我们感谢宜君宋，博士和Hattar Kuruvilla赵三实验室生物系，美国约翰霍普金斯大学的建议和帮助的成员。由美国国立卫生研究院拨款DC007395和DC009946支持。

第1部分。眼电图记录设置录音装置由记录电极，参比电极，空气输送管，标本阶段，和解剖显微镜，所有挂靠在一个法拉第笼的空气表。 Micromanipulators用于放置电极和空气输送管。通过持续的空气流冒泡蒸馏水，增加湿度，然后通过空气输送管，并在标本的合格。一个60毫米的培养皿填充到6-8毫米的深度与Sylgard是用来作为试样的安装面。一个良好，一个通道被掏空，在安装盘的Sylgard提供一种手段，参比电极的电气连接通过改良林格液标本。 记录电极和参比电极连接到放大器。从放大器的信号被发送到数字转换器，然后到计算机上。如Axograph或pClamp软件，可以用来控制协议的刺激，记录信号，并为随后的反应分析。示波器连接后放大器可方便的同时放置记录电极的电势的实时监控，并在EOG录音。 气味刺激的交付是由Picospritzer，这是连接到同一台计算机用于信号采集控制。 Picospritzer空气压力设置为10磅。可以使用一个单一的空气罐和稳压器，送风空气表和Picospritzer。第二个空气罐和稳压器是用来提供空气加湿空气流，因为这需要一个较低的压力和大量的气流。前提供气味刺激，Picospritzer输出连接到气味瓶。气味瓶，然后连接到空气输送管。 第2部分：准备电极记录电极是在拉玻璃毛细管与改良林格液（135毫米氯化钠，氯化钾5毫米，1毫米氯化钙 ，氯化镁2 1.5毫米，10毫米的HEPES，pH值7.4，过滤消毒）充满chlorided银线。参比电极是chlorided银线。 <ol><li>电极支架安装到银线。为记录电极，一到两厘米的导线应突出从电极支架年底。可以留下更多的线为参比电极。 </li><li>要添加氯化银大衣的银线，位置在0.1 M氯化钠的导线和电极支架连接到了1.5-9 V直流电源的正极。电气连接电源负端，应以0.1 M的NaCl溶液。允许chloriding反应进行10分钟。要平衡的任何记录和参比电极之间的静电荷，简要地谈谈前电极上安装录音装置。 </li><li>使用微量拉马拉玻璃毛细管。在毛细管尖端的开放应该是直径在5-10微米左右。 </li><li>使用钻石铅笔得分和折断的钝端的毛细血管，所以它是比银线长1〜2厘米。消防波兰丁烷火炬砍完。 </li><li>熔体改良林格液0.5％琼脂糖。使用移液管的电极尖端拉进少量的熔化的琼脂糖溶液。 </li><li>填写拉毛细管约1 / 2的修改林格氏液（已加热和拉有一个很长的细结束，为此非常有用的注射器）方式。轻轻一抖毛细管驱逐任何气泡。商店，直到他们准备用来填充少量的改良林格在底部的解决方案的存储JAR电极。一旦组织样本准备和录制，安装一个以上的记录电极丝的填充毛细管。 </li></ol>第3部分：准备气味的解决方案气味戊酯和庚醛唤起大的反应，从而很好的选择EOG兴奋剂。 <ol><li>在离心管中，准备了一系列的气味稀释在二甲基亚砜（DMSO）。作为一个剂量反应曲线的起点，准备10倍稀释，从5米至5 × 10 -6 M。让新鲜稀释的每一天。 </li><li>进一步淡化水的气味混合4.9 MLS 2盎司瓶的水用硅胶塞在DMSO 100μL稀释股票的50倍。让我们的解决方案，在瓶至少30分钟的平衡。请注意，每个瓶子的气味的蒸气浓度是未知的，但在液相中的浓度的气味功能会有所不同。 </li><li>两个18表针通过硅塞插入到Provide输入和输出端口。端口应插瓶是在不使用时。 </li></ol>第4部分：记录眼电图和分析数据<ol><li>牺牲CO 2安乐死麻醉过量或斩首鼠标。取下的皮肤覆盖的头骨和弧矢头，沿中线平分。 </li><li>一个半头，内侧安装，安装盘上。小心地取出隔揭露鼻甲。 </li><li>将安装到录音阶段组织的菜。对齐舞台上鼻甲的录音位置，以便在显微镜下为中心。 </li><li>转到提供鼻甲表面加湿空气的空气罐上。位置的空气输送管，以便它是从录音的位置相距约10毫米。流速〜600毫升/分钟。 </li><li>在1 kHz的低通滤波器，增益100X设置放大器DC模式（交流放大，就会诱发EOG信号的文物）。 </li><li>安装记录和参比电极上micromanipulators。 </li><li>下入井安装盘和参比电极覆盖与修改林格氏液，这是电气连接到组织。 </li><li>上鼻甲IIB或III表面，小心地记录电极。电极应刚好触及的嗅觉上皮细胞的表面！当电极与上皮细胞接触（即完成一个电路）直线基线会显示在示波器。 </li><li>将气味瓶空气输送管的侧端口。 </li><li>在计算机上，启动一个刺激协议。数据采集​​的采样率应该是2 kHz或更高。该软件就会触发气味脉冲，并开始录制。 </li><li>一个典型的刺激协议可能是一个100毫秒的时间内单脉冲，配对100毫秒脉冲1秒的间隔隔开，或10秒的持续脉冲。 </li><li>允许这样的组织是微创适应之间的一些协议的时间。一分钟是足够的液体浓度的醋酸戊酯和庚醛10 -3 M;，允许在较高浓度时5分钟。提供高的气味浓度（如在一个剂量反应曲线结束）后残留的气味可能会留在管。用95％乙醇清洗风管和干燥，然后才能继续与其他组织样本。 </li><li> Axograph软件提供了用于测量EOG信号的关键参数的工具。这些参数包括响应的幅度，延迟，峰值时间，终止时间常数。它可能是可取的数字滤波器在25赫兹的痕迹，然后再进一步分析。 </li></ol>代表性的成果<img src="/files/ftp_upload/1850/1850fig1.jpg" alt="图1" /> 图1。眼电图分析参数的眼电图的几个参数比较小鼠之间的反应，包括响应振幅，潜伏期（当刺激是管理和响应开始之间的时间），上升时间（开始之间的时间特别有用响应和峰值），峰值时间（从刺激开始到高峰期的响应时间），和终止的时间常数（τ，由一个单一的指数方程拟合响应衰减阶段）。动力学参数，如延迟，上升时间和终止时间常数的比较，最好是分析前正常化的峰值振幅的响应。 <img src="/files/ftp_upload/1850/1850fig2.jpg" alt="图2" /> 图2。代表EOG不同的刺激下协议信号。戊酯浓度的增加（一）从鼠标EOGs的响应刺激的例子。在面板顶部的黑线表示的气味刺激的时机和持续时间。在传说中的浓度溶液的浓度。 （二）从5只，平均剂量反应关系。误差棒是95％置信区间。峰值幅度的下降，往往是观察到的气味浓度非常高。 （三）在配对脉冲刺激EOG的一个例子。一个单一的短脉冲的气味会引起持续几秒钟的适应。 （四）在10秒EOG的一个例子持续气味的刺激。眼电图显示在连续的气味介绍脱敏。

