这项工作得到了中国国家自然科学基金 (31472037 号) 的资助。在本条中提及商号或商业产品, 纯粹是为了提供具体资料, 并不意味着建议。

1. 仪器和昆虫的制备
<ol><li>
制备钨电极和刺激溶液
	<ol>
<li>固定一个新的钨线 (直径为0.125 毫米, 长度为75毫米) 成一个机器人, 并锐化它在 10% (瓦特/v) 亚硝酸钠 (纳米2) 溶液中的一个注射器在 10 v 在一个显微镜 (40X 放大倍数) 的电源提供约1分钟。</li>
		<li>将锐钨丝反复浸入10% 纳米2溶液中, 约4毫米, 5 V < 1 分钟 (图 1A)。</li>
		<li>在显微镜下经常检查削尖钨尖的直径, 直到它足够细, 才能穿透蝗虫下颚嗅触角的角质层 (图 1B)。</li>
		<li>准备刺激方案。稀释矿物油中的化学刺激物质。稀释 1-壬醇和壬酸在10% 稀释。稀释 E-2-hexenal 和己醛在 10-2, 10-3, 10-4和 10-5。</li>
		<li>准备带刺激的巴斯德管: 将滤纸条 (长度2厘米, 宽度0.5 厘米) 插入巴斯德管, 将稀释后的刺激方案 (每10µl) 添加到滤纸条上, 然后用吸管尖 (1 毫升) 塞巴斯德管。</li>
	</ol>
</li>
	<li>
准备昆虫
	<ol>
<li>后蝗虫 (飞蝗) 与新鲜小麦幼苗在拥挤的条件下, 在相对湿度 60%, 温度为 28-30 摄氏度, 和光周期 18:6 h (光线: 暗)。选择1至3天龄的蝗虫若虫,用细剪刀取下天线, 以避免记录时出现干扰。</li>
	</ol>
</li>
	<li>
蝗虫上颌下颚支架的研制
	<ol>
<li>使用玻璃滑块 (25 毫米 x 75 毫米) 作为上颌下颚支架 (英里) 的基础。将塑料件 (高度为1毫米, 宽度为10毫米, 长度35毫米) 到玻璃滑轨的一角, 用双面胶带, 最后用双面胶带固定在塑料件顶部的盖玻璃 (18 毫米 x 18 毫米)。将一小块红色橡胶胶带放到盖玻璃上作为防滑层。塑料件和盖玻璃构成蝗虫下颚的平台。平台的高度大约是1.5 毫米。</li>
		<li>安装一根钨线 (直径为0.125 毫米, 长度为36毫米), 其距离与平台内边缘平行1.5 毫米。用双面胶带将导线两端固定在平台上。</li>
	</ol>
</li>
</ol>2. 蝗虫上颌瓣的制备
<ol><li>将离心管 (1.5 毫升) 垂直切成两半, 并切断底部。把蝗虫放进准备好的管子里。离开腹侧区域和蝗虫头暴露。用双面胶带将组件固定在玻璃滑轨上 (图 2A)。</li>
	<li>把右上颌下颚到平台上。</li>
	<li>把钨线放在下颚的第四段。将胶粘剂腻子放在钨线的两侧, 大约2毫米从上颌下颚 (图 2A和2B)。</li>
</ol>3. 单触角录音
<ol><li>在显微镜下用低倍率 (100X) 将蝗虫上颌下颚准备。调整准备位置, 直到下颚垂直于记录电极 (图 3A)。</li>
	<li>使用机器人将参考电极 (钨电极) 插入蝗虫眼中。将记录电极 (钨电极) 移动到与上颌下颚接近的机器人 (图 3B和3C)。</li>
	<li>将气味传递装置调整至上颌下颚约1厘米 (图 3B)。</li>
	<li>打开录音软件自动钉32。设置记录参数, 如下所示: 500 µV 的记录刻度;过滤器的高截止度在300赫兹, 低截止在200赫兹;和 pretrigger 在十年代。</li>
	<li>将录音电极连接至10x 通用交流/直流放大器。</li>
	<li>将显微镜切换到高放大倍数 (500X)。将录音电极插入上颌下颚上的 basiconic 触角的底部, 并微妙地调整记录电极以获得良好的自发峰值 (图 3D)。</li>
	<li>打开刺激控制器, 以提供一个连续的气流在20毫升/秒. 将刺激时间设置为1秒, 在刺激脉冲启动前十年代开始, 记录信号为十年代。</li>
	<li>使用10x 通用交流/直流放大器来放大信号。把信号送进 IDAC 4。用自动道钉32软件分析信号。交流信号是带通滤波在200到300赫兹之间. 使用自动尖峰32区分峰值与槽幅的噪声。计算神经元的响应, 作为动作电位频率 (峰值每秒) 在自发频率上的增加。使用 GraphPad 棱镜7进行统计分析。</li>
</ol>

