这项工作是支持由加拿大卫生研究所（CIHR）批准机构韦恩南Sossin。阿玲A.法拉是从全宗德拉RECHERCHE EN桑特魁北克（FRSQ）和康拉德哈灵顿奖学金博士后奖学金获得者。作者想感谢乔安娜探条，玛格丽特黑斯廷斯和玛格丽特Labban与视频拍摄和图形概述帮助马德琳里士满池帮助。

1。昆虫细胞单层培养的制备及保养<ol><li>在组织培养罩的Sf9细胞培养和维护。 </li><li> Sf9细胞来源于草地夜蛾卵巢细胞（Sf21细胞），可作为冷冻细胞购自Invitrogen Grace的媒体。 </li><li>广场8毫升Grace的昆虫培养基（1X），其中30％小牛血清（FBS），已在一个倾斜的脖子75 厘米2细胞培养瓶中加入。 </li><li>在37℃水浴融化Sf9细胞冷冻管迅速来回移动管（约1-2分钟）。 </li><li> Sf9细胞转移到75厘米的2细胞培养瓶和岩石板，用手轻轻地均匀地分发细胞。 </li><li>孵育1-2小时在27℃直至细胞附着。 </li><li>删除旧的介质，含30％FBS和10毫升freshGrace的昆虫媒介取代。 </li><li>孵育27℃直至细胞融合。 </li><li>为了保持单层培养，Sf9细胞融合烧瓶中删除旧培养基，加入含10％胎牛血清的Grace的昆虫媒介的5毫升。 </li><li>烧瓶的侧轻轻敲击几次分离Sf9细胞。 </li><li>使用10 mL的吸管和一个移液器，移液器媒体一次轻轻的向上和向下，喷洒烧瓶壁吹打下来。将细胞15毫升无菌Polysterene管。 </li><li> Grace的昆虫媒介的8毫升与10％FBS（1:5稀释维持数期增长）在新的75厘米 2细胞培养瓶和岩石板手轻轻的Sf9细胞悬液，加入2毫升。 </li><li>孵育27℃直至细胞融合。 </li></ol> 2。 PKCS荧光标记在Sf9细胞中的表达<ol><li>对于实时成像实验，文化的Sf9细胞上的35毫米MatTek玻璃底部文化disheswith一个14毫米，盖玻片厚度为0.16至0.19毫米的玻璃表面。 </li><li> PKCS荧光蛋白标记的表达的Sf9细胞，利用Cellfectin II转染试剂在下面详细的修改制造商的以下建议。 </li><li>第1天：电镀细胞之前，对待每个MatTek菜Grace的昆虫媒介为至少30分钟，含10％胎牛血清。 </li><li> Sf9细胞计数从第11步使用hemacytometer（以上）。 </li><li>板0.05 × 10 6细胞上的每一个总体积150μLMatTek玻璃盖玻片。允许细胞附着在室温下30分钟。 </li><li>加入2毫升Grace的补充慢慢地在同一时间，以避免细胞分离到每道菜1毫升含10％胎牛血清。 </li><li>孵育27℃过夜。 </li><li>从这点上，利用昆虫细胞与质粒DNA转染的制造商的建议。 </li><li>第2天：转染的Sf9细胞与荧光标记的使用PKCS Cellfectin二转染试剂。我们经常合作与增强型绿色荧光蛋白（EGFP）和单体红色荧光蛋白（MRFP）（先前已经描述2,5质粒的构建的细节）标签系统PKCS表示。一次在一个荧光蛋白的表达产量约70-80％，表达细胞转染后72小时。两个荧光蛋白的共表达减少约30％，共同表达细胞转染效率。当共同表达EGFP标记的蛋白激酶C和MRFP标签的蛋白激酶C，使用最佳的蛋白表达MRFP标签蛋白的2倍更多的质粒DNA。 </li><li>现场演奏成像48-72小时后转染（最佳蛋白的表达，等待72小时）。 </li></ol> 3。 海兔感觉神经元中表达的荧光标记的PKCS <ol><li> 海兔的神经元细胞培养制备已在视频文章赵和他的同事4。 </li><li> PKCS荧光蛋白标记的表达在海兔感觉神经元，用显微注射过程的详细。 </li><li>准备在蒸馏水中的1％快绿（FCF）原液，过滤器使用注射器和一个28毫米的注射器过滤器（0.20微米）。分装并储存于-20 ° C </li><li>微量注射的感觉神经元可进行1天或2天，后分离的神经元，但我们发现，等待2天，可以更好的细胞黏附盖玻片。 </li><li>第2天，感觉神经元的隔离后，解冻快速绿色等分，并再次使用的注射器和注射器过滤器的28毫米（0.20微米）过滤。 </li><li>准备在蒸馏水中的质粒DNA溶液中含有0.5％的快速绿色。可用于高0.4μg/μL的质粒DNA的浓度，但它是不建议去以上这些价值观。 </li><li>过滤用1 ml注射器和4毫米的注射器过滤器（0.20微米）的质粒DNA /快速绿色解决方案。 </li><li>离心15分钟用一个麦克风的质粒DNA在16110 XG /快速的绿色解决方案rocentrifuge。 </li><li>准备使用电极拉马的神经元显微注射（萨特火焰山/布朗微管拖轮，P - 97型）电极是雪亮的。使用一箱长丝，拉1毫米外径，内径0.75毫米和100毫米的长度内丝（世界精密仪器TW100F - 4）采用薄壁玻璃毛细管玻璃微电极。对于拉动显微注射电极的更多信息，请参阅萨特电极食谱</li><li>填写每个电极与2μL质粒DNA /使用microloader枪头（Eppendorf公司CA32950 - 050），快速的绿色解决方案。 </li><li>转让MatTek玻璃底培养皿中含有海兔的神经元文化，以显微注射站。 </li><li>显微注射站包括以下内容：一个自制的大玻璃舞台举行振动隔离表（动力学系统隔振工作站），与外部的卤素灯照明的立体范围，显微（萨特赫胥黎墙型微操作机器人的培养皿它允许在三个轴粗，超细定位），一个电极连接到气动泵（世界精密仪器的Pico - PV820气动泵）的持有人。气动泵的压力输入连接到一个压缩的氮气罐。 </li><li>一个微电极插入微电极支架。 </li><li>一个立体显微镜下观看，插入到细胞核内的微尖。 </li><li>提供短期的氮压力脉冲（持续时间10至100毫秒，20磅/英寸2），直到原子核变得uniformely绿色。 </li><li> 4小时在室温下孵育细胞，并保持在4℃，直到使用。为了达到最佳的PKC易位，执行生活成像实验20 - 24小时后注射。 </li></ol> 4。可视化在生活Sf9细胞，并在海兔感觉神经元PKC的易位<ol><li>成像，我们使用的Zeiss LSM510倒置共聚焦显微镜与AXIOVERT 200。 </li><li> Sf9细胞和海兔感觉神经元以下除外：Sf9细胞与x63油浸物镜成像，而海兔感觉神经元与X40油浸物镜成像成像协议。 Sf9细胞成像维持在26-27 摄氏度的温度控制腔， 而海兔感觉神经元是在室温保持18-20摄氏度左右成像</li><li>我们用一个30兆瓦的氩激光（488nm激发）50％的激光输出图像与EGFP蛋白激酶C和一个氦氖激光（543nm激发）MRFP蛋白标记的图像的PKC标签。氩和氦氖激光线衰减到4％，和50％的传输输出分别成像和针孔前调整到一个。 </li><li>重要的是有稳定成像舞台上的培养皿，以避免运动或振动。 </li><li>删除从盘留在菜总量的1毫升10毫升吸管轻轻使用1毫升培养基。 </li><li>在细胞的细胞核中可以看出中间部分图片。 </li><li>设置了10-20共聚焦图像，拍摄一张照片，每30秒的时间序列。 </li><li>启动时间序列。 </li><li> 2张照片（后30秒的时间点）后，添加1毫升的药物（2X浓度）的使用P1000的吸管细胞之上轻轻落降。该药物是不断增加约30秒。 </li><li>有些药物诱导快速移位，可立即显示下列药物治疗（在60秒的时间点），而其他诱发一个缓慢的易位，这是唯一的最佳治疗后（1电影和电影2）5-10分钟。 </li><li>如果药物需要被冲走，使用2 × 10 mL的吸管做洗，上下吹打洗涤缓冲液，轻轻一个吸管的菜一边和对方pipeting媒体。 </li><li>如果洗一直有效，如果该药物的影响是可逆的的，蛋白激酶C在30-60秒内将恢复到细胞质。 </li><li>作为LSM文件保存的电影。 </li><li>美国国立卫生研究院使用Image J软件打开LSM的文件和量化的图像前和后的药物治疗。定量已在别处1。 </li></ol>

