Name: using single sensillum recording to detect olfactory neuron responses of bed bugs to semiochemicals
Uploaded: 2016-01-18T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals at JoVE.com

The project was supported by Award AAES 461Hatch/Multistate Grants ALA08-045 and ALA015-1-10026 to N.L.

1.准备仪器，刺激方案和臭虫的<ol><li>制备在20ml的瓶子50％KNO 2溶液（重量/体积）。 </li><li>通过反复浸渍钨电极和流出溶液的锐化2钨微电极KNO 2溶液在5 V。 <ol><li>大致通过浸渍钨丝和移出KNO 2溶液的约10毫米，在2骤降/秒的速度约5分钟，从而可以大大消耗钨丝的前端锐化钨丝。 </li><li>微妙锐化电极浸渍在进出溶液的金属丝端头为约1毫米，在2骤降/秒的速度进行至少1分钟，以便使了微细且鲜明点电极。检查电极尖端直径在显微镜下频频，直至达到0.2-0.5微米，这应该是好到足以击穿的臭虫嗅觉感受器的角质层。 注意:当手动锐厄宁所述电极，所述钨丝送入KNO 2溶液的浸渍速度不是恒定的所有时间。随着越来越多的实践中，这是很容易保持一个相对恒定的速度在锐化电极。磨刀时间也是不确定取决于电极应该怎么罚金。这里，电极尖端〜0.2微米的直径足以刺穿嗅觉感受器。 </li></ol></li><li>每个从整齐化合物二甲亚砜（DMSO）中的化学刺激稀释至1:10体积/体积的初始浓度作为原液。创建一系列十进​​制稀释液根据多少剂量在实验用DMSO需要，再次，从每个原液每个化学。这里，使用10％的（+） - β蒎烯和桉叶油。 </li><li>将不喂食物，七天后饲养的成年臭虫（男性或女性）从英尺。在T使用迪克斯殖民地（从海恩斯在肯塔基大学博士赠送）他尝试在培养皿。 注意:对于放置在培养皿臭虫没有确切的数字。它可以是几个或很多。 </li></ol> 2.臭虫触角准备<ol><li>麻醉冰（2-3分钟）的臭虫。 </li><li>解决这两个天线和虫体在显微镜盖玻片用双面胶带，并用细剪刀去除腿部。 </li><li>用一个小针轻轻触碰天线，以便把它们粘在磁带上稳步推进。 </li><li>休息盖玻片针对牙科蜡的一个小球（〜1厘米直径）以方便操纵，并将其调整到一个适当的角度（〜90°）为记录电极（ 图1）。 </li><li>一旦固定，将臭虫立体显微镜下，打开冷光源，调整光照强度，直到天线清晰呈现，并聚焦显微镜上的臭虫天线在高放大倍率的第二鞭毛（720X） 。 注意:在实验中使用照明的强度是不定量，这真的取决于实验者的眼睛的感觉照度。 </li></ol> 3.单感器记录<ol><li>连接前置放大器（10X）与信号采集控制器，其与所述计算机进行信号记录和可视化连接。打开电脑，并启动该软件， 例如 ，AutoSpike32并单击"录制"模式从菜单栏中。然后选择"波"，以开始录制波信号。 注:平线从左侧重复运行到显示器的，现在应该是可见的。这里，记录窗口持续40秒。最大波记录为10秒。选择的采样速率是96000和数字采样率是240有0％偏移和无滤波，没有整流为记录信号。在该软件的所有这些参数的设置可以根据需要进行修改。 </li> &lt;利&gt;打开连接到前置放大器，其用于呈现调色模式从触角感器的神经元的反应的扬声器。 </li><li>将参比电极进入稳定臭虫的腹部。 注意:参考电极举行通过金属站在磁力附着到空气表。 </li><li>后参考电极被连接到臭虫的腹部，将记录电极，其连接到前置放大器和操纵由显微，朝向臭虫的天线的后端。 </li><li>当记录电极与天线的右顶端接触，开关在显微镜并定位在低倍率的电极。 </li><li>调整记录电极，同时逐渐增大放大倍数，直到两个电极和触角感器是在同一平面上，并在显微镜下清晰可见。 注意:这个时候，显微镜通常LY最高放大倍率。 </li><li>将记录电极到使用显微感器的轴，去得更深一些，如果比动作电位的背景噪声高。 </li><li>一旦清楚动作电位是从所记录的感器观察，填充微量吸有10％（+） - β蒎烯。使用微量沉积由10％（+），10微升等分试样β - 蒎烯到滤纸条（〜3×15毫米）放置在玻璃巴斯德吸管内。 <ol><li>加载的吸移管连接至刺激控制器的脉冲流管的出口与吸移管的尖端放入小孔面向天线管。 </li></ol></li><li>当所有这些连接已经稳定，抑制了刺激控制的脚踏开关来实现刺激（0.5升/分）的0.5秒粉扑进入连​​续加湿空气流。动作电位的记录将被同时启动时footswitch是郁闷。录制过程将持续10秒开始刺激前1秒。 </li><li>计数动作电位离线两个500毫秒期间，一前一刺激后。减去500毫秒后的刺激来自于前500毫秒记录的自发活动期间在穗率的任何变化，并通过它们乘以2转换成计数尖峰/ s的常规规模。 </li></ol> 4.更换刺激的SSR <ol><li>在吸移管β蒎烯到臭虫触角并记录该特定加臭剂的响应10秒钟，然后将吸管除去 - 一旦脚踏开关被触发，提供10％的（+）。 </li><li>标号另一个新的吸移管与0.001％桉叶油素进行测试。放置一小片滤纸到其上10微升的刺激得到了应用，到新的吸管。 </li><li>等待2-5分钟，直到刺激是完全vapori捷思在玻璃吸管。连接吸管到脉冲流管的出口。 </li><li>将移液管尖端到面向天线管的小孔。踩下脚踏开关，启动10秒的录音。 </li><li>断开吸管，并准备另一吸管0.01％桉。 </li><li>测试桉叶油素的剂量（0.001％至10％）的触角感受器的所有其余观察的剂量依赖性反应。从最测试稀释到最低稀释剂量。 </li></ol>