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作者没有什么可透露的。

昆虫依靠瓣来检测食物的气味, 他们的瓣被认为在物种13,27中扮演着重要的角色。瓣是一种简单的嗅觉器官, 越来越受到人们的重视, 作为探索 chemosensation28neuromolecular 网络的一个吸引人的模式。
昆虫 labellar 和下颚 SSRs 已成功地在果蝇、按蚊按蚊和淡色致倦13,14,15,16,17 , 但很少被报告的形式的视频演示16,29。相比之下, 触角 SSRs 的视频数据可用于果蝇、脐 orangeworm 蛾 (Amyeloistransitella)、蝗虫美洲和床虫 (Cimex 臭虫)16,30,31,32,33。
蝗虫下颚触角 basiconica 有一个特殊的结构, 不同于蝗虫触角触角和许多其他昆虫触角。使用此处描述的方法, 可以记录和区分蝗虫下颚触角 basiconica 子类型 pb1 和 pb2 产生的动作电位 (图 4和图 5)。
关键步骤是记录电极的穿透。记录电极应插入触角和先进的基础, 直到获得良好的信号。此外, 在将记录电极插入触角的底部时, 防止下颚的圆顶崩溃是很重要的。为了达到这一目的, 我们建立了一个平台, 包括一个特殊的蝗虫上颌下颚持有人 (英里), 并使用钨线压缩第四段的下颚。这一程序的许多重复表明, 这是有效的。根据触角神经元在几个气味中的反应模式, 首次在蝗虫上颌下颚上发现了触角 basiconica 的两种亚型, 即 pb1 和 pb2。
该出版物中概述的技术的局限性是, 它可以用来记录大昆虫 (如蛾、甲虫和蝗虫), 而不是记录小昆虫 (如苍蝇和蚊子), 它们有自己的平台和技术13,14,15,16,17。这种技术与现有方法相辅相成。
最后, 详细介绍了一种从昆虫下颚触角 basiconica 的 SSR 的高效协议。该协议可为研究人员在 mouthpart 昆虫嗅觉的分子和细胞机制方面提供有用的技术。该方法与气相色谱法相结合, 可用于确定有利食品资源提取物中天然 electrophysiologically 活性配体。

动物已经进化出一系列的化学感受器官来感知外源化学线索。在昆虫中, 最重要的化学感受器官是触角和瓣。在这些器官, 几种类型的化学感受毛, 称为化学感受触角, 是支配的化学感受神经元 (国别战略说明) 在头发。国别战略说明在化学感受触角识别特定的化学线索, 通过信号转导从化学刺激到电位, 随后转移到中枢神经系统1,2,3.
国别战略说明表达各种化学感受受体 [例如, 气味受体 (口服)], 离子型受体 (IRs) 和味觉受体 (GRs) 在他们的细胞膜上, 编码外源化学线索相关的不同类型的 chemosensation4,5,6。国别战略说明的特性是阐明昆虫 chemoreception 细胞和分子机制的关键。现在单触角记录 (SSR) 是一种广泛使用的技术, 用于国别战略说明昆虫的触角触角的特征, 包括蝇7、蛾8、甲虫9、蚜虫10、蝗虫11和蚂蚁12。然而, 很少有研究将 SSR 应用于昆虫瓣13、14、15、16、17, 因为它们的触角的特殊结构使电生理记录困难18。
成群的蝗虫 (直翅目) 经常造成严重的农作物损害和经济损失19。瓣被认为在蝗虫20、21、22、23、24的食物选择中起着重要作用。采用扫描电镜 (SEM) 对两种化学感受触角进行了研究。通常, 350 触角 chaetica 和 7-8 触角 basiconica 在蝗虫的每个圆顶瓣18被观察。触角 chaetica 是味觉触角, 感觉非挥发性化学线索, 而触角 basiconica 有嗅觉功能, 感知挥发性化学线索。
在蝗虫瓣, 触角 basiconica (约12µm) 的毛插座直径比触角 chaetica (约8 µm)18,25大得多。瓣上触角 basiconica 的表皮壁比触角触角18厚得多。另外, 下颚的圆顶在高度灵活的角质层之内有液体内容。这些特征意味着, 微电极的穿透和良好的电生理信号的获得比触角触角更困难。本文提出了一种用于蝗虫下颚触角 basiconica 的详细、高效的 SSR 协议。

