支持这项研究是由美国国立卫生研究院DC052256和DC006015和NSF 0324769 Qg和T32 - DC008072符合BS。

1。制备 此过程是生物安全2级，因此，显微注射过程中的生物危害罩以下所有的准备工作 。 <ol><li>准备和预热了37 ° C水床:装满水的塑料储物袋 - 清空加热块孵化器的密封和设置。这将使小鼠恢复注射后。 </li><li>填写一个生物危险废物容器的10％漂白水浸泡的所有从这个过程中浪费的一个合适的音量。 </li><li>准备一个小型开放的菜70％的乙醇和在生物安全柜的灭菌ddH2O和地方第二盘。 </li><li>喷涂用70％乙醇的身体和针与柱塞拉出所有的方式，用33号针头消毒一个5μL能力汉密尔顿注射器。反复抽吸乙醇和从盘弹出所有的方式至少5倍。 </li><li>吸入和弹出至少5倍无菌水冲洗注射器墨盒。安全放置注射器抛开文化罩，让其干燥。 </li><li>删除病毒从-80 ° C冰箱储存和冰的地方，让解冻的股票。显微注射需要在10 9传染性单位/ ml滴度的病毒每鼠标股票2μL 。慢病毒可以从商业渠道获得，或在实验室中根据既定的协议（洛伊丝等，2002 ）。 </li><li>准备注射阶段提供罩表面以上提出了一个区域 - 可以使用一个10cm的培养皿中的Kimwipe表盖。 </li><li>安排注射站附近的酒精擦拭。 </li></ol> 2。嗅上皮微量注射慢病毒颗粒 所有动物在此方法中执行的程序是根据IACUC批准的指引。 家鼠应变只C57BL/6J这里演示的是在所有的实验中使用的动物模型 。 <ol><li>低温笼鼠标小狗（P0 - P3），并把截开5分钟的手套上冰小狗麻醉鼠标幼仔。 </li><li>虽然等待小狗将完全麻醉，消毒汉密尔顿注射器吸病毒的股票2μL。 </li><li>将鼠标小狗注射阶段顶部。从头顶轻轻拉动皮肤，向颈部顶部耳。这应提供在鼻腔讲授皮肤表面，将缓解注射。 </li><li>通过约3mm的皮肤表面以下的鼻腔顶部插入针到达嗅上皮。 Microinject0.5μL病毒解决方案，每拍，选择每边两个站点，左，右（每鼠注射=所需的病毒2μL）。注射部位应错开彼此相隔。针对适当的注射部位，搜索背头骨表面的嗅球的轮廓。此行是在出生时几乎没有可见通过半透明的皮肤和颅骨。为了使每边两个注射，目的只​​是延髓一个灯泡在中轴大纲一次注射。使第二个灯泡大纲横向插入，但确保内侧角注射，这样您就可以到达嗅上皮，位于腹部下方的灯泡。避免穿透嗅球！ </li><li>吸干任何残留的病毒解决方案或的血液可能会逃脱从注射部位或使用Kimwipe鼻孔开口。立即处置Kimwipes在稀释的漂白液的生物危险容器。 </li><li>广场上的水床孵化器注入鼠标。 </li><li>重复步骤1-4，所有被注入小鼠记住你将需要大约每鼠的病毒库存2μL。 </li><li>让他们返回到原来的筑巢笼前，小鼠恢复30分钟的水床孵化器。 </li><li>在漂白液的生物危险处理处置显微注射阶段，消毒纸巾和任何额外的清洁纸产品。 </li><li>清洁汉密尔顿注射器反复抽吸和乙醇弹射，然后水做的准备步骤。允许注射器干。 </li><li>为了获得最大的转导效率，再次重复这个过程在4个小时。感染OSNs可能在数为3天可视化整个细胞的长度。 </li></ol> 3。浮动嗅球部分的免疫组织化学<ol><li>牺牲所需的时间点注入鼠标和灌注4％多聚甲醛的修复组织。解剖出的OB。孵育过夜，然后在多聚甲醛30％蔗糖PBS中过夜。嵌入在OCT包埋剂干冰。 </li><li>日冕的OB的部分获得在使用低温恒温器的60微米的厚度。灯泡部分扫成10cm的培养皿用5ml 1X PBS（pH值7.4）。部分应保持在PBS浮动允许华侨城到Dissolve。转移到一个50ml的锥形瓶中的浮动部分和PBS。 </li><li>允许部分定居瓶底部。关闭PBS真空不吸吮任何部分。用20ml PBS冲洗组织切片。