这项工作是由美国国立卫生研究院DC009613，人类前沿科学计划和莫雷护理奖学金（JR）的支持。

1。录像设置的记录室被安装在尼康TE2000U Eclipse的倒置显微镜相衬嵌合的光学上的气动工作台和电屏蔽使用一个法拉第笼。有机玻璃录音室包括两个部分，由阻挡层部分分离，并粘附到硅烷化的载玻片。该室的部分之一是用来解决细胞，而另一种是用于在录制过程中，以尽量减少刺激曝光过早曝光的结算，但尚未使用的单元格，以加臭剂。的记录设置包括的吸入移液管（见下文），其安装在一个沉淀的吸液管保持和接地电极。使用一台显微吸入吸液管的位置。激励信号的解决方案应用于使用三重桶的玻璃的方管，每个为0.7毫米×0.7毫米的方管。的三重管玻璃连接到机动化的快速步骤灌注系统，其安装在一个机械手（述之三，华纳仪器SF-77B）和输入的记录室，在〜45°的角度。三重桶玻璃的端部被斜切再次使得最后面（开口）的三重管玻璃是垂直的，以允许接近定位的移液器45°的角度处。 的吸入移液管填充有标准的哺乳动物Ringer溶液和接地电极的电极被连接到1GΩ探头在电压钳位模式的膜片钳放大器（PC-501A，华纳仪器）。从放大器（低通滤波的在5千赫）的模拟信号被显示在数字示波器在实时监测的信号，并还连接到一个8极贝塞尔滤波器（克罗恩音乐-海特），其中该信号是低通滤波在50 Hz。这两个信号（在5千赫和50赫兹的低通滤波的）被馈送到一个A / D转换器（剑桥电子设计微1401 mkII使用的），其连接到PC上的数据采集。使用信号3 ACQ数据被记录uisition软件（剑桥电子设计）的采样频率为10 kHz。首先记录的受体电流一起叠加后的动作电位，然后单独的受体电流（参照图2），该信号被记录在两个不同的带宽。灌注系统使用林格氏液（对照组）的溶液，加臭剂的刺激或任何所希望的组合物的溶液之间快速切换，并控制由PC和信号3软件。这些解决方案都包含在60毫升的注射器，由医疗级静脉流动调节器和流量控制。通常情况下，流率被设定为1毫升/分钟。 记录移液管上的吸入控制在以下的方式:吸液管保持器的侧端口的连接线又被连接到一个高度可调的贮油的油。提高或降低水库确保吸液管压力和因此林格氏流的通过在p的前端ipette是最小的，稍微积极。额外的吸力或压力可应用于通过管与吹嘴的另一端连接到空域水库吸记录移液管的尖端的一个孤立ORN进入细胞体。 实验是在37℃下进行，以便密切模拟本机的细胞生理状态的。将温度控制通过的解决方案，通过量身定制的加热单元，其由陶瓷电阻器，通过该不锈钢管中含有的解决方案被传递10。使用热电偶温度计克（Fluke）的温度监测。 2。解决方案哺乳动物Ringer氏溶液（毫摩尔）:140的NaCl，5氯化钾的MgCl 2，1，2的CaCl 2，0.01 EDTA，10 HEPES，10葡萄糖。用NaOH将pH调节至7.5。使用了10％的稀释的林格氏溶液测量结电流。菌种NT解决方案:苯乙酮和丁子香酚，每天在林格氏液从20毫米DMSO股票。 3。使电极<ol><li>将在萨特P-97微吸管拔出器的unfilamented硼硅玻璃毛细管拉微量长锥形。 </li><li>移液管转移到一个特制的的微量移液器支架安装在尼康E200的Eclipse的显微镜，这是又配有一个十字线的目镜和一个可移动的安装在舞台上的钻石刀。 </li><li>观察，20x的目标下的吸移管的前端，作为指南，使用分划板;轻轻划线的吸移管以90°的角度使用吸移管的宽度为10μm，（外径）的量身定制的钻石刀。 </li><li>进一步移动的钻石刀向尖部，并逐步将用金刚石刀的前端上的压力。吸头在被刻划的地步，要打破干净。 </li></ol>另外步骤3.13.4可以结合起来的情况下直接切吸头，吸液管可以拉动所需的尺寸，但它可以证明很难可靠地拉移液管这么大的齿顶圆直径和干净切白边。 <ol start="5"><li>下一个40倍的目标，火抛光的前端的移液管的内直径为5μm，使用电加热的灯丝。一旦完成后，微管是准备用于记录电流从鼠标ORNs。应该是开放的吸液管电阻约1MΩ时，充满了林格。 </li></ol> 4。嗅觉受体神经元的分离<ol><li>牺牲一个鼠标跟随机构的指导方针和法规。移开磁头，剥离的皮肤覆盖在颅骨内平分头，沿中线。 </li><li>解剖体视显微镜下，将两个半头，拉断的鼻中隔和删除的嗅觉鼻甲。将所有的组织培养皿中的葡萄糖Containing哺乳动物的林格氏液。 </li><li>剥离脱落上皮的底层的软骨从两个鼻甲的嗅觉和组织转移到含有250μL林格氏液的Eppendorf管中。我们使用2鼻甲组织，以获得合适的细胞密度的大小，我们的录音室。存储的其它组织，在4℃，供以后使用。 </li><li>涡的Eppendorf管中含有的嗅觉上皮细胞两次简要以中等速度为1秒。此步骤导致从上皮ORNs机械分离。 </li><li>放置的记录室中含有的解离ORNs Ringer溶液。卸下大块的组织也存在于暂停用细镊子。 </li><li>我们，ORNs解决与振铃前20分钟开始连续灌流和进行的录音。 </li></ol> 5。录音<ol><li>降低吸入油线电极连接到记录室和REGUL吃的贮油腔的高度，在移液管尖建立微正压。这是可以做到通过观察如细胞碎片远离（正压）或朝向（负压）的吸移管移动。尽量不污染的移液管的前端与细胞碎片。 </li><li>扫描为一个孤立的的ORN能够识别其典型的双极形态使用的倍率为20-40x的记录室。移动的记录电极，为ORN电池主体的接近。轻轻开始抽吸，使该单元体的移液管的尖端进入。小心地，慢慢地继续适用于吸入，直到整个细胞体被吸入到吸入移液管的尖端，留下的枝晶的浴溶液和纤毛暴露。吸的ORNs和吸管形状的图像，看到7,11。确保，一旦没有吸，ORN不移动或吸管的。如果是这样，相应地调整高度的贮油腔。 </li><li>使用显微操作，从部分包含结算ORNs到记录部分的腔室中移动的吸入移液管。在前面的解决方案交换管的管，小心地将吸吸液管。的吸入移液管应放置在靠近到足以3长筒管的开口，使得ORN露出到层流，从而避免混合湍流，并实现快速的溶液交换。溶液交换实现步进横跨吸入移液管的尖端的流动溶液的并行数据流之间的接口。的溶液交 ​​换过程中的时间通常是20毫秒左右，诱发通过步进细胞不同的离子组合物之间的解决方案（参见图2）从交界处电流测量。解决方案交付重力。 </li></ol> &lt;跨度类="的pdflinebreak"&gt; 6。代表性的成果<img alt="图1。" src="/files/ftp_upload/3862/3862fig1.jpg" /> 图1:A）双极型受体在嗅球神经元。 B）信号转导机制中的纤毛，最终导致兴奋性电流涌入。随后的去极化触发在电池主体的动作电位的产生。 <img alt="图2。" src="/files/ftp_upload/3862/3862fig2.jpg" /> 图2示出的溶液交 ​​换的速度和可靠性。一个移液管加强从正常到10％的稀林格氏液为100毫秒交界处的电流，从而从不同的离子浓度。黑色的轨迹是在大街愤怒的5项试验中，红色追踪它的SD。在解决方案交流演示的变化不大，SD解决方案交换的可靠性试验，试验和缺乏任何一种解决方案中多余的噪音。 <img alt="图3。" src="/files/ftp_upload/3862/3862fig3.jpg" /> 图3示出了从ORN使用吸入移液管技术的丁子香酚诱导响应。 ORN被暴露于100μM的丁子香酚，1秒，如所指示的记录的栏上面。另外，在图3A（黑线）受体电流在0 - 50赫兹的带宽过滤，只显示受体电流。 图。图3B示出的相同的记录，现在过滤在宽的带宽为0 - 5000赫兹（红色迹线），还显示动作电位（受影响，由箭头表示）。插图示出在一个扩展的时间刻度的可视化受影响更清楚地在发病的响应相同的跟踪。 <img alt="图4。" srC =的"/ files/ftp_upload/3862/3862fig4.jpg"/&gt; 图4。剂量反应（A）的丁子香酚响应ORN家庭。丁子香酚，用于浓度范围从0.3μM至100μM，刺激的持续时间为1秒，并过滤，在0 - 50赫兹的痕迹。的渐进的加臭剂的浓度增加导致更大范围和更快的响应，这也终止的更慢，一旦加臭剂曝光终止。 ORNs可以显示在中间的加臭剂的浓度（ 图4B）在长刺激的振荡响应模式。这里一个acetephenone（3μM），响应ORN刺激持续8秒。在发病的刺激经过快速和瞬态的峰值响应一系列观察到较小的，经常性的反应。记录带宽为0 - 50赫兹。