<table border="0" cellpadding="0" cellspacing="0" style="width:1145px;" width="1145">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">Tungsten wire</td>
			<td style="width:140px;">ADVENT</td>
			<td style="width:217px;">W559504</td>
			<td style="width:485px;">Used for making the electrode and fixing the palp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NaNO2</td>
			<td>Sigma-aldrich</td>
			<td>563218-25G</td>
			<td>Used for sharpening the tungsten wire</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AC Power Supply</td>
			<td>Syntech</td>
			<td>A2-70</td>
			<td>Providing the voltage in sharpening the tungsten wire</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Stereoscope</td>
			<td>Motic</td>
			<td>SMZ-163</td>
			<td>Used for observing the sharpening of tungsten wire</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope</td>
			<td>Olympus</td>
			<td>W-51</td>
			<td>Used for observing the sensilla on locust maxillary palp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Intelligent Data 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 height="40">
			<td height="40" style="height:40px;">Stimulus controller</td>
			<td>Syntech</td>
			<td>CS-55</td>
			<td>Used for controlling the stimulus application</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electronic micromanipulator</td>
			<td>C.M.D.T</td>
			<td>CFT-8301D</td>
			<td>Used for minor movement of the recording electrode</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Micromanipulator</td>
			<td>Narishige</td>
			<td>MN-151</td>
			<td>Used for minor movement of the reference electrode</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Speaker</td>
			<td>EDIFIER</td>
			<td>R101T06</td>
			<td>Connected with IDAC-4 and providing sound for the signal</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Magnetic base</td>
			<td>PDOK</td>
			<td>PD-101</td>
			<td>Used to hold the electrode, and stimulus delivery tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vibration Isolation Table</td>
			<td>TianHe</td>
			<td>HAP-100-1208</td>
			<td>Used for isolating the vibration from the equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass slide</td>
			<td>CITOGLAS</td>
			<td>ZBP-407</td>
			<td>Used for making the base for the MPH</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blu-tack</td>
			<td>Bostik</td>
			<td>Blu-tack-45g</td>
			<td>Fixing the tungsten wire</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Pasteur tube</td>
			<td>YARE</td>
			<td>WITEG</td>
			<td>Placing the filter paper containing stimuli solutions</td>
		</tr>
	</tbody>
</table>

single sensillum recordings for locust palp sensilla basiconica

蝗虫口器的瓣被认为是传统的味觉器官, 在蝗虫的食物选择中起着重要作用, 特别是通过触角 chaetica 检测非挥发性化学线索 (以前命名的终端触角或冠触角)。现在越来越多的证据表明, 这些瓣也有嗅觉功能。气味受体 (LmigOR2) 和气味结合蛋白 (LmigOBP1) 已分别在神经元和附属细胞, 在触角 basiconica 的瓣。单触角记录 (SSR) 用于记录气味受体神经元的反应, 是筛选特异气味受体活性配体的有效方法。SSR 用于下颚触角气味受体的功能研究。位于瓣圆顶上的触角 basiconica 的结构与天线上的结构有一定的不同。因此, 当执行气味引起的 SSR 时, 一些特定的建议可能有助于获得最佳结果。本文介绍了一种针对昆虫下颚触角 basiconica 的 SSR 的详细、高效的协议。