重复定居和吸尘步骤。用10ml PBS悬浮组织和浮动部分转移到免疫锥形瓶15ML。一旦组织切片相继落户，互不干扰组织关闭PBS上清真空。与悬浮部分0.3％的Triton X - 100的1X PBS，pH值7.4（PBS - TX）允许洗涤剂穿孔的细胞膜。孵育10分钟，在室温。关闭真空上清，用PBS - TX重复孵化两次。 </li><li>两个小时在室温下孵育封闭液10毫升（5％马血清的PBS - TX）的组织切片。 </li><li>阻断溶液冲洗，用PBS - TX 15分钟，在室温下，每年至少两次。 </li><li>准备一个初级抗体溶液在PBS - TX 2ML。在本实验中使用的抗体试剂和仪器节。 </li><li>更换第二PBS - Tx的封锁与初级抗体溶液清洗。孵育上一个缓慢摇动的平台（〜30rpm）在4 ° C为24-48小时，以确保彻底渗透到60微米的浮动部分。 </li><li>的主要抗体溶液孵育后，可仔细用吸管检索和存储在4 °重用在两周内两次没有任何重大损失的染色强度</li><li>清洗部分为20分钟，在室温下轻轻旋转（〜30rpm）在PBS - TX的3倍。 </li><li>准备在PBS的TX二级抗体稀释液2ml。真空最后的清洗，并添加二次抗体溶液组织切片。包装铝箔染色瓶挡光，这可能会导致荧光团的漂白之外。将包裹上一个缓慢摇动的平台，并在4 ° C过夜孵育的小瓶。 </li><li>丢弃吸尘距离的二次抗体溶液。一次20分钟，在室温下用PBS - TX洗净节。冲洗20分钟，在室温下用PBS液（pH 7.4）两个额外的时间。安装玻璃上使用Fluoromount G.的幻灯片染色的组织切片</li><li>通过荧光显微镜正感染的OSN轴突和轴突总站染色切片的画面。 </li><li>要构造一个一个标记的OSN的轴突和终端乔木三维视图，通过一系列光学部分目标OB组织切片，使用激光共聚焦显微镜（沿Z轴）60微米的深度。 </li><li>随后可以预计这Z的部分图像叠在一起，重建一个360度旋转的角度单个标记的OSN的轴突，包括乔木终端的全部细节。任何一个单一的旋转角度可以被捕获并作为2D格式的演示图像文件（如JPG，TIFF）导出。 </li></ol> 4。代表性的成果这里介绍的方法，我们可以在体内的慢病毒转导的嗅觉感官神经元。我们已与GFP和mCherry荧光记者迄今为止可视感染OSNs，以及与一个myc基因通过免疫组织化学标记的融合蛋白。这种技术提供了大量感染OSNs。尽管如此，转导偶尔出现，足以提供单独标记OSNs，从而使单细胞形态分析。轴突和轴突终末内的OB组织切片成像，共聚焦显微镜（图1）原生环境之中。整个OSN轴突终端乔木，通常可以在40 -50μm的光学部分迷住了。被感染的OSN轴突终端的高倍率允许在肾小球层轴索乔木形态的详细分析。 <img src="/files/ftp_upload/2951/2951fig1.jpg" /> 图1。单一的嗅觉感官神经元轴突和其终端乔木轨迹。A）的一个P7的嗅神经层（ONL）鼠标的嗅球冠状切面免疫染色为OMP（红色）和肾小球层（GL）与两个OMP的免疫染色Vglut2（灰色）。一个单一的嗅觉感官神经元轴突表达GFP（绿色）内ONL移动，并渗透到肾小球结构。 B）轴突终端凉棚内的肾小球形态明确地强调通过GFP的表达。 Vglut2灰色合作染色照明的总glomular面积内GFP阳性轴突阐述终端ABOR。 C）慢病毒感染OSNs OE中的表达GFP（绿色）。嗅上皮cytostructure用DAPI染色（蓝色）。 GFP的表达，允许OSN的胞体和OE中的细胞过程的可视化。 d）一个OSN的轴突和Rap1GAP2表达其终端形态击倒通过的shRNA。 Rap1GAP2的shRNA慢病毒携带鼠标下的U6启动子和GFP下一个双表达盒CMV启动子的OE。感染的嗅觉轴突终端展示一个更加简化的终端乔木相比只能控制的GFP。酒吧=20μm的。

