使用<em&gt; Flywalk</em&gt;范式

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantification of Drosophila Grooming Behavior

果蝇美容行为的量化

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...