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小鼠进行处理和安乐死的动物护理和使用委员会，美国约翰霍普金斯大学认可的方法。

在本议定书中所描述的设置，在嗅觉上皮细胞表面的气味刺激之间的组织筹备工作保持一致，与野生型和突变型小鼠相比，尽管确切的气味浓度和动态是未知的。几个因素，特别是录音的位置和加湿空气的流速，导致的眼电图的变化。应注意记录从相同鼻甲相近的立场，以尽量减少变化。这可以很容易地实现始终如一地记录从同一侧的头部和保持显微镜的足迹，气味输送管，空气表之间的组织样本不变micromanipulators。此外，组织样本应立即放入加湿空气流后，剥离，以防止过度干燥的组织。 对小鼠的眼电图的录音也可以进行准备鼠标上的液体灌注仪器鼻甲7，14，15，或离开头部完好，鼻甲16，17以上的钻了一个小洞，插入电极。每个眼电图记录的变化有其自身的优势：空气相记录在本议定书中所述的组织筹备工作需要设置少量的，是最容易进行的录音使用的液体灌注设备方便的药理试剂的使用，虽然许多气味的疏水性气味传递的复杂性，最后，在这头是原封不动的录音，可以使用“人工嗅”实验，虽然电极放置较充分暴露时鼻甲是很难。

