笔者想承认儿童研究所成像核心使用共聚焦显微镜和张健博士的建议，安装使用琼脂糖珠的部分。这项工作是由美国国立卫生研究院授予DC010387。

1。内耳的分离和固定<ol><li>在Corti器的蜂窝图案分析，安乐死适当上演胚胎或成人。 </li><li>解剖奥迪胶囊，含有膜的内耳。鼠标，可以轻松完成对胚胎的胚胎（五）14.5天以上，其中E0.5检测阴道塞的一天。解剖奥迪胶囊是第一个完成斩首和开放的头骨中线。移除大脑揭露每个耳朵（图1A）。内耳可掏出完整的使用镊子（如杜蒙＃5，图1B）。如果成像Corti器，小心去除与奥迪胶囊相关的所有神经细胞和结缔组织是没有必要的。 </li><li>修复整个内耳由4％多聚甲醛浸泡在温和的摇摆（PFA）4 ° C 1.8毫升螺丝帽NUNC小瓶冷冻管，或其他适当大小的容器中过夜。煤灰的解决方案应该是在2 0 DDH取得了新的，然后储存在-20 ° C，直到需要。 </li><li>第二天，洗出三个1X磷酸盐的变化固定液缓冲液（PBS）。组织可存储天至数月，直到准备进行处理，在无菌1X PBS。成人组织，除垢在10％EDTA（0.27 M）在4 ° C为5天，其次是在1X PBS冲洗和存储。 </li></ol> 2。切割vibratome节<ol><li>准备在1X PBS 4％低熔点琼脂糖。添加低熔点琼脂糖1X PBS和微波，直到琼脂糖溶液中。该解决方案不泡过的护理 - 利用微波定期和漩涡混合。保持在55℃水浴，直到准备使用的解决方案。 </li><li>使用镊子，取出内耳1X PBS液和一个剥离一个双向嵌入模具的底部的位置。吸取了任何多余的1X PBS从模具。填充模具用大量液体4％低熔点琼脂糖（图2A），以支付组织。 </li><li>内耳切片在适当的平面位置。我已获得逼近奥迪胶囊的位置，如果它一直在原地留在头部，并在横向平面切片Corti器的横截面。 E14.5胚胎到成人，位置，以便它视为横向与外侧的半圆形运河可见（图2B），内耳。角的内耳使奥迪胶囊后形成的线大约是在一个45度角，从切片（图2A，B）的拟平面。当奥迪胶囊被认为奥迪胶囊背部分是在顶部，耳蜗底回应位于底部（箭头，图2b）和前半规管（ASC）在顶部（图2B）。 </li><li>琼脂糖将设置在室温下30分钟内。转移剥走模具含有包埋组织，以4℃，直到准备第。如果切片将被推迟到第二天，加入1X PBS覆盖在模具和存储在一个潮湿的纸毛巾浸泡在1X PBS框琼脂糖。 </li><li>剥离的塑料模具。使用刀片，切出琼脂糖框，其中包含的内耳组织。照顾，以确保截面直和平行块的对面，这将是表面上粘琼脂糖块。由于琼脂糖不渗透的组织，不从周围的组织修剪过多琼脂糖，这样的组织将是很好的支持。 </li><li>使用强力胶块切割面。 </li><li>在第40至100微米的厚度vibratome。与1X PBS，0.1％TritonX - 100填补了组织培养皿中保存的部分。一个有代表性的部分是通过耳蜗螺旋图所示。 2C。 </li><li>使用镊子，解剖远离组织琼脂糖。琼脂糖不会穿透组织，但也只是坚持到内耳的外表面。 </li><li>如果需要，池琼脂糖，切片组织一个大洞的开放使用吸管，以防止组织过度剪切。如果执行多个不同组织的多种抗体的污渍，部分可以在16 - 或24孔组织培养皿举办。 </li></ol> 3。抗体染色vibratome部分继续与抗体染色。在整个安装过程部分，在溶液中自由浮动。下面是一个例子Corti器的支柱细胞S100的标记抗体染色协议，但该协议可以用于任何抗体。除非另有说明，所有的清洗是在室温下进行。 <ol><li>在一个多井的组织培养皿中，PBT温和的摇摆（1X PBS，1％的牛血清白蛋白，0.1％Triton X一100）孵育30分钟的自由浮动的部分。 </li><li>吸取关闭旧的解决方案和块自由FLoating节30分钟的温和的摇摆PBT +正常山羊血清（50μL农工商超市，PBT / 1毫升）。 </li><li>吸取关闭旧的解决方案，并添加在PBT +农工商超市（PBT +农工商S100的1:200稀释）稀释抗体。在4℃孵育过夜彗星与温和的摇摆。 </li><li>吸取过初级抗体溶液。洗3倍，5分钟。与PBT，然后4X，30分钟。与PBT。 </li><li>座与PBT +农工商超市，30分钟。 </li><li>孵育与荧光标记的二抗稀释PBT +农工商超市（山羊提出）。孵育过夜，4 ° C，或者几个小时，放疗，温和的摇摆。 </li><li>吸取过二次抗体溶液。洗3倍，5分钟，然后4X，30分钟。与PBT。 </li><li>关闭最后PBT吸取清洗和适当的水安装包含核染液的介质更换。 </li></ol> 4。安装在幻灯片上部分和共聚焦显微镜为了防止盖玻片压碎厚，安装个别路段时，一个间隔必须在显微镜载玻片和盖玻片之间插入。下面是两个替代方法安装厚vibratome部分： <ol><li><ol type="a"><li>在广场周围的组织切片置于真空润滑脂的边缘，可以创建一个间隔。因此，当放下盖玻片，盖玻片边缘将密封的真空润滑脂。按盖玻片边缘用拇指到幻灯片上，坚定不移地坚持它。如果周围盖玻片边缘整齐，均匀，是目前真空润滑脂，幻灯片将被封闭，足够为一个倒置的共聚焦显微镜的使用。 </li><li>另外，可以创建一个间隔使用已知的网目尺寸的琼脂糖层析树脂（如阿菲 - 凝胶蓝胶，Bio - Rad公司）。吸取1μL树脂到每个将被放置盖玻片的四个角落的显微镜幻灯片。吸取之一的组织切片和树脂的四个点中安装媒体。盖玻片。如果使用的是一个倒置的共聚焦显微镜，密封的显微镜玻片上，盖玻片，使用指甲油。 </li></ol></li><li>获得使用共聚焦显微镜切片染色vibratome的光学部分。为了量化Corti器内的各种细胞类型，在定义的步长（1.5至3微米）获得一个Z - ​​Stack的。部分应该反染核染料（步骤3.8）。共聚焦图像采集后的任何电脑屏幕上的图像可以检查。细胞可以算作手动图像观察原子核的外观和失踪审议通过Z - Stack的顺序。 </li></ol> 5。代表性的成果： 一个代表共聚焦的Z - Stack通过vibratome的部分，S100蛋白，这是目前在支柱产业和Deiters细胞，核染色（图3A - F）的counterstained染色。每个小组在3微米的步骤采取的共聚焦图像，与面板的一个共聚焦图像获得最接近vibratome节和面板F的共聚焦图像vibratome节内获得最内部表面。 Corti器的形态出现最小中断。特别是，支柱细胞的细胞质扩展不破。指出，通过在Z - Stack（图3A至3F）的外观和支柱细胞核消失，无论是内部和外部的支柱细胞数量可算（见编号，图3A - F）。由于少数每人共聚焦图像的细胞类型，这种方法可以用来计数Corti器内的大多数细胞类型。 <img src="/files/ftp_upload/2793/2793fig1.jpg" alt="图1"/> 图1。完好，周15岁，成人内耳在奥迪胶囊包裹。 （一）正确的滴耳胶囊（括号内）包裹在颅骨，从内部头部的制高点看，大脑取出后。前路是正确的。 （二）完整，15周龄后，从头骨解剖内耳。左侧内耳侧面可见;右内耳的内侧表面可见。标签表明前半规管（ASC），合作（耳蜗），OW（卵圆窗）的位置。 <img src="/files/ftp_upload/2793/2793fig2.jpg" alt="图2"/> 图2：（一）所有在琼脂糖嵌入在剥离的模具中心，从15周龄成年内耳。拟截面表示。 （b）关闭图像嵌入到前15周龄，整个内耳。周围骨的一部分已被解剖了，所以膜迷路可以更清楚地查看。被跟踪的横向半圆形运河（LSC），前半规管（ASC），和耳蜗（CO）（白色曲线）。耳蜗（箭头）基转，并打算平面部分（虚线）表示。 （三）代表的CROS -通过耳蜗。在一个截面通过人工耳蜗管，尔蒂（OC）的器官是盒装，血管纹（SV）括号内，Reissner变分的膜（RM）表示。 <img src="/files/ftp_upload/2793/2793fig3.jpg" alt="图3"/> 图3。尔蒂在野生型小鼠在P21的器官。 （AF）的顺序焦系列，在3微米步长，通过一个S100抗体染色Corti器，可视化的支柱细胞和Deiters，“细胞。从开始到结束的一系列内部和外部的支柱细胞计数的编号。缩写：IHC（内毛细胞），aIHC（相邻的内毛细胞），OHC（外毛细胞），PC（支柱细胞），DC（Deiters&#39;细胞）。

<ol>
	<li>Lim, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+structure+of+the+organ+of+Corti:+a+review.">Functional structure of the organ of Corti: a review.</a> Hear Res. 22, 117-146 (1986).</li><li>Raphael, Y., Altschuler, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+innervation+of+the+cochlea.">Structure and innervation of the cochlea.