这项工作是支持的格兰特在科研创新领域的援助的介观Neurocircuitry“（第22115002号）和”综合脑科学网“（221S0003号），教育，文化，体育，科学部，和技术，日本和格兰特援助青年科学家（B）（第21700344号），日本学术振兴科学（JSPS）到香港

1。幼虫准备<ol><li>维持飞线OK6-GAL4 UAS-CHR 2或OK6-GAL4，UAS-NpHR2在塑料小瓶含标飞食品。 </li><li>传播酵母膏含有全反式视黄醛（ATR）在适当的浓度CHR 2（1毫米，10毫米为NpHR2（“NpHR”的简称）的苹果汁琼脂平板。 </li><li>从小瓶拿起第二或第三龄幼虫，并把它们上的ATR含板。 </li><li>后他们在黑暗中一段适当的时间（CHR 2，2天NpHR为1日的在25°C。） </li></ol> 2。显微镜设置<ol><li>将CCD相机（XCD-V60，索尼）传统的共聚焦显微镜（在本例中，奥林巴斯FV1000）与C型底座附件（放大倍率0.35X）。 </li><li>来自物镜的光路上的CCD摄像头，关闭激光扫描期间，如果有一个快门沿，小心取出。由于此步骤取决于显微镜应对设置，请联系技术支持，如果必要的显微镜制造商。 </li></ol> 3。解剖<ol><li> ATR-喂养幼虫水冲洗，以去除残留的食物从体内。 </li><li>将幼虫一个SYLGARD涂层菜，背侧（背侧有两个气管导管，纵向运行，一个背中线两侧的）。 SYLGARD的厚度约为5mm。 </li><li>插入昆虫针（奥斯特利茨昆虫针，Φ0.10mm，不锈钢）之间的气管进入尾管钳（＃5 INOX由杜蒙，瑞士，FST）。标签，约10mm长，弯曲或切成足够短（约2毫米长），以避免碰撞而损坏的表面上的物镜。然后把第二昆虫针嘴钩，黑色前端的爪状结构附近的幼虫入目。 </li><li>添加的 Ca 2 +生理盐水（氯化钠140毫米，氯化钾2毫米， 氯化镁 6毫米，HEPES-NaOH溶液5毫米，蔗糖36毫米（pH7.1））保持幼虫湿润。 </li><li>靠近尾巴的地方做一个小切口显微剪（MB-50-7，Napox，日本）。 </li><li>从切口，作一纵切口沿背中线向头部。要小心，不要损坏腹神经索（VNC）和轴突。 </li><li>做一个小切口在头侧面。 </li><li>将4个引脚的解剖体壁在每个角落。体壁应足以看到一个体壁的节段性模式被拉长，但没有太多的阻碍蠕动。 </li><li>除去内脏除了大脑和VNC和与Ca 2 +生理盐水冲洗样品。 </li><li>调整腹神经索的方向是左右对称的，通过改变昆虫体壁的标签的位置和方向。要修复腹神经索的位置，通过组织之间的大脑和嘴钩SYLGARD插入一根针。 </li> &lt;LI&gt; 2毫米，更换缓冲的Ca 2 +林格液（氯化钠130毫米，氯化钾5毫米的2 2MM，氯化镁，氯化钙2毫米，HEPES-NaOH溶液5毫米，蔗糖36毫米（PH7.3））。 </li></ol> 4。激光照明成像<ol><li>附加4干共聚焦显微镜的物镜（4倍UPlanSApo，NA 0.16，奥林巴斯，日本），并在舞台上设置的幼虫准备。 </li><li>获取的透射图像的解剖准备的红色激光（633nm波长），激光共聚焦显微镜定位腹神经索。对于这种扫描，确保不使用488nm的或559nm激光，从而引起不​​必要的刺激的CHR2或NpHR分别。 </li><li>定义感兴趣的区域（ROI）的透射图像上。缩放模式可能是有用的投资回报率的详细测定。 </li><li>更换过滤器组与488nm的或559nm光显微镜照亮。 </li><li>用卤灯照亮了准备，以可视化的体壁。钍与CCD相机连接到共聚焦显微镜，可以监测评估准备。 </li><li>记录的体壁的运动切换的激光束和关闭，同时监测动议。 </li></ol>