<table><tbody><tr><td>Air delivery tube</td><td>equipment</td><td>Custom Made</td><td></td><td>The barrel of a 1-mL syringe with a T-fitting can be used as a substitute</td></tr><tr><td>Air table</td><td>equipment</td><td>Newport Corp.</td><td>LW3030B-OPT</td><td></td></tr><tr><td>Amplifier</td><td>equipment</td><td>Warner Instruments</td><td>DP-301</td><td></td></tr><tr><td>Computer and Data Acquisition Software</td><td>equipment</td><td>Axograph 4.9.2 on Apple Macintosh</td><td></td><td>Updated versions of Axograph for Mac OS X and Windows are available from http://axographx.com/.</td></tr><tr><td>Butane torch</td><td>equipment</td><td></td><td></td><td>A cr&amp;#232;me br&amp;#251;l&amp;#232;e torch works well</td></tr><tr><td>Digitizer</td><td>equipment</td><td>Axon Instruments</td><td>Digidata 1322A</td><td></td></tr><tr><td>Dissecting Scope</td><td>equipment</td><td>Scienscope</td><td>SSZ</td><td></td></tr><tr><td>Electrode holder</td><td>equipment</td><td>Harvard Apparatus</td><td>64-1021</td><td></td></tr><tr><td>Magnetic Holding Devices (12 mm)</td><td>equipment</td><td>World Precision Instruments, Inc.</td><td>M10</td><td></td></tr><tr><td>Micromanipulators</td><td>equipment</td><td>World Precision Instruments, Inc.</td><td>M3301R M3301L</td><td></td></tr><tr><td>Micropipette Puller</td><td>equipment</td><td>Sutter Instrument Co.</td><td>P2000</td><td></td></tr><tr><td>Oscilloscope</td><td>equipment</td><td>Tektronix, Inc.</td><td>5110</td><td></td></tr><tr><td>Picospritzer III</td><td>equipment</td><td>Parker Hannifin Corporation</td><td></td><td></td></tr><tr><td>Silicone tubing</td><td>equipment</td><td>Nalge Nunc international</td><td></td><td></td></tr><tr><td>Specimen stage</td><td>equipment</td><td>Custom Made</td><td></td><td>Any small solid object can be used to elevate the mounting dish. Immobilize the dish with modeling clay.</td></tr><tr><td>18 gage needles</td><td>material</td><td>BD Biosciences</td><td>305195</td><td></td></tr><tr><td>2 oz. glass bottles</td><td>material</td><td>VWR international</td><td>16152-201</td><td></td></tr><tr><td>Glass capillaries</td><td>material</td><td>World Precision Instruments, Inc.</td><td>TW150F-6</td><td></td></tr><tr><td>Silicone stoppers size 16D</td><td>material</td><td>Chemware</td><td>D1069809</td><td></td></tr><tr><td>Silver wire</td><td>material</td><td>World Precision Instruments, Inc.</td><td>AGW1010</td><td></td></tr><tr><td>SylGuard 184</td><td>material</td><td>Dow Corning</td><td>SYLG184</td><td>From World Precision Instruments</td></tr><tr><td>Agarose</td><td>reagent</td><td>Invitrogen</td><td>15510-027</td><td></td></tr><tr><td>Amyl acetate</td><td>reagent</td><td>Aldrich</td><td>W504009</td><td></td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>reagent</td><td>Sigma-Aldrich</td><td>C-1016</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>reagent</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>HEPES</td><td>reagent</td><td>Fisher Scientific</td><td>BP310</td><td></td></tr><tr><td>Heptaldehyde</td><td>reagent</td><td>Aldrich</td><td>H2120</td><td></td></tr><tr><td>Magnesium chloride hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;+6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>reagent</td><td>Sigma-Aldrich</td><td>M9272</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>reagent</td><td>JT Baker</td><td>3624-05</td><td></td></tr><tr><td>flowmeter</td><td>equipment</td><td>Gilmont Instruments</td><td>GF-2260</td><td></td></tr></tbody></table>

analyzing responses of mouse olfactory sensory neurons using the air-phase electroolfactogram recording