</a> Brain Res Bull. 60, 397-422 (2003).</li><li>Chung, W. S., Shin, C. H., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmp2+signaling+regulates+the+hepatic+versus+pancreatic+fate+decision.">Bmp2 signaling regulates the hepatic versus pancreatic fate decision.</a> Dev Cell. 15, 738-748 (2008).</li><li>Sun, X., Mariani, F. V., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+FGF+signalling+from+the+apical+ectodermal+ridge+in+limb+development.">Functions of FGF signalling from the apical ectodermal ridge in limb development.</a> Nature. 418, 501-508 (2002).</li><li>Trinh, L. A., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+regulates+epithelial+organization+during+myocardial+migration+in+zebrafish.">Fibronectin regulates epithelial organization during myocardial migration in zebrafish.</a> Dev Cell. 6, 371-382 (2004).</li><li>Yin, C., Kikuchi, K., Hochgreb, T., Poss, K. D., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand2+regulates+extracellular+matrix+remodeling+essential+for+gut-looping+morphogenesis+in+zebrafish.">Hand2 regulates extracellular matrix remodeling essential for gut-looping morphogenesis in zebrafish.</a> Dev Cell. 18, 973-984 (2010).</li><li>Shim, K., Minowada, G., Coling, D. E., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sprouty2,+a+mouse+deafness+gene,+regulates+cell+fate+decisions+in+the+auditory+sensory+epithelium+by+antagonizing+FGF+signaling.">Sprouty2, a mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by antagonizing FGF signaling.</a> Dev Cell. 8, 553-564 (2005).</li></ol>

IACUC威斯康星医学院规定的准则和法规的规定进行了动物实验。

vibratome切片，免疫组织化学和激光共聚焦成像的过程，允许尔蒂与器官组织的损伤最小的可视化。显然，从内部在vibratome部分地区采取的共聚焦图像显示对细胞的形态，只有通过固定工件有限的优秀保存。接近两个削减了vibratome部分表面的共聚焦图像往往无法区分来自内部区域的图像，虽然偶尔蜂窝中断，如在休息的支柱细胞的细胞质扩展，观察（未显示）。这种方法很简单，主要设备的物品 - ...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>徕卡VT1000S振动刀片切片机</td><td>徕卡</td><td></td><td></td></tr><tr><td>蔡司LSM510激光扫描共聚