<ol>
	<li>Farah, C. A., Nagakura, I., Weatherill, D., Fan, X. &. a. m. p. ;. a. m. p., Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+role+for+phosphatidic+acid+in+the+translocation+of+the+novel+protein+kinase+C+Apl+II+in+Aplysia+neurons.">Physiological role for phosphatidic acid in the translocation of the novel protein kinase C Apl II in Aplysia neurons.</a> Mol Cell Biol. 28, 4719-4733 (2008).</li><li>Zhao, Y., Leal, K., Abi-Farah, C., Martin, K. C., Sossin, W. S. &. a. m. p. ;. a. m. p., Klein, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M.Isoform+specificity+of+PKC+translocation+in+living+Aplysia+sensory+neurons+and+a+role+for+Ca2+-dependent+PKC+APL+I+in+the+induction+of+intermediate-term+facilitation.">M.Isoform specificity of PKC translocation in living Aplysia sensory neurons and a role for Ca2+-dependent PKC APL I in the induction of intermediate-term facilitation.</a> J Neurosci. 26, 8847-8856 (2006).</li><li>Farah, C. A., Weatherill, D., Dunn, T. W. &. a. m. p. ;. a. m. p., Sossin, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKC+differentially+translocates+during+spaced+and+massed+training+in+Aplysia.">PKC differentially translocates during spaced and massed training in Aplysia.</a> J Neurosci. 29, 10281-10286 (2009).</li><li>Zhao, Y., Wang, D. O., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aplysia+sensory-motor+neuronal+cell+cultures.">Preparation of Aplysia sensory-motor neuronal cell cultures.</a> J Vis Exp. , (2009).</li><li>Manseau, F., Fan, X., Hueftlein, T., Sossin, W. S., Castellucci, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-independent+PKC+Apl+II+mediates+the+serotonin+induced+facilitation+at+depressed+synapses+in+Aplysia.">Ca2+-independent PKC Apl II mediates the serotonin induced facilitation at depressed synapses in Aplysia.</a> J. Neuroscience. 21, 1247-1256 (2001).</li></ol>

我们已经描述了一种技术，实时图像PKCS荧光标记的易位在Sf9细胞， 并在海兔感觉神经元。 Sf9细胞提供了一个简单的系统形象PKC的易位，因为培养和转染他们是相当平直向前。相比之下，微量注射海兔感觉神经元需要一些时间来掌握，最多几个月。有关这项技术的困难，包括电极堵塞。过滤注射液和离心它通常会帮助，但有时气泡在电极形成却使。我们发现，使用microloader枪头填补?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sf9 frozen cells</td>
<td>Spodoptera frugiperda ovarian cells</td>
<td>Invitrogen</td>
<td>B825-01</td>
<td>&nbsp;</td>
<tr>
<td>Grace&#x2019;s Insect Medium, Supplemented (1X), liquid</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11605-094</td>
<td>Use to culture Sf9 cells</td>
</tr>
<tr>
<td>Corning 75cm2 canted neck cell culture flask</td>
<td>Tool</td>
<td>Corning</td>
<td>430720</td>
<td>Use to culture Sf9 cells</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>CanSera</td>
<td>CS-C08-500</td>
<td>Supplement Grace's media with FBS prior to use</td>
</tr>
<tr>
<td>Syringe</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309604</td>
<td>Use to filter Fast Green solution</td>
</tr>
<tr>
<td>Syringe</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>309602</td>
<td>Use to filter plasmid DNA / Fast Green solution</td>
</tr>
<tr>
<td>Filter</td>
<td>Tool</td>
<td>Corning</td>
<td>431229</td>
<td>Use to filter Fast Green solution</td>
</tr>
<tr>
<td>Filter</td>
<td>Tool</td>
<td>Corning</td>
<td>431212</td>
<td>Use to filter plasmid DNA / Fast Green solution</td>
</tr>
<tr>
<td>Glass Bottom Dish</td>
<td>Tool</td>
<td>Mattek</td>
<td>P35G-1.5-14-C</td>
<td>Use to culture Sf9 cells prior to transfection and imaging</td>
</tr>
<tr>
<td>Cellfectin II Reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10362-100</td>
<td>Used to express fluorescently tagged PKCs in Sf9 cells</td>
</tr>
<tr>
<td>Fast Green FCF</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F-7252</td>
<td>Use to visualize microinjection of plasmid DNA solution into Aplysia sensory neurons</td>
</tr>
<tr>
<td>Thin wall glass capillaries</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>TW100F-4</td>
<td>Use to make microelectrodes for microinjection</td>
</tr>
<tr>
<td>Microloader pipette tip</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>CA32950-050</td>
<td>Autoclave before using to fill up the microelectrodes for microinjection</td>
</tr>
<tr>
<td>PV820 Pneumatic PicoPump </td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>SYS-PV820</td>
<td>Part of microinjection station</td>
</tr>
<tr>
<td>Microelectrode holder</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>MPH6S</td>
<td>Use to hold microelectrode </td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Tool</td>
<td>Sutter</td>
<td>MP-85</td>
<td>Use to position microelectrode in the three-axis</td>
</tr>		</tbody>
		</table>
		</div>