基于不同反应动力学的化学气味 (10% 1-壬醇和10% 壬酸), 确定了蝗虫上颌下颚的两个触角亚型 (pb1 和 pb2)。pb1 神经元的峰值比壬酸大 1-壬醇, 而 pb2 的神经元比壬醇酸显著降低1壬的活化 (图 4)。己醛和 E-2-Hexenal 能诱发蝗虫下颚26的开放反应。己醛是一个丰富的寄主植物绿叶挥发性, 这可能有助于进一步确认食品来源26。在 pb1 神经元中的峰值在 E-2-hexenal 刺激的时候比 pb2 的长, 而它们的持续时间也会延长 (图 4)。pb1 和 pb2 中的神经元对己醛的反应也同样强劲 (图 4)。在刺激后的 5 s 期间和5秒之间所有峰值的平均变化比较表明, 对 1-壬醇的反应明显高于 pb1 壬酸, 但在 pb2 (图 5) 中相反。触角的两种亚型神经元对 E-2-hexenal 和己醛反应剂量依赖性, 它们对这两种醛的反应模式不同 (图 6A和6B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57863/57863fig1.jpg"> 图1。电极制备.(A) 本面板显示电极锐化装置的一般观点。含有10% 纳米2 (左) 的注射器用于锐化电极 (右)。(b) 本小组对电极提示 (a: 适用; b: 不合适) 进行仔细查看。<a href="/files/ftp_upload/57863/57863fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57863/57863fig2.jpg"> 图2。蝗虫上颌下颚保持架 (米/秒).(a) 在显微镜下将每小时和一只蝗虫安装在玻璃滑梯上。(B) 这个小组显示了在平台上用钨线固定的蝗虫上颌下颚的特写。<a href="/files/ftp_upload/57863/57863fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57863/57863fig3.jpg"> 图3。单触角录音.(A) 本小组展示了电生理学设置的观点。(B) 本小组对在显微镜下安装的蝗虫制剂有密切的看法。(C) 该图像显示蝗虫上颌下颚的放大率为100X。(D) 此图像显示下颚的放大倍数为500X。箭头指示 basiconic 触角。<a href="/files/ftp_upload/57863/57863fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57863/57863fig4.jpg"> 图4。蝗虫上颌下颚单触角记录的反应痕迹.在这个小组中, pb1 代表下颚触角 basiconica 的亚型 1;pb2 代表下颚触角 basiconica 的亚型2。痕迹上方的条形指示刺激持续时间 (1 秒)。对于这些录音, 所有气味在10% 稀释使用除了 E-2-hexenal 和己醛, 稀释到1%。这个数字已经从张等上改了。26.<a href="/files/ftp_upload/57863/57863fig4large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57863/57863fig5v2.jpg"> 图5。壬酸和 1-壬醇刺激的 pb1 和 pb2 神经元中棘突的平均数比较.在刺激前后5秒内, 计算出峰值的平均数。在 pb1 中, 反应于 1-壬醇的神经元中峰值的平均值明显高于响应壬酸的神经元峰值 (n = 11 瓣;方差分析与后验 t 检验;p < 0.0001), 与 pb2 (n = 10 瓣;方差分析与后验 t 检验;p = 0.0110)。误差线代表 SEM。这个数字已经从张等上改了。26.<a href="/files/ftp_upload/57863/57863fig5v2large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57863/57863fig6v3.jpg"> 图6。pb1 和 pb2 的神经元形态与 E-2-hexenal 和己醛反应剂量依赖性.(A) 本小组显示 pb1 神经元的模式 (SEM;n = 12 瓣)。(B) 本小组显示 pb2 神经元的模式 (SEM;n = 10 瓣)。这个数字已经从张等上改了。26.<a href="/files/ftp_upload/57863/57863fig6large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>

Watch this Scientific Journal Video about Single Sensillum Recordings for Locust Palp Sensilla Basiconica at JoVE.com

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

本文介绍了触角 basiconica 瓣昆虫口器的单触角记录的一种详细、高效的协议。

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 ...

single-sensillum-recordings-for-locust-palp-sensilla-basiconica

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.1K 次观看.  China Agricultural University. 本文介绍了触角 basiconica 瓣昆虫口器的单触角记录的一种详细、高效的协议。

视频: Single Sensillum Recordings for Locust Palp Sensilla Basiconica

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

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

单机在体内</em从大鼠视交叉>录音

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

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 ...

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

单果蝇 Ommatidium解剖和影像

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

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 ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

一个程序植入微丝的有组织阵列的单机录音清醒，举止动物

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

电生理记录从<em&gt;果蝇</em&gt; Labellar味感器

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

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 ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

通过RNA定位蝗虫天线中的气味受体基因<em&gt;原位</em&gt;杂交

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...