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作者有没有透露。

<p class="jove_content"&gt;抽吸移液器技术是一个电的方法，该方法被用于记录气味诱导慢受体电流和快速的双相动作电位同时从ORN。由于细胞的质膜不违反，此方法离开确保没有改变的加臭剂的反应，由于细胞质离子浓度或稀释的细胞内因素变化的细胞内环境不受干扰。细胞可以记录从较长的持续时间（在小鼠在青蛙和1小时至4小时）。这种技术的另一个优点是，因为在单元主体内的记录移液器，即纤毛和树突状旋钮?...

<table border="1"><tbody><tr><td> 的材料的名称 </td><td> 类型 </td><td> 公司 </td><td> 目录/ 型号 </td><td> 评论 </td></tr><tr><td>航空表</td><td>设备</td><td>新港</td><td></td><td></td></tr><tr><td>气泵</td><td>设备</td><td>新港</td><td> ACGP </td><td></td></tr><tr><td>用移液管拔轮器</td><td>设备</td><td>萨特</td><td> P-97 </td><td></td></tr><tr><td>高硼硅玻璃</td><td>设备</td><td> WPI </td><td> 1B150-4 </td><td></td></tr><tr><td>尼康的Eclipse倒置显微镜</td><td>设备</td><td>尼康</td><td> TE2000U </td><td>配备汞灯，GFP过滤器和目标的20倍和5倍，至少</td></tr><tr><td>放大器的PC-501A </td><td>设备</td><td>华纳</td><td> 64-0008 </td><td>探头1GΩ </td></tr><tr><td>钻石刀</td><td>设备</td><td>定做</td><td></td><td></td></tr><tr><td> A / D数字化仪Mikro1401 </td><td>设备</td><td>剑桥电子设计</td><td></td><td></td></tr><tr><td>过滤单元3382 </td><td>设备</td><td>克罗恩海特公司</td><td></td><td></td></tr><tr><td>信号</td><td>软件</td><td>剑桥电子设计</td><td></td><td></td></tr><tr><td>成型的Ag / AgCl颗粒</td><td>设备</td><td> WPI </td><td> 64-1297 </td><td></td></tr><tr><td>吸管架</td><td>设备</td><td>华纳</td><td> 64-0997 </td><td>自定义修改，以适应 探头</td></tr><tr><td>录音室</td><td>设备</td><td>定做</td><td></td><td></td></tr><tr><td>微操作机器人 MP85-1028 </td><td>设备</td><td>萨特仪器</td><td>微操作机器人 MP85-1028 </td> &lt;TD&gt; </td></tr><tr><td>矿物油</td><td>解</td><td>西格玛</td><td> 330779-1L </td><td></td></tr><tr><td>示波器TDS 1001 </td><td>设备</td><td>泰克公司</td><td></td><td></td></tr><tr><td>三管方玻璃管</td><td>设备</td><td>华纳</td><td> 64-0119 </td><td> 0.6 mm内径，长5厘米</td></tr><tr><td>阀</td><td>设备</td><td>利公司</td><td></td><td></td></tr><tr><td> Valvelink 8.2 </td><td>设备</td><td>自动化科学</td><td></td><td></td></tr><tr><td> SF-77B灌注快一步</td><td>设备</td><td>华纳</td><td></td><td></td></tr></tbody></table>