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The authors have nothing to disclose.

的单感器记录技术已被广泛使用在测试昆虫的神经反应，如果苍蝇，蚊子和臭虫到环境中的不同的化学刺激。这些化学刺激往往溶解并稀释在一个共同的溶剂中，以制备不同剂量的治疗。然而，不同的溶剂可以产生完全不同的释放速度的刺激。在一些广泛的研究昆虫，例如果蝇 ， 冈比亚按蚊 ， 致倦库蚊和伊蚊已经过去的研究 通常使用石蜡油作为溶剂以溶解的刺激，因为这些昆虫是相对不敏感的石蜡油17-20。石蜡油也被用于在臭虫较早单感器记录研究中，出于同样的原因14。然而，最常用的溶剂可能不是最好的为每个昆虫物种。在CA臭虫，既石蜡油和DMSO中，向其中臭虫还表现出不敏感本身已被用来溶解在不同的研究14,15的刺激，但同样的剂量刺激在DMSO中稀释的出现上引起更强的神经反应臭虫的感受器。例如，DMSO溶解的R - （+） - 柠檬烯和S - （ - ） - 苎烯≥70尖峰/从臭虫触角Dγ感器秒产生神经反应，而石蜡油溶解柠檬烯引起的只有神经反应≤25尖峰/从Dγ感器秒。这种减少在神经反应是在已稀释用石蜡油，可能是由于石蜡油用DMSO相比较慢的释放速率刺激相当普遍。这个较慢的释放速率降低刺激递送到感器的表面上的量，并可能导致关于昆虫某些化学信息素的敏感性误导结论。 两个关键STEPS用于进行单感器记录是:1）样品制备和2）信号记录。对于样品制备，由于臭虫具有很强的腿和积极移动天线，这是非常重要的是要除去所有的腿和紧紧贴天线到双面胶带。在信号记录过程中，有时，它在位置上不可能用于电极点进感器轴上。如果是这样的情况下，电极可刺穿感器，它总是给出了一个非常干净和清楚的信号用很少的背景噪声的后端。 由于有收容在D和C型感器多个神经元，因此，通常很难利用SSR期间所产生的动作电位的振幅和形状的单个神经元之间进行区分。然而，它仍然是可能的，看看在臭虫的反应的基础上的所有神经元在同一个S合并的发射频率不同的刺激的差异ensillum。理论上，臭虫与强刺激而不受其它刺激与弱刺激在相同剂量下对某些刺激敏感。因此，进一步的研究整合行为测试，他们对这些刺激的神经反应的信息将提供关于生态相关的化学信息素的臭虫有意义的信息。 在这项研究中，我们也采用了SSR技术测试嗅觉感受器的神经反应，以不同剂量的刺激。我们观察到在臭虫的神经反应的剂量依赖性模式不同的化学物质。但是，考虑到环境复杂臭虫住，臭虫在其正常的环境中所遇到的挥发物的实际剂量将是非常低的。其结果是，该引出在低剂量强神经反应向下至1:10 5体积/体积和1:10 4体积/体积更可能是生物学上有意义的臭虫比其它化学品吨的信息化学物质帽子的功能仅在高剂量。因此，那些在低剂量时充当化学信息素很可能在臭虫的化学感受发挥重要的作用，帮助他们找到一个主机或避免不良因素的影响，并因此在有希望的臭虫引诱剂或驱虫剂的使用提供了在筛选有用的指导实验室和现场试验。

