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。

<ol>
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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。

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Air delivery tube</td>
<td>equipment</td>
<td>Custom fabricated</td>
<td>&nbsp;</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</td>
<td>LW3030B-OPT</td>
<td>&nbsp;</td>
<tr>
<td>Amplifier</td>
<td>equipment</td>
<td>Warner</td>
<td>DP-301</td>
<td>&nbsp;</td>
<tr>
<td>Computer and Data Acquisition Software</td>
<td>equipment</td>
<td>Axograph 4.9.2 on Apple Macintosh</td>
<td>&nbsp;</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>&nbsp;</td>
<td>&nbsp;</td>
<td>A cr&egrave;me br&ucirc;l&egrave;e torch works well</td>
</tr>
<tr>
<td>Digitizer</td>
<td>equipment</td>
<td>Axon Instruments</td>
<td>Digidata 1322A</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Scope</td>
<td>equipment</td>
<td>Scienscope</td>
<td>SSZ</td>
<td>&nbsp;</td>
<tr>
<td>Electrode holder</td>
<td>equipment</td>
<td>Harvard Apparatus</td>
<td>64-1021</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Holding Devices (12 mm)</td>
<td>equipment</td>
<td>World Precision Instruments</td>
<td>M10</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulators</td>
<td>equipment</td>
<td>World Precision Instruments</td>
<td>M3301R M3301L</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette Puller</td>
<td>equipment</td>
<td>Sutter Instrument Co.</td>
<td>P2000</td>
<td>&nbsp;</td>
<tr>
<td>Oscilloscope</td>
<td>equipment</td>
<td>Tektronix </td>
<td>5110</td>
<td>&nbsp;</td>
<tr>
<td>Picospritzer III</td>
<td>equipment</td>
<td>Parker Instrumentation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Silicone tubing</td>
<td>equipment</td>
<td>Nalge Nunc</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Specimen stage</td>
<td>equipment</td>
<td>Custom fabricated</td>
<td>&nbsp;</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>Becton Dickinson</td>
<td>305195</td>
<td>&nbsp;</td>
<tr>
<td>2 oz. glass bottles</td>
<td>material</td>
<td>VWR International</td>
<td>16152-201</td>
<td>&nbsp;</td>
<tr>
<td>Glass capillaries</td>
<td>material</td>
<td>World Precision Instruments</td>
<td>TW150F-6</td>
<td>&nbsp;</td>
<tr>
<td>Silicone stoppers size 16D</td>
<td>material</td>
<td>Chemware</td>
<td>D1069809</td>
<td>&nbsp;</td>
<tr>
<td>Silver wire</td>
<td>material</td>
<td>World Precision Instruments</td>
<td>AGW1010</td>
<td>&nbsp;</td>
<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>&nbsp;</td>
<tr>
<td>Amyl acetate</td>
<td>reagent</td>
<td>Aldrich</td>
<td>W504009</td>
<td>&nbsp;</td>
<tr>
<td>Calcium chloride (CaCl2)</td>
<td>reagent</td>
<td>Sigma</td>
<td>C-1016</td>
<td>&nbsp;</td>
<tr>
<td>Dimethyl sulfoxide (DMSO)</td>
<td>reagent</td>
<td>Sigma</td>
<td>D5879</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>reagent</td>
<td>Fisher</td>
<td>BP310</td>
<td>&nbsp;</td>
<tr>
<td>Heptaldehyde</td>
<td>reagent</td>
<td>Aldrich</td>
<td>H2120</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium chloride hexahydrate (MgCl2+6H2O)</td>
<td>reagent</td>
<td>Sigma</td>
<td>M9272</td>
<td>&nbsp;</td>
<tr>
<td>Sodium chloride (NaCl)</td>
<td>reagent</td>
<td>JT Baker</td>
<td>3624-05</td>
<td>&nbsp;</td>
<tr>
<td>flowmeter</td>
<td>equipment</td>
<td>Gilmont</td>
<td>GF-2260</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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

16.4K 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

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

科研

神经科学

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

闪光光解笼状化合物在嗅感觉神经元的纤毛

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

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 ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

细胞内记录，感官字段映射，并在水蛭养殖鉴定神经元，医用水蛭</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

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 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 ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

通过计算机跟踪检测大鼠嗅觉行为<em&gt;果蝇</em&gt;在四象限嗅

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

嗅球的四次免疫染色用于嗅觉感觉轴突分子识别码的可视化

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...