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。 </li></ol> 2。 10μM全反式视黄醛的强化食品<ol><li>在17.6毫升的95％乙醇中溶解100毫克的全反式视黄醛，使20mM的视网膜。保持全反式视黄醛避光，在任何时候。 </li><li>标准的玉米面飞的食物在微波炉融化，放凉，直到温暖的触摸。 </li><li>混合50微升的20mM全反式视黄醛的10毫升粉煤灰食品到小瓶中。 </li><li>让瓶冷却，并保持避光。 </li></ol> 3。设备<ol><li>枪头：标准1000μL枪头附近的尖被切断，创造〜2.25毫米的孔径。 </li><li>平台（参见图1）。 <ol><li>一个聚甲醛基地，17厘米X 25厘米，被修建在每一个角落，以适应¼“不扩散核武器条约”冷却液软管接头的螺纹孔。 </li><li>由Delrin制成，甲垂直支架，安装的碱的中心。整体尺寸为25毫米X 40毫米X 65毫米（宽X深X高）。运行一个10毫米宽的凹槽的保持器的长度，在底部具有翼形螺钉。 A平台安装的支架的顶部，25毫米×40毫米×10毫米，用3.5毫米直径的孔与在支架中的槽对齐。 </li></ol></li><li>发光二极体（LED）阵列（参见图1）。 <ol><li>冷却液软管，四臂〜18厘米长，都贴有平台BASE使用的冷却液软管连接器。冷却剂软管仅被用作结构支撑和不用于冷却的目的。 </li><li>切槽，到最后一块冷却液软管，每个臂，每个臂的末端贴上了散热片，以适当的距离。 </li><li>使用预切的热胶带安装在每个散热片上的蓝色LED反叛的三星级。一个CARCLO 18°三镜头贴在每个三星级。 </li><li>的BuckPuck DC驱动器和电源连接到LED三连星。我们已经安排我们的设置，与每个BuckPuck供电两个三连星系列。 </li><li>所有四个LED照明的三连星在700 mA时产生的辐射为713瓦/米2，在我们的平台上。 </li></ol></li><li>摄像头：摄像头是装在一个小三脚架和重点顶部的平台上。 </li></ol> 4。行为检测<ol><li>简单地说，麻醉苍蝇冰。 </li><li>将个人苍蝇在枪头，用胶带将关闭两端部的前端。 </li><li>苍蝇后语惊醒梦中人，并正在积极探索枪头，取出磁带，然后将移液管在垂直支架的凹槽中。的指旋螺丝，用于固定枪头，并关闭底部的尖端。 </li><li>一样的飞走探索枪头（一般为30 - 60秒），启动相机记录之前出现的飞行从尖端到这个平台上。 </li><li>后飞已经出现在平台上，等待1-2塞科和打开的蓝色LED。使用一个定时器来手动测量的时间，直到飞启动飞行。 </li></ol>

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	<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>

没有利益冲突的声明。

<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 ...

optogenetic-stimulation-of-escape-behavior-in-drosophila-melanogaster

Research

JoVE Journal

Neuroscience

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

17.3K 次观看.  Stanford University. 基因编码的optogenetic的工具，使非侵入性的操作，在特定的神经元果蝇脑。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。

视频: Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (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

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

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

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

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

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

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

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

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 ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

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

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

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

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

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

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

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

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

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

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

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

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

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 ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic 在行为小鼠海马θ振荡中的作用

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...