常见的臭虫臭虫臭虫L（半翅目:Cimicidae），作为临时外寄生虫，是一种责任吸血昆虫，这意味着它们的生存，发育和繁殖需要从主机的血液来源，包括人类和动物1,2。虽然病毒的传播很少被报道，由于C.臭虫 ，由为患产生刺骨的滋扰，严重影响主机的生理和心理上3。引进和广泛使用化学杀虫剂，特别是滴滴涕，降低侵染的风险，由20世纪50年代的侵扰末端是在如此低的水平，他们不再是严重的公众关注。然而，许多可能的因素导致在床虱种群到回潮全世界，如杀虫剂的使用减少了，公众意识的下降，增加的行进活性，和耐药性的发展对杀虫剂4-9。&lt;/ P&gt; 化学线索的环境中检测到，并通过嗅觉器官，如天线和上颌触须认可昆虫。对昆虫触角的嗅觉感受器在检测这些化学信号发挥了至关重要的作用。化学分子穿过角质层表面气孔进入触角角质层。气味结合蛋白在触角淋巴液绑定到这些化学分子和他们运到气味受体10。臭气物质受体和它们的共受体从神经膜的非选择性阳离子离子通道，这将在去极化一旦这些化学分子被气味受体11识别。 单感器记录（SSR）的开发，以检测所引起的化学或非化学刺激的应用的动作电位的胞外变化。通过将记录电极浸入感器淋巴液和参考电极入昆虫身体（通常是复眼或腹部）的其他部分，所述神经元的响应的燃烧率，以刺激可记录12。变化尖峰的数目代表该昆虫对特定刺激的敏感性。不同的身份和浓度的化学刺激将引起不同的神经反应，用不同的燃烧率和时间的结构，并且因此可以用于研究昆虫到特定化学品的编码处理。 对于常见的臭虫，有性形式分享嗅觉感受器的相同模式的触角:九槽的PEGÇ感器，29发丝般E（E1和E2）感器，而每个Dα，Dβ，Dγ顺利盯住一对感器13,14。作为多个神经元已经于每种类型感器的已经确定，这是不容易区分从安置在同一感器不同的神经元的动作电位，所以对于这个实验中托塔动作电位升数字被用于前和刺激后500毫秒周期期间计数的脱线。然后，从刺激前动作电位的数目中减去，并以量化在每秒 15尖峰每个单独感器中的变化的燃烧率乘以二的刺激后动作电位的数目。