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没有利益冲突的声明。

神经活动的时间和地方的扰动是一种宝贵的神经回路网络的动态分析技术。在这个协议中，我们提出了一种使用激光的光遗传学方法来操纵神经活动。激光的高方向性使得更多的本地化的光遗传学刺激比使用汞或氙气灯的宽视场刺激。虽然激光照明已经应用于光遗传学在以前的研究中，需要特殊的设置，如玻璃纤维，微操作机器人，激光源，在以往大多数研究。在这个协议中，我们使用了传统的激光共聚焦显微镜，用于局部照明。由于共聚焦显微镜系统被广泛使用，此方法将打开机会申请更高分辨率的光遗传学在许多实验室。 该协议有两个关键点：幼虫解剖和激光功率。首先，如果脑，腹神经索或运动神经受损杜里纳克夹层，幼虫会表现出较少或没有自发蠕动。因此，精确度是必不可少的，尤其是当弹簧剪刀和背侧切口，去除内脏用钳子。已有报道21详细解剖。其次，如果有足够的刺激来实现的，为约0.1〜1毫瓦/毫米2 CHR 2或1〜10毫瓦/毫米2用于NpHR的必需的激光功率。我们评估了激光功率的总光功率低于物镜除以估计面积照明。我们测量的总光功率使用功率计（mobiken，日本三和MI TECHNOS）。我们估计的区域的照明，如圆形的常数倍的波长的激光，衍射极限大致给照明面积的平方。不仅对样品的实际照明功率可以调整激光输出功率，而且还通过改变扫描速度和麻木呃重复。我们扫描激光20-100微秒/像素的63倍。此外，光遗传学蛋白质和量ATR的表达水平也刺激效率的关键。因此，如果的幼虫没有显示光遗传学响应，有以下几点应检查和纠正：1）激光功率（通过优化显微镜系统），2）光遗传学蛋白表达水平的（检查基因型和饲养温度），和3）量的ATR（调整浓度ATR和哺乳期）。 NpHR需要较高浓度的ATR比CHR2确实原因不明13。 CHR2运作良好，在幼虫饲养食物中含有较小的ATR（ 比如 0.1毫米）比（1毫米）。由于喂养幼虫为1毫米的ATR CHR2光反应是足够的，我们建议这个浓度一审在CHR2实验。 ATR浓度从1毫米随后被滴定。 ATR暴露时间，我们建议这里描述的强大的光遗传学控制的持续时间。我们经常喂养幼虫时，ATR的时间更短，没有观察到光遗传学反应。 在这个协议中，激光照明和图像采集操作由一台单独的计算机。所以，清晰的激光照排的时空格局CCD相机检测数据分析的关键。如果激光点或线CCD图像暗淡，你应该优化卤素灯和增益值的CCD相机以可视化的激光照射，同时保持体壁可见的力量。 在这个协议中示出的时空照明幼虫电机电路上提供的信息，然而，该方法具有一定的局限性。至于“空间”方面，记录位置的照度低倍率的CCD图像用于偷拍体壁变动。因此，照明面积，可在分部层面分配，而不是在细胞水平。至于“吨依赖于激光共聚焦显微镜系统和扫​​描条件emporal“方面，开关上的激光扫描的激光共聚焦显微镜和照明时间之间的时滞，因此，需要一些测试，通过试验和错误来调整照明的定时。由于照明的定时可以确定使用适当的软件，如ImageJ的CCD图像电影，关键的因素在时空分辨率的CCD相机成像的帧速率，我们通常设置为7.5-15帧每秒的帧速率。 进展optogenetic工具为我们提供了机会修改此协议。我们使用第三龄幼虫拥有OK6-GAL4和UAS-CHR2 [H134R]或UAS NpHR2的。光遗传学工具，而不是CHR2 [H134R]或NpHR2等CHR2 T159C/E123T的] NpHR3的或拱可以提高效率的活动的控制22。此外，使用等GAL4线或其他发育阶段进行分析，可以提供更多的信息与电机有关的电路，TS。 在这个协议中，由透射图像的体壁的收缩用CCD照相机监测运动活性。与钙敏感的荧光分子（ 例如 GCaMP）运动神经元的活性，也可以直接监测的同时操纵与CHR 2或NpHR的神经活动。在这种情况下，蓝色光照射在一个广泛的区域和一个高度敏感的CCD照相机（ 例如 EMCCD相机），而不是使用卤素灯，本文中前面描述的普通的CCD摄像机。为了改善空间分辨率的刺激，同时监测体壁的运动，使用一个额外的物镜来自上述样品和监测体壁由低放大倍率透镜的高倍率的物镜（ 例如，40倍），可能是一个更高级的方法：照明腹神经索以下的样品（ 例如 4倍）。这种双镜头系统，可以使我们能够刺激神经系统，在更高的空间分辨率。

