1。气味稀释<ol><li>大多数气味溶于石蜡油。然而，DMSO或乙醇也可以被用来作为对特定气味的替代溶剂。准备适当稀释（例如1:10体积：体积，V：V）从纯粹的玻璃小瓶气味。大多数的气味稀释，在室温下稳定，但极易挥发的化合物，它是更好的每周工作稀释。每个感器的响应，以不同浓度范围内不同的气味。对于果蝇 ，一个有用的查表的，适当的浓度，使用一个给定的感器，可以发现在6，7。 对于视频实验中，我们用石蜡油作为溶剂的控制和醋酸甲酯（10 -6 V：v在石蜡油）和1 -辛烯-3 - 醇 （10-7 V：v在石蜡油）对果蝇和按蚊录音，分别为。 </li><li>用剪刀，切成3毫米× 5厘米条层析纸，使他们适合巴斯德吸液管内。 </li><li>移液器30μL的滤纸条上所需的气味，玻璃吸管插入。切〜3厘米的空气管路，插入吸管的开口端，关闭它的连接器。连接器是用来密封吸管，然后将连接到一个空气泵空气管路时，时间，在实验过程中提供的气味。 </li></ol> 2。气味输送系统<ol><li>使用的小钻，切10毫升塑料血清移液器（如在4毫升），并创建两个孔（例如，在-1.5 mL和-0.5毫升商标）将用来存放移液器含有的气味。插入一个200μL枪头刺耦合，并引入到10毫升吸管的钝端的耦合。吸管将被用作气味交付系统的一部分。 </li><li>附加磁性支架上的吸管，用吸管钳和附近的显微镜，它的位置。 </li></ol> 3。锐化电极<ol><li>为了加强电极，准备0.5米的氢氧化钾溶液（KOH）和过滤器以去除细颗粒物（如：采用45微米过滤器）。 20毫升的注射器和一个小孔（直径为2毫米），墙上贴着一针针尖（从尖〜1厘米），在其中插入电线（图1A）。 </li><li>填充注射器，用0.5 M KOH和打击，在显微镜下它的立场上，使针尖放置在视野（图1B，C），。到注射器上墙（图1B）的小孔，插入电线，确保导线是不能直接在注射器入口前。此线连接到直流电源（例如野生Heerbrugg MTR32，见方法）或交流电源（如野生Heerbrugg - LEP 990018）极的阴极。连接到显微镜右侧的手动显微电极支架轴，并附加电缆与鳄鱼夹的电极支架轴的基础。连接电缆的直流电源或交流电源（图1A）的另一极阳极。 </li><li>插入电极支架的钨丝（约5厘米长），并且将它附加到电极支架轴操纵。设置为6 V的电源，插入注射器反复磨练线的一角，小心监控，在显微镜下在这个过程中的提示（图1C）。要得到一个理想的录音的电极，其长度在溶液中的90％，为1分钟，并慢慢地将其拉出。然后插入只有约50％的电极，以进一步薄30秒，并重复削尖（〜10倍）的导线尖端。 电极尖端应足够细，进入了感​​器，但没有这么细，弯曲，当它触动它在录音（步骤6和7）。虽然看解剖显微镜下的电极尖端，而它被削尖，是一个很好的迹象，其厚度仅看它记录在高倍显微镜下将给出一个明确的想法，一个给定的电极是否适合用于记录。 </li></ol> 4。昆虫准备： 果蝇天线<ol><li>构建一只苍蝇抽吸。剪下一块空气管路足够长的时间挂在你的脖子，舒适（约90 - 120厘米）。剪下200μL枪头的尖端和管的一端插入。在另一端，位置1〜1.5厘米× 1.5厘米的网状片，以便它创建了一个物理屏障，但并不妨碍空气管流出。剪下1毫升枪头和管开放的顶部位置更广泛的提示，阻断之间的网格。这部分将用于拿起和操纵成人醋蝇（图2）。 </li><li>以载玻片，放置了一块大约在长边的中间牙科用蜡。它positio顶部NA玻璃盖略微倾斜向上（〜30），确保蜡是不是直接放在最上面的一部分，这将防止在显微镜下可视化飞醋。拉玻璃电极垂直拉拔。其尖端应薄且具有足够的灵活性，以适应在第二和第三的触角段之间，并保持录音天线稳定。另一块蜡玻璃电极的位置，放在玻璃盖的一侧，远远不够，笔尖的时候，降低到达角落的玻璃盖（图3A）。 </li><li>工作所需的基因型成蝇瓶或小瓶，拿起一个成年人的飞醋，使用飞抽吸​​。虽然女性通常是因为他们的更大的大小，男性也可以使用。 1毫升的小费上放置一个200μL枪头，以避免逃跑的飞行。吹入管，使飞对200μL枪头年底推。修剪刀片的枪头远离苍蝇本身几毫米宽的一端，然后插入一些蜡，以防止飞打了退堂鼓。在显微镜下，再次下调头附近的飞醋，注意不损害动物。有了一个小吸头，推蜡力飞头，使眼睛的一半左右，从尖（图3B）挤出。确保它的腿不出来为好，否则可能会移动和干扰录音。 </li><li>山飞一块蜡，其头部朝上，并将其放置在前面的玻璃盖的幻灯片上。将头微微对玻璃的一角，使触角延伸和休息上的玻璃。下之间的第二次和第三次的触角段（图3B）的玻璃毛细管的尖端。 </li><li>天线的不同部分会进入到不同类型的感受器。根据特定的实验需要，天线将需要面向不同的，允许访问到不同类型的感受器。要记录在我们的例子从大basiconic感器的阿里斯塔向下压在玻璃上（图3B）。 </li></ol> 5。昆虫准备： 按蚊上颌触须<ol><li>使用电动吸引器（图2E），40至60 3-5天（混合男性和女性）老蚊子收集的小塑料箱（图2F）。蚊子应该已经在正常条件下饲养，在昆虫的孵化器或insectary即在25-28℃，湿度70-80％。将冰〜15分钟的小动物的塑料保持架，直到它们被寒冷的麻醉。