<table><tbody><tr><td>Tungsten wire</td><td>A-M SYSTEMS</td><td>#716500</td><td>Used for preparing the electrode</td></tr><tr><td>KNO2</td><td>Sigma</td><td>#310484</td><td>Used for sharpening the tungsten wire</td></tr><tr><td>AC Power Supply</td><td>BK Precision</td><td>1653A</td><td>Providing the voltage in sharpening the tungsten wire</td></tr><tr><td>Leica Z6 APO Microscope</td><td>Leica</td><td>10447424</td><td>Used for observing the sensilla on antennae</td></tr><tr><td>Simulus controller</td><td>Syntech</td><td>CS-55</td><td>Used for controlling the stimulus application</td></tr><tr><td>4-Channel USB Acquisition Controller</td><td>Syntech</td><td>IDAC-4</td><td>Real-time on screen display of all signals before and during recording</td></tr><tr><td>Light Source</td><td>SCHOTT</td><td>A20500</td><td>Providing light sources for observation</td></tr><tr><td>Micromanupulator</td><td>Leica</td><td>115378</td><td>Used for minor movement of electrode</td></tr><tr><td>Speaker</td><td>Juster</td><td>95a</td><td>Connected with Acquisition Controller IDAC-4 and providing sound for the signal</td></tr><tr><td>Magnetic stand</td><td>Narishige</td><td>GJ-1</td><td>Used to hold the reference electrode, stablized bed bug and stimulus delivery tube</td></tr><tr><td>TMC Vibration Isolation Table</td><td>TMC</td><td>63-500</td><td>Used for isolating the vibration from the equipments</td></tr><tr><td>Coverslip</td><td>Tedpella</td><td>2225-1</td><td>Used for holding the bed bug</td></tr><tr><td>Double-sided Tape</td><td>3M</td><td>XT6110</td><td>Used for stablizing the bed bug on the coverclip</td></tr><tr><td>Dental Wax</td><td>Dentakit</td><td>DK-R012</td><td>Used for supporting the coverclip where bed bug is stablized&amp;nbsp;</td></tr></tbody></table>

using single sensillum recording to detect olfactory neuron responses of bed bugs to semiochemicals

昆虫嗅觉系统在环境中的检测信息化学物质中起重要作用。特别是，触角感器其中内部容纳单个或多个神经元，被认为是使在响应化学刺激的重大贡献。暴露于刺激后可直接记录动作电位的嗅觉感器，单感器记录（SSR）技术提供了强大的方法来研究昆虫化学刺激的神经反应。为臭虫，其是臭名昭著的人类寄生虫，多种类型的嗅觉感受器的已经表征。在这项研究中，我们证明了臭虫嗅觉感受器的神经反应两个化学刺激和剂量依赖反应使用SSR方法其中之一。这种方法使研究人员能够进行早期筛查有关臭虫的嗅觉感受器个人化学刺激，这将提供有价值的信息，为devel软件包opment新的臭虫引诱或驱虫剂和效益的臭虫的控制力度。