live-imaging of pkc translocation in sf9 cells and in aplysia sensory neurons

蛋白激酶CS（PKCS）丝氨酸苏氨酸激酶，发挥核心作用，在规范的多种细胞过程，如细胞的生长和学习记忆。有四种已知脊椎动物中的PKC亚型的家庭：古典PKCS（α，βI，βII和γ），新颖的I型PKCS（ε，η），小说II型PKCS（δ和θ），非典型PKCS（ζ和ι ）。古典PKCS被激活的Ca 2 +和diacylclycerol（DAG），而小说PKCS是由DAG的激活，但独立的Ca 2 +。既不钙2 +和 DAG的非典型PKCS被激活。在海兔californica，我们的模型系统来研究记忆的形成，有三个中枢神经系统特定PKC亚型从每个主要类别之一，即传统的PKC的美国总统轮船我的小说类型我PKC的美国总统轮船II和非典型PKC的美国总统轮船三。 PKCS脂激活蛋白激酶和外信号激活古典与新颖的PKCS已经经常与PKC从胞浆易位到质膜相关。因此，在活细胞中实时蛋白激酶C移位可视化已成为一个宝贵的工具，阐明导致PKC激活的信号转导通路。例如，这项技术已允许我们建立不同的PKC亚型转运不同条件下的调解不同类型的突触可塑性和激活PKC的美国总统轮船II，5 - 羟色胺（5HT）需要DAG和磷脂酸（PA）的生产易位1-2。更重要的是，相同的神经元的能力，可视化多次允许我们，例如，在精致的细节 3 PKC反应来衡量的脱敏。在这个视频中，我们展示了准备的Sf9细胞培养的每一步，海兔感觉神经元的文化已在另一视频第4条所述，表示在Sf9 细胞，并在海兔感觉神经元和PKC的易位的生活成像PKCS荧光标记反应不同的活化剂，使用激光扫描显微镜。

Watch this Scientific Journal Video about Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons at JoVE.com

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

在这段视频中，我们证明在使用PKCS荧光标记的活细胞PKC移位可视化。

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

live-imaging-pkc-translocation-sf9-cells-aplysia-sensory

Research

JoVE Journal

Neuroscience

11.9K 次观看.  McGill University. 在这段视频中，我们证明在使用PKCS荧光标记的活细胞PKC移位可视化。

视频: Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

神经调节和线粒体运输：在较长的时间里，海马神经元的实时成像

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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 ...

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. ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

运动神经元细胞外识别的肌肉运动游泳池<em&gt;海兔夜蛾</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

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 ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

一种方法为Target和隔离气道支配感觉神经元的小鼠

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...