在果蝇幼虫发生正向蠕动运动，肌肉收缩的传播，从后到前分部。实现此议案通过顺序激活运动神经元内腹神经索沿纵向的体轴。要检查电路，这背后的图案的传播活动，当地的神经活动的扰动可能是一个信息的方法。虽然药理实验可以控制特定的物质，如神经递质，在神经组织​​中，其效果的药理药物可以产生的几乎所有的部分是相似的，因为幼虫的腹神经索沿纵向轴线的类似neuromeres数据是重复的结构。或者，人们可以表示光遗传学或对温度敏感的探针分子在一个小的段数。然而，这些具体GAL4线是非常困难的产生。在这里，我们提出了一个方法操纵神经元几赛格目采用激光照明。一个可以利用的空间密闭的共聚焦显微镜的照明，扰动的局部区域​​中的神经元的活性。与探头由神经元特异性GAL4行的子集的表达模式相结合，此激光照射方法大大提高了扰动的空间分辨率。 ，因为这个方法只需要一个传统的共聚焦显微镜，购买额外的实验室设备通常是没有必要的。 使用这种技术，我们研究运动 ​​神经元活性的作用，在传播过程中，电机的输出13。通过阻断蠕动运动过程中在几个分部的运动神经元的活动，我们可以测试是否运动神经元本身的活性是必需的为电动机输出信号的传播。运动神经元的本地和短暂封锁拘捕蠕动的传播。封锁被删除后，pagation出现在段波已被逮捕。这种现象表明，没有运动神经元激活，繁殖波不能沿腹神经索的任何进一步取得进展，从而对蠕动运动的运动神经元的活化是必要的。之前我们已经报道了详细的数据和讨论这种现象在稻田等人 （2011）13。在这里，我们描述交互干扰神经活动的方法同时监测解剖幼虫的运动。可以使用各种GAL4线和系列活动操纵的时空模式，逻辑电路，电机电路通过扰动的响应性能。

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 <tr>
 <td>Name</td>
 <td>Company</td>
 <td >Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>BX61WI Microscope</td>
 <td>Olympus Corporation</td>
 <td>BX61WI</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluoview FV1000 confocal system</td>
 <td>Olympus Corporation</td>
 <td>FV1000</td>
 <td></td>
 </tr>
 <tr>
 <td>CCD camera</td>
 <td>Sony</td>
 <td>XCD-V60</td>
 <td></td>
 </tr>
 <tr>
 <td>all-trans retinal</td>
 <td>Sigma</td>
 <td>R2500-500MG</td>
 <td>200 mM stock in 95% EtOH</td>
 </tr>
 <tr>
 <td>Silpot184</td>
 <td>Dow Corning Toray</td>
 <td>3255981</td>
 <td></td>
 </tr>
 </tbody>
</table>

optogenetic perturbation of neural activity with laser illumination in semi-intact drosophila larvae in motion