动物一旦停止移动，只有4-6动物转移的阶段，在显微镜下在任何一个时间内，保持其余的动物，在程序上冰。虽然女性通常是因为他们对CO 2的敏感性，男性也可聘用。选择女性或男性的蚊子，其天线结构（羽毛般的女性，男性长丝）来看，并删除它们的翅膀和腿，用细镊子固定。保持在一个小塑料杯，他们在底部的湿纸，以防止他们从desiccating。 </li><li>把两条双面胶带（〜1厘米的长度），在该中心，并在载玻片两侧平行于对方（〜1厘米），（图3C）。用细镊子，地方对中央磁带之一蚊子解剖显微镜下，侧身把它坚持它的身体和一只眼睛，在磁带上。调整这样既触须延伸到磁带上（图3D）平行的位置上颌骨的触须。弦细（如人的头发）放置在基地和触须（图3D）的尖端修复上颌骨的触须。端磁带存储作为弦细，而中央的磁带需要被替换后，每一个记录（图3C）。 </li></ol> 6。记录果蝇 <ol><li>幻灯片下放置在低倍率的显微镜和天线位置大致在中间领域的视野（FOV）（图4B）（图4A）。轻轻放下，使参比电极电极位于飞和记录电极靠近天线（图4C）眼睛附近。增加在中间的视野（图4D）的放大倍率和重新定位的天线。 </li><li>广场的气味传递给设备密切飞的头，指着在天线。 </li><li>在低倍率（图4C），参比电极插入飞的眼睛。下而不触及其表面上的天线顶端的记录电极。 </li><li>切换到高放大倍率，控制与显微记录电极，并把它插入到选定感器（图4D）。沿感器长度的任何一点是录制好的​​。一旦感器内，电极可推更远（有时所有的方式通过）以获得更好的信噪比。一旦电极感器，可以检测细胞的自发活动。 </li></ol> 7。录音按蚊 <ol><li>放置在低倍率（10倍）的视野和头顶部的中间位置，大约在上颌骨的触须的显微镜下的载玻片（图4E）。旋转舞台，直到在直角与记录电极的触须。 </li><li>调整电极的高度，使参比电极定位略高于眼睛的蚊子和记录电极附近的上颌骨的触须。 </li><li>将气味传送装置，使其尽可能接近的触须。 </li><li>参比电极插入眼睛，在较低的放大倍率（10倍），并切换到更高的放大倍率（100X），触须（图4F）的记录电极插入PEG感器。一旦电极感器，可以检测细胞的自发活动。 </li></ol> 8。代表性的成果根据感器和录音质量，可以区分不同数量的嗅觉神经元在一个单一的跟踪。 果蝇在大basiconic感器，例如，2至4尖峰幅度不同的细胞应该会出现在录制过程中，10 。 我们的视频实验中，果蝇AB2感器显示两个细胞，一个细胞（图5，蓝色的尖峰）和B细胞（图5，绿色尖峰） 。石蜡油（图5A）申请的过程中无论是细胞被激活，而只有在一个细胞响应10-6稀释醋酸甲酯（图5B ）。 在按蚊的上颌触须，沟槽PEG感器包含三个单元格，但只有两个很容易受到歧视（图5C，蓝色和绿色的峰值，分别）。在视频实验中，我们显示的B细胞如何响应一个10-7稀释1 -辛烯-3 -醇（图5D ）。 <img src="/files/ftp_upload/1725/1725fig1.jpg" alt="图1" /> 图1。电极卷笔刀 （二）（A）一般查看电极机设备。的注射器，注射器开幕电极尖端含有0.5 M KOH（左）用于增强电极（右）。（c）关闭。 <img src="/files/ftp_upload/1725/1725fig2.jpg" alt="图2" /> 图2。如何准备飞抽吸和蚊子吸气泵 （A）启动材料：。。空气线塑料管，两个切割枪头，和网状（二）（四）一旦完成飞抽吸（三）年底，是用来捕捉醋蝇的详细细节飞抽吸另一端（E）电动吸引蚊子收集由一个主体和一个可拆卸的塑料保持架（F）可拆卸的蚊子塑料保持架。 <img src="/files/ftp_upload/1725/1725fig3.jpg" alt="图3" /> 图3。准备录制一个飞醋（ 果蝇 ）和疟疾的蚊子（ 按蚊 ） （二）（一）飞行装醋的图片前，它定位在显微镜下幻灯片上。特写玻璃毛细管保持在天线的飞醋头（C）一种通过蚊子传播的图片上安装幻灯片（d）关闭蚊子头长鼻和磁带上的触须坚持。 <img src="/files/ftp_upload/1725/1725fig4.jpg" alt="图4" /> 图4。录制从飞醋（ 果蝇 ）和疟疾的蚊子（ 按蚊 ） （一）电设置（B）的飞行准备关闭安装在显微镜上。请注意各自的位置，记录电极（左），气味传送系统（中间移液器），和记录电极（右）（C）下10倍的目标动态图像（D）飞天线100X的目标;大basiconic感器（箭头），（五）非感官毛（箭头）之间穿插的10X查看记录安装一只蚊子（F）的蚊子的触须和PEG感器（箭头）高倍镜视野。 。 <img src="/files/ftp_upload/1725/1725fig5.jpg" alt="图5" /> 图5。从果蝇的录音和按蚊 （一） 果蝇的AB2感器房子两个感觉神经元;。一个单元格（蓝色尖峰）和B细胞（绿色尖峰）（二 ）10-6醋酸甲酯的应用过程中的A和B 细胞 （C） PEG 按蚊感器设有两个感觉神经元的一个单元格（蓝色尖峰）和B细胞（绿色尖峰）（四）10-7 1 -辛烯-3 -醇PEG感器的应用。