动物依赖于许多关键的行为，如寻找食物来源，避免天敌，确定交配和其他社会互动conspecifics，嗅觉。 electroolfactogram（EOG）记录是一个内容翔实，易于进行，和可靠的方法检测水平在嗅觉上皮的嗅觉功能。自1956年描述的青蛙1 Ottoson眼电图，眼电图记录已经应用在许多脊椎动物，包括蝾螈，兔，大鼠，小鼠和人类（斯科特和斯科特-约翰逊，2002年，文献2审查） 。在小鼠基因改造的最新进展，重新燃起兴趣的眼电图记录嗅觉功能的生理特性，在基因敲除和基因敲除小鼠。 EOG录音已成功应用，证明核心作用的嗅觉信号转导元件 3-8，以及最近的特点一定的监管机制的贡献OSN 的反应9-12。 气味检测发生在嗅上皮表面的纤毛OSNs，信号转导级联导致离子通道的开放，产生的电流流入的纤毛和去极化膜 13 。 EOG是负电位，细胞外记录在嗅觉上皮细胞表面的嗅觉刺激后，从2在录音领域的个别反应OSNs所造成的潜在变化的总和。因此的眼电图的振幅和动力学的比较基因改造和其他实验操作的影响底层的OSN的气味反应的分子信号如何提供有价值的信息。 在这里，我们描述了一个关于编制小鼠嗅觉鼻甲空气相EOG记录。简单地说，牺牲鼠标后，嗅觉鼻甲接触，沿中线由一分为二的头部和消除隔​​。鼻甲准备，然后放置在录音设置，并记录电极放置在嗅觉上皮细胞的表面，内侧鼻甲之一。参比电极是电气连接的组织，通过缓冲溶液。吹的上皮细胞表面保持湿润的一个加湿空气的连续流。膨化成加湿空气流刺激上皮的气味解决方案的蒸气。反应记录，并作进一步的分析数字化。

Watch this Scientific Journal Video about Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording at JoVE.com

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

electroolfactogram（EOG）记录翔实，易于进行，和可靠的评估水平在嗅觉上皮的嗅觉功能。此协议介绍录音设置，鼠标组织编制，数据的收集，和数据分析的基本。

鼠标采用的空气相Electroolfactogram录制的嗅觉感官神经元响应分析

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

analyzing-responses-mouse-olfactory-sensory-neurons-using-air-phase

Research

JoVE Journal

Neuroscience

16.6K 次观看.  Johns Hopkins University. electroolfactogram（EOG）记录翔实，易于进行，和可靠的评估水平在嗅觉上皮的嗅觉功能。此协议介绍录音设置，鼠标组织编制，数据的收集，和数据分析的基本。

视频: Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

多电极阵列的犁鼻上皮录音

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

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 ...

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

在小鼠嗅球成像气味诱发的活动，利用光学反射和自体荧光信号

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

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 ...

Extracellular Multi-Unit Recording from the Olfactory Nerve of Teleosts

Recent studies have shown that ocean acidification affects olfactory-driven behavior in fish. This may be due in part to a reduction in olfactory sensitivity in high PCO2/low pH water. To assess the effects of ocean acidification, or olfactory sensitivity in marine fish in general, we propose that extracellular multi-unit recording from the olfactory nerve is the method of choice. Although invasive, it is sensitive, robust, reproducible and independent of external salinity (unlike the electro-olfactogram [EOG], for example). Furthermore, it records a primary sensory input into the CNS, prior to any central processing. We show that this method can show a reduction in olfactory sensitivity that is both temporary and odorant-dependent, using a range of amino acids to construct concentration-response curves and calculate the thresholds of detection.

Extracellular multi-unit recording from the olfactory nerve is a sensitive, robust and reproducible method for assessing olfactory sensitivity in marine fish. It records primary sensory input and is independent of external salinity.

特勒奥茨的嗅觉神经的细胞外多单元记录

Recent studies have shown that ocean acidification affects olfactory-driven behavior in fish. This may be due in part to a reduction in olfactory ...

环境科学

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 ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

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, ...

Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice

Source: Jarriault, D., et al. <a href="https://app.jove.com/v/52652">Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor.</a> J. Vis. Exp. (2015).
This video demonstrates electrophysiological recording of olfactory sensory neurons (OSNs) in mice using a perforated patch-clamp technique. The olfactory epithelium, containing OSNs with fluorophore-tagged odor receptors, is isolated from a mouse and secured in a recording chamber. A recording pipette is clamped to the dendritic knob of an OSN, and nystatin within the pipette perforates the membrane to form electrical connectivity with the cell. Finally, another pipette is used to deliver an odor stimulant, and the electrophysiological activity of the patched cell is recorded.

小鼠嗅觉感觉神经元的穿孔膜片钳记录

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

鼠标采用的空气相Electroolfactogram录制的嗅觉感官神经元响应分析

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