单感器录音昆虫化学生态学和神经生理学的研究使用了强大的侦查手段。调查昆虫不同的挥发性化合物，特别是那些想到的神经反应是生态环境关系到昆虫的生存和发展，不仅为我们提供了宝贵的见解昆虫嗅觉过程中，同时也开启了充满希望的新途径，它可能会导致到有用的新试剂的发展，防治病虫害。 常见的臭虫，作为臭名昭著的城市害虫，无疑吸引了众多研究者的注意。在与臭虫的各个研究领域，它们的嗅觉机制是对臭虫化学生态学至关重要。以前的研究已经明确描述的数量和不同类型的床上嗅觉感受器的分布错误的天线。 如图2A所示，臭虫天线具有四个段（SC，PE，F1和F2）。多数嗅觉感受器的呈现在第二鞭毛（F2）的后端，但它们的分布是对于每种类型的明显不同:对D感器，即Dα，Dβ和Dγ，只有沿的内侧位于触角（图2C），而C和E（E1和E2）感器上找到天线（图2B）的两侧。因此，为了确保我们记录对D感器的神经反应，仔细定位天线是必不可少的。 由于臭虫份额有性形式的感受器类型相同的模式和他们的触角内侧包含了所有类​​型的感器，针对这方面的使它更容易分别记录各种不同类型的感受器的化学反应在天线上E（ 图3A）。在单感器录音，不同的嗅觉感受器表现出神经信号具有明显不同的动作电位种类和幅度（ 图3B）。例如，E感器已知具有内的一个或两个神经元，而D型产品感器房子比E或Ç感器更多的神经，产生更复杂的动作电位比其他作为结果。从C感器的神经反应的振幅比其他感器类型的小得多。 一旦电极连接已经建立，从各类型感器的神经反应，以每个刺激可以基于他们的身份和强度进行记录。对于一些刺激到臭虫极为敏感，神经反应能超越刺激的终止非常强，最后几秒钟。例如，响应于10％（+） - β蒎烯，臭虫显示一个巨大的射速（≥200尖峰/秒），相比单独溶剂作为刺激（ 图4A和B）控制超持续时间动态变化的强烈反应。不同的刺激可引发来自相同感器和不同浓度的相同的刺激的完全不同的神经反应可能产生相当不同的烧成频率。以剂量依赖性方式，如图5，增加桉叶油素的浓度升高，从30尖峰/秒击发频率在0.001％至240尖峰/秒，在10％以上。 <img alt="图1" src="/files/ftp_upload/53337/53337fig1.jpg" /> 图1示出的固定程序为臭虫的示意图。该臭虫稳定在与天线固定在磁带上的盖玻片。组装的样品，然后放置一磁阶段上。的样品c中的取向和高度一个被调整到臭虫天线和记录电极之间的合适的角度。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53337/53337fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53337/53337fig2.jpg" /> 图2.分配上的臭虫触角嗅感器。臭虫天线（A）扫描电极显微镜（SEM）图像。该天线具有四个段，所述Sculpus（SC），Pedecel（PE），所述第一鞭毛（F1）和所述第二鞭毛（F2）。大部分的嗅感器位于F2，尽管少数嗅感器也被发现在F1中，这被认为是相关的聚集信息素检测为臭虫16的它们的功能。 （二）F2的外侧的SEM图像，里面的C和E olfac保守党感器。 （ 三）F2的内侧，该产品被发现家中所有不同类型的嗅觉感受器的SEM图像:D（Dα，Dβ，Dγ），C和<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53337/53337fig2large.jpg" target="_blank">E，请点击此处查看该图的放大版本。 。</a> <img alt="图3" src="/files/ftp_upload/53337/53337fig3.jpg" /> 图 不同类型的臭虫触角嗅觉感受器的3. 典型的 神经信号，对臭虫的触角每种类型的嗅觉感受器的（A）高分辨率SEM照片。 （B）暴露于刺激前不同的嗅觉感受器的典型的神经信号。 Dα，Dβ，Dγ和C感器，其中容纳多个嗅感觉神经元（嗅觉神经元），表现出更复杂的动作电位比E1和E2感器，其中合作ntain只有一个或两个嗅觉神经元。从C感器动作电位的幅度较其他类型的感器要小得多。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53337/53337fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53337/53337fig4.jpg" /> 图4. 代表神经响应于刺激该臭虫敏感。（A）中的信号迹线示出了嗅觉感器（Dγ）与溶剂，这是作为在单感器记录控制的典型神经反应。该信号线被设定刺激的0.5秒粉扑前开工1秒。该信号线继续发起的刺激粉扑后记录为10秒。 （二）信号跟踪显示嗅觉的极强的神经反应感器（Dγ）向植物的刺激，10％（+） - β蒎烯。的（+），粉扑后β - 蒎烯递送至Dγ感器，嗅觉神经元容纳这感器内被激发高频和持久的时间动态。白条信号路径上述指示刺激曝光之前的1秒时间间隔，跟踪上述的红色条对应于递送的刺激​​粉扑到嗅觉感器，和黑条的跟踪上述指示终止后记录的信号刺激粉扑。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53337/53337fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53337/53337fig5.jpg" /> 嗅觉神经元的 图5。代表性的 剂量依赖性反应的刺激。使用其他植物的刺激，桉树作为EXAmple，所述Dγ感器显示的剂量依赖性响应于不同浓度的桉树。随着浓度从0.001％提高到10％，发射频率从30穗/秒上升到240尖峰/秒。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53337/53337fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals at JoVE.com