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用于生物识别的食物来源，潜在的队友，和食肉动物的嗅觉线索。嗅觉感觉神经元（OSNs）之间的外部刺激，并进一步处理信息的大脑中心的第一个接力中心。在果蝇和按蚊 ，OSNs方便，可以监视他们的电活动而刺激的气味喷。 单感器记录（SSR）技术的解释，该视频已被广泛应用于从OSNs记录和研究它们的电反应，大量的气味6， 7。嗅觉受体（ORS）6，11...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Paraffin oil</td>
<td>Odors</td>
<td>Fluka</td>
<td>76235</td>
<td>&nbsp;</td>
<tr>
<td>High purity odors (&gt;98%)</td>
<td>Odors</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>Methyl acetate 
#296996 
1-octen-3-ol 
#74950 </td>
</tr>
<tr>
<td>Filter paper strips</td>
<td>Odors</td>
<td>Fisherbrand</td>
<td>05-714-1</td>
<td>Chromatography paper</td>
</tr>
<tr>
<td>Connectors</td>
<td>Odors</td>
<td>Cole-Parmer</td>
<td>EW-06365-40</td>
<td>1/16x1/8"</td>
</tr>
<tr>
<td>Glass vials</td>
<td>Odors</td>
<td>Agilent Technologies</td>
<td>5182-0556</td>
<td>&nbsp;</td>
<tr>
<td>Air line plastic tubing</td>
<td>Odor Delivery</td>
<td>Python Products</td>
<td>500PAL</td>
<td>&nbsp;</td>
<tr>
<td>1 serological pipette</td>
<td>Odor Delivery</td>
<td>Corning</td>
<td>4101</td>
<td>10 mL</td>
</tr>
<tr>
<td>Plastic tubing</td>
<td>Odor Delivery</td>
<td>Cole-Parmer</td>
<td>EW-06418-0</td>
<td>0.050"x0.090"OD</td>
</tr>
<tr>
<td>Disposable borosilicate glass Pasteur pipettes</td>
<td>Odor Delivery</td>
<td>FisherBrand</td>
<td>13-678-20A</td>
<td>5-3/4 inches</td>
</tr>
<tr>
<td>Programmable stimulus controller</td>
<td>Odor Delivery</td>
<td>Syntech</td>
<td>CS-55</td>
<td>&nbsp;</td>
<tr>
<td>Anti-vibration table</td>
<td>Electrophysiology Equipment</td>
<td>TMC</td>
<td>63533</td>
<td>36&#x201D;Wx30&#x201D;Dx29&#x201D;H</td>
</tr>
<tr>
<td>Faraday cage</td>
<td>Electrophysiology Equipment</td>
<td>TMC</td>
<td>MI8133303</td>
<td>&nbsp;</td>
<tr>
<td>Inverted microscope</td>
<td>Electrophysiology Equipment</td>
<td>Nikon</td>
<td>E600FN ECLIPSE</td>
<td>Recording microscope</td>
</tr>
<tr>
<td>10x and 100x objectives</td>
<td>Electrophysiology Equipment</td>
<td>Nikon</td>
<td>10x Plan Fluor 100x L Plan</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>Electrophysiology Equipment</td>
<td>Nikon</td>
<td>EZ645</td>
<td>electrode sharpening/insect prep microscope</td>
</tr>
<tr>
<td>Magnetic stands</td>
<td>Electrophysiology Equipment</td>
<td>Newport</td>
<td>MODEL 150</td>
<td>&nbsp;</td>
<tr>
<td>IDAC</td>
<td>Electrophysiology Equipment</td>
<td>Syntech</td>
<td>IDAC-4</td>
<td>&nbsp;</td>
<tr>
<td>Acquisition software</td>
<td>Electrophysiology Equipment</td>
<td>Syntech</td>
<td>Autospike</td>
<td>&nbsp;</td>
<tr>
<td>1 macromanipulator</td>
<td>Electrophysiology Equipment</td>
<td>NARISHIGE</td>
<td>MN-151</td>
<td>Joystick manipulator 