<ol>
	<li>Chen, H., Kohno, K., Gong, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+ablation+of+mature+olfactory+sensory+neurons+mediated+by+diphtheria+toxin+receptor.">Conditional ablation of mature olfactory sensory neurons mediated by diphtheria toxin receptor.</a> J Neurocytol. 34, 1-2 (2005).</li><li>Doi, K., Nibu, K., Ishida, H., Okado, H., Terashima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+gene+transfer+in+olfactory+epithelium+and+olfactory+bulb:+a+long-term+study.">Adenovirus-mediated gene transfer in olfactory epithelium and olfactory bulb: a long-term study.</a> Ann Otol Rhinol Laryngol. 114, 629-633 (2005).</li><li>Lois, C., Hong, E. J., Pease, S., Brown, E. J., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transmission+and+tissue-specific+expression+of+transgenes+delivered+by+lentiviral+vectors.">Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors.</a> Science. 295, 868-872 (2002).</li><li>Zhao, H., Otaki, J. M., Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+gene+transfer+in+olfactory+neurons+in+vivo.">Adenovirus-mediated gene transfer in olfactory neurons in vivo.</a> J Neurobiol. 30, 521-530 (1996).</li></ol>

该协议是生物安全2级标准。已经建立了相应的所有适当的工作区，保护人身安全，和病毒废物遏制措施。根据IACUC指引，所有动物的程序进行。

微量注射慢病毒基因结构的永久转会到OSN的基因组DNA的结果。这种方法可以让我们的候选基因进行短期或长期的操作，通过过表达或shRNA介导击倒。此外，我们可以在现有的转基因小鼠，观察气味受体种群的合作本地化荧光标签单OSNs。显微注射是必需的慢病毒转导OSNs。我们曾试图进入鼻腔冲洗慢病毒悬浮导OSNs没有任何成功。因此，我们执行microinjections通过背鼻子表面，以提供病毒的OE OSN的细胞?...

显微注射:新生小鼠显微注射使用5μL注射器汉密尔顿（汉密尔顿＃7647-01）装有33号针头（汉密尔顿＃7762-06）。 免疫组织化学试剂:一抗:兔多克隆抗体的GFP（分子探针＃A - 6455），鸡对OMP的抗体（自定义）（陈等人，2005年），豚鼠对VGlut2多克隆抗体（Millipore公司＃AB2251）。二抗:CY2共轭驴抗兔（杰克逊Immunolab＃711-225-152），共轭Cy3标记的驴抗鸡（杰克逊Immunolab＃703-165-155）和Cy5标记的山羊抗豚鼠（杰克逊Immunolab＃106-175-008）。被安装在玻片Fluoromount G（南方生物技术＃0010-01）50ng/mL的DAPI染色染色解决方案。 共焦成像:Z - Stack的图像，使用奥林巴斯Flouview FV1000共聚焦显微镜，到使用奥林巴斯FV10 - ASW的2.01焦采集和分析软件的3D预测收集和处理的图像。

lentivirus-mediated genetic manipulation and visualization of olfactory sensory neurons in vivo

一个精确的嗅觉电路的发展依赖于嗅觉的感觉神经元（OSN）轴突在嗅球（OB），突触目标准确的预测。 OSN的轴突的生长和定位的分子机制不能很好地理解。操纵基因的表达和随后的单一的OSN的轴突和他们的终端乔木形态的可视化迄今已具有挑战性。要在规定的时限内的单细胞水平上研究基因的功能，我们开发了基于慢病毒的技术，操纵在体内OSNs的基因表达。慢病毒颗粒，通过显微注射到嗅上皮（OE）的OSNs。表达盒，然后永久地整合到转OSNs基因组。绿色荧光蛋白表达确定感染OSNs，并概述了他们的整个形态，包括轴突终端乔木。由于显微注射和记者检测之间的周转时间短，基因功能研究，可以集中在一个很窄的发展时期。使用这种方法，我们三天内检测GFP的表达，只要3个月以下注射。我们已经取得的过度表达和shRNA慢病毒显微注射介导击倒。这种方法提供了详细的形态在体内的单个细胞水平OSN胞体和轴突，从而使候选基因在嗅觉发展功能特性。

Watch this Scientific Journal Video about Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo at JoVE.com

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

我们提出了一种慢病毒基因操纵和单一的嗅觉感官神经元轴突的可视化技术及其终端树枝状<em&gt;在体内</em&gt;。

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 ...

lentivirus-mediated-genetic-manipulation-visualization-olfactory

Research

JoVE Journal

Neuroscience

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

15.6K 次观看.  University of California, Davis. 我们提出了一种慢病毒基因操纵和单一的嗅觉感官神经元轴突的可视化技术及其终端树枝状在体内。

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

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

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. ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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 ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

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 ...

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.

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Subtitles

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