果蝇幼虫的运动是一个灿烂的模型系统在神经系统的发育和生理，由于遗传辅助功能相关的神经元件在电路1-6。光遗传学7,8幼虫的神经电路的应用，使我们能够操纵神经元的活动在空间和时间上的图案的方式9-13。通常情况下，试样大致汞灯时照明或LED，所以目标神经元特异性控制的二进制的基因表达系统，如Gal4的UAS系统14,15。在这项工作中，“子基因的分辨率提高空间分辨率，局部照明系统实施常规的共聚焦显微镜的使用激光的腹神经索中的神经元的一个子集。在监测半完整的幼虫体壁的运动，我们交互激活或抑制神经用16,17 channelrhodopsin O活动ŗhalorhodopsin 18-20。由时间和空间上的限制照明的神经组织中，我们可以操作在一个特定的行为相电路中的特定神经元的活性。这种方法是有用研究本地腹神经索和神经装配在电机的输出的时空图象的活动之间的关系。

<html>本地和瞬态运动神经元的激活与CHR 2。 我们在运动神经元中表达CHR 2。从每个VNC段项目体壁的一个对应段落新兴的运动神经元的轴突。当我们用的蓝色激光，刺激的一个或两个分部的VNC中的相应节段的肌肉表现出收缩（ 图1）。 本地和瞬态抑制运动神经元与NpHR。 我们表示NpHR，在运动神经元中。当我们在蠕动肌肉的收缩与黄色激光刺激少数（1〜2）段的VNC，传播波涉嫌在体壁的相应节段（ 图2）。然后重新启动波从被捕段时，我们关掉激光照排13。 50513fig1.jpg“ALT =”图1“FO：内容宽度=”5.5英寸“FO：SRC =”/ files/ftp_upload/50513/50513fig1highres.jpg“/&gt; 图1。本地运动神经元激活CHR2。A.本地激活圆角准备计划。腹神经索在运动神经元中表达CHR2几个分部的蓝色光的照度，在相应节段的肌肉收缩（箭头）。B.传输图像的解剖幼虫。局部照明的腹神经索（箭头头部）诱导体壁肌肉（箭头）的节段性收缩。 <img src="/files/ftp_upload/50513/50513fig2.jpg" alt="图1" fo:content-width="5.5in" fo:src="/files/ftp_upload/50513/50513fig2highres.jpg"/> 图2。局部抑制运动神经元与NpHR A.计划对当地抑制圆角准备。通过黄色的-l几个分部腹神经索表示NpHR，的运动神经元，肌肉收缩过程中自发地发生蠕动运动（在顶部的箭头）放宽飞行照明（在底部的箭头）。B.传输图像的解剖幼虫。腹神经索（箭头元首）局部照明放宽自发承包体壁段（箭头）。

Watch this Scientific Journal Video about Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion at JoVE.com

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

在这里，我们描述了一个协议，光遗传学操纵运动元活动，同时监控电机输出的变化（肌肉收缩）半完好<em&gt;果蝇</em&gt;幼虫使用在传统的激光共聚焦显微镜。这项技术使研究人员能够实现的神经活动几neuromeres在局部扰动阐明电机电路的动态。

光遗传学的扰动激光照射半完好的神经活动<em&gt;果蝇</em&gt;幼虫运动

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

optogenetic-perturbation-neural-activity-with-laser-illumination-semi

Research

JoVE Journal

Neuroscience

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

11.5K 次观看.  The University of Tokyo. 在这里，我们描述了一个协议，光遗传学操纵运动元活动，同时监控电机输出的变化（肌肉收缩）半完好果蝇幼虫使用在传统的激光共聚焦显微镜。这项技术使研究人员能够实现的神经活动几neuromeres在局部扰动阐明电机电路的动态。

视频: Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

在 C 在体内激光干切断线虫

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

科研

神经科学

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

局部和全局的方法评估热痛觉<em&gt;果蝇</em&gt;幼虫

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

欲求联想嗅觉学习<em&gt;果蝇</em&gt;幼虫

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

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 ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

神经活动的传播的展开海马准备具有穿透微电极阵列

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

新的分析冷痛觉在<em&gt;果蝇</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.