Used for positioning reference electrode
</td>
</tr>
<tr>
<td>1 micromanipulator</td>
<td>Electrophysiology Equipment</td>
<td>EXFO</td>
<td>PCS-6000</td>
<td>Used for positioning recording electrode</td>
</tr>
<tr>
<td>Crocodile clip</td>
<td>Electrophysiology Equipment</td>
<td>Pomona</td>
<td>AL-B-12-0</td>
<td>&nbsp;</td>
<tr>
<td>Electric cable</td>
<td>Electrophysiology Equipment</td>
<td>Pomona</td>
<td>B-36-0</td>
<td>Test Cable Assembly</td>
</tr>
<tr>
<td>2 electrode holders</td>
<td>Electrophysiology Equipment</td>
<td>Syntech</td>
<td>N/A</td>
<td>Electrode holders (set of 2) for tungsten wire electrode</td>
</tr>
<tr>
<td>AC probe</td>
<td>Electrophysiology Equipment</td>
<td>Syntech</td>
<td>N/A</td>
<td>Universal single ended probe (10xAC)</td>
</tr>
<tr>
<td>Tungsten electrodes</td>
<td>Electrophysiology Equipment</td>
<td>Microprobes</td>
<td>M210</td>
<td>straight tungsten rods, 0.005&#x201C;x3&#x201C;</td>
</tr>
<tr>
<td>Potassium hydroxide</td>
<td>Electrophysiology Equipment</td>
<td>Sigma-Aldrich</td>
<td>221473</td>
<td>&nbsp;</td>
<tr>
<td>Syringe</td>
<td>Electrophysiology Equipment</td>
<td>BD</td>
<td>301625</td>
<td>20 mL</td>
</tr>
<tr>
<td>Power supply</td>
<td>Electrophysiology Equipment</td>
<td>WILD HEERBRUGG 6V 40W</td>
<td>e.g MTR32</td>
<td>&nbsp;</td>
<tr>
<td>Vertical puller</td>
<td>Insect prep</td>
<td>Narishige</td>
<td>PB-7</td>
<td>&nbsp;</td>
<tr>
<td>Razor blade</td>
<td>Insect prep</td>
<td>VWR</td>
<td>55411-050</td>
<td>&nbsp;</td>
<tr>
<td>Dental wax</td>
<td>Insect prep</td>
<td>Patterson</td>
<td>091-1503</td>
<td>&nbsp;</td>
<tr>
<td>Microscope slide</td>
<td>Insect prep</td>
<td>FisherBrand</td>
<td>12-550A</td>
<td>&nbsp;</td>
<tr>
<td>Cover glass</td>
<td>Insect prep</td>
<td>FisherBrand</td>
<td>12-541A</td>
<td>18X18 #1.5</td>
</tr>
<tr>
<td>Polypropylene mesh</td>
<td>Insect prep</td>
<td>Small Parts inc.</td>
<td>CMP-0500-B</td>
<td>&nbsp;</td>
<tr>
<td>Glass electrode</td>
<td>Insect prep</td>
<td>Frederick Haer & Co.</td>
<td>27-32-0-075</td>
<td>Capillary tubing borosilicate 1.5mm OD x 1.12mm ID x 75 mm</td>
</tr>
<tr>
<td>Double-sided tape (3M)</td>
<td>Insect prep</td>
<td>3M</td>
<td>MMM6652P3436</td>
<td>Double-sided tape (3M)</td>
</tr>
<tr>
<td>Forceps</td>
<td>Insect prep</td>
<td>Fine Science Tools</td>
<td>021x0053</td>
<td>Dumont #5 Mirror Finish Forceps</td>
</tr>
<tr>
<td>Small plastic cup</td>
<td>Insect prep</td>
<td>VWR</td>
<td>89009-662</td>
<td>7 x 5.7 (23/4 x 21/4)</td>
</tr>
<tr>
<td>Electric aspirator</td>
<td>Insect prep</td>
<td>Gempler&#x2019;s</td>
<td>RHM200</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