odorant-induced responses recorded from olfactory receptor neurons using the suction pipette technique

动物采样的臭味通过化学感受系统位于鼻腔周围环境。化学感受信号的复杂行为的影响，如食物的选择，捕食，同种和配偶识别和其他社会有关的线索。位于嗅觉受体神经元（ORNs）中嵌入鼻腔的嗅觉上皮细胞背侧部分。这些双极神经元的轴突发送到嗅球（见图1，Reisert钊1，最初发表于普通生理学杂志"）和扩展的单枝晶的纤毛辐射到的粘液覆盖的嗅觉上皮细胞的边界上皮细胞。的纤毛包含的信号转导机制，最终导致兴奋性通过睫状转导通道，环核苷酸门控通道（CNG）的Ca 2 +活化的Cl -的信道（ 图1）流入的电流。随之而来的depola三丙触发在电池主体2-4的动作电位的产生。 在这个视频中，我们描述了使用的"吸吸液管技术"，记录的气味引起的反应ORNs。该方法最初发展到记录从杆感光体5和jove.com修改，以记录从鼠标锥感光体6，可以发现在该方法的一个变种。后来被改编吸入吸液管技术也记录ORNs 7,8。简要地说，在解离的嗅觉上皮细胞和细胞分离后，整个细胞的ORN体被吸入到的记录吸管的前端。枝晶和纤毛仍然暴露于沐浴液，从而解决方案的变化，例如，使气味或药物受体阻滞剂的应用。在此配置中，还没有进入到细胞内环境获得（的没有全细胞电压钳）和细胞内的电压保持自由变化。这一切都OWS受体电流缓慢，起源于发射的细胞体9纤毛和快动作电位的同时记录。动力学这两个信号之间的差异在允许它们被分开使用不同的过滤器设置。这种技术可以用在任何野生型或基因敲除小鼠，或选择性地从记录ORNs也表达GFP标记的特定子集ORNs，例如表示一个给定的加臭剂的受体或离子通道。

Watch this Scientific Journal Video about Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique at JoVE.com

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

嗅觉受体神经元（ORNs）气味信号转换成一个受体的电流，从而触发动作电位，被输送到第二个神经元在嗅球中。这里，我们描述的吸入移液管的技术，同时记录从小鼠ORNs电流和动作电位的加臭剂诱导的受体。

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 ...

odorant-induced-responses-recorded-from-olfactory-receptor-neurons

Research

JoVE Journal

Neuroscience

10.7K 次观看.  Monell Chemical Senses Center. 嗅觉受体神经元（ORNs）气味信号转换成一个受体的电流，从而触发动作电位，被输送到第二个神经元在嗅球中。这里，我们描述的吸入移液管的技术，同时记录从小鼠ORNs电流和动作电位的加臭剂诱导的受体。

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

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

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

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 ...

科研

神经科学

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

一个NMDA受体电导的大鼠中脑多巴胺能神经元的动态钳技术中的应用

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

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 ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二:外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

针对嗅球神经元采用组合在体内电穿孔和GAL4基于增强陷阱斑马鱼线

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

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. ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

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 ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

受体激活的测量通过荧光素酶活性:哺乳动物的嗅觉受体的高通量分析

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...