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

使用单一感器录音来检测臭虫的嗅觉神经元的反应，以信息化学物质

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

using-single-sensillum-recording-to-detect-olfactory-neuron-responses

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

9.8K 次观看.  Auburn University. Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

视频: Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

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

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

从蛾的嗅觉系统的电生理测量

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

Electrophysiology of Scorpion Peg Sensilla

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the mid-ventral surface of all scorpions are two appendages called pectines, which have dense fields of mechano- and chemosensory peg sensilla5,6. One method for assessing chemoresponsiveness of these sensilla uses a tungsten electrode for extracellularly recording neural activity within a sensillum as a volatile odorant is introduced to the sensory field5,7. The limitations of this method include slow data collection and uncontrolled stimulant introduction to, and removal from, the peg field. To overcome these limitations, we developed a new tip-recording technique that uses nonpolar mineral oil as a medium through which to deliver water-based tastants to individual peg sensilla8,9. We have successfully applied this method to obtain sensillar chemoresponses to citric acid, ethanol, and salt. Here we describe the experimental protocol for such a study9. We think this new method may be useful for studying the response properties of other arthropod chemosensory systems, including those of insects10, 11 and crustaceans12.

This article describes an electrophysiological method for isolating chemical stimulation to individual sensilla via extracellular, tip-recordings under mineral oil.

毒蝎钉感器电

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

从使用吸吸液管技术的嗅觉受体神经元的气味引起的反应记录

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

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 ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

应用一种适应微流控嗅觉芯片的神经活性成像对雄性线虫神经元的信息素反应

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

电生理记录<em&gt;果蝇</em&gt; Trichoid Sensilla响应低挥发性气味

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially for the detection of non-volatile chemical cues through sensilla chaetica (previously named terminal sensilla or crested sensilla). There is now increasing evidence that these palps also have an olfactory function. An odorant receptor (LmigOR2) and an odorant-binding protein (LmigOBP1) have been localized in the neurons and accessory cells, respectively, in the sensilla basiconica of the palps. Single sensillum recording (SSR) is used for recording the responses of odorant receptor neurons, which is an effective method for screening active ligands on specific odorant receptors. SSR is used in functional studies of odorant receptors in palp sensilla. The structure of the sensilla basiconica located on the dome of the palps differs somewhat from the structure of those on the antennae. Therefore, when performing an SSR elicited by odorants, some specific advice may be helpful for obtaining optimum results. In this paper, a detailed and highly effective protocol for an SSR from insect palp sensilla basiconica is introduced.

This paper describes a detailed and highly effective protocol for single sensillum recordings from the sensilla basiconica on the palps of insect mouthparts.

蝗虫下颚触角 Basiconica 单触角记录

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially ...

一种增强昆虫味觉神经元活性分析的新方法

Base Recording: A Technique for Analyzing Responses of Taste Neurons in Drosophila

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential food source, compounds from the food source enter through the pore and activate neurons within. For over 50 years, these responses have been recorded using a technique called tip recording. However, this method has major limitations, including the inability to measure neural activity before or after stimulus contact and the requirement for tastants to be soluble in aqueous solutions. We describe here a technique that we call base recording, which overcomes these limitations. Base recording allows the measurement of taste neuron activity before, during, and after the stimulus. Thus, it allows extensive analysis of OFF responses that occur after a taste stimulus. It can be used to study hydrophobic compounds such as long-chain pheromones that have very low solubility in water. In summary, base recording offers the advantages of single-sensillum electrophysiology as a means of measuring neuronal activity - high spatial and temporal resolution, without the need for genetic tools - and overcomes key limitations of the traditional tip recording technique.

作者聚焦：化学接收的进步 – 从昆虫气味受体到非编码 RNA

A rarely used method of electrophysiological recording, base recording, allows analysis of features of taste coding that cannot be examined by conventional recording methods. Base recording also allows the analysis of taste responses to hydrophobic stimuli that cannot be studied using traditional electrophysiological methods.

基础记录：一种分析果蝇味觉神经元反应的技术

Insects taste the external world through taste hairs, or sensilla, that have pores at their tips. When a sensillum comes into contact with a potential ...

使用单一感器录音来检测臭虫的嗅觉神经元的反应，以信息化学物质

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