single sensillum recordings in the insects drosophila melanogaster and anopheles gambiae

昆虫的嗅觉寻找食物，配偶，天敌和产卵场所3，。昆虫的嗅觉感觉神经元（OSNs）被称为感受器的感觉毛，其中包括嗅觉器官表面封闭。每个感器的表面覆盖着微小的孔隙，通过气味传递和化解感器淋巴液的液体，沐浴安置在一个给定的感器OSNs的感官树突。通过OSN树突表达气味受体（OR）的蛋白质，它在昆虫气味门控离子通道4，5的功能。互动的ORs要么增加气味或减少通过OSN基础射速。这在动作电位形成的神经元活动体现的质量，强度和时间特征的气味6，7的第一个代表性。 鉴于方便地访问这些感觉毛，它可以执行从单一OSNs引入感器淋巴结记录电极，参比电极放置在昆虫的眼睛或身体的淋巴结外的录音。在果蝇 ，一至四个OSNs感器的房子，但每个OSN通常会显示一个特征的尖峰幅度。穗排序技术使人们有可能分配扣球个别OSNs的反应。这种单一感器记录（SSR）技术监控潜在OSNs 1，2，8受体活性产生的尖峰电脉冲感器淋巴液和参比电极之间的区别。在尖峰数量变化的气味代表的气味编码在昆虫细胞的基础。这里，我们描述的制备方法，目前在我们的实验室用来执行对果蝇和按蚊的SSR，并显示在感器特定方式的气味引起的代表痕迹。

Watch this Scientific Journal Video about Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae at JoVE.com

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

在使用单一感器录音昆虫气味的嗅觉感官神经元的电生理反应，可测。在这个视频文章，我们将演示如何执行单一飞醋的触角感器录音（。<em&gt;果蝇</em&gt;）和疟疾的蚊子的上颌触须（<em&gt;按蚊</em&gt;）。

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 ...

single-sensillum-recordings-insects-drosophila-melanogaster-anopheles

Research

JoVE Journal

Neuroscience

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

25.1K 次观看.  Rockefeller University. 在使用单一感器录音昆虫气味的嗅觉感官神经元的电生理反应，可测。在这个视频文章，我们将演示如何执行单一飞醋的触角感器录音（。果蝇）和疟疾的蚊子的上颌触须（按蚊）。

视频: Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

从巨纤维通路的电生理记录 D。果蝇</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

科研

神经科学

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 ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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 ...

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 ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

染色昆虫行为检测:麻醉的交配行为<em&gt;果蝇</em

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

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 ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

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 ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

毛细管馈线分析测量的摄食<em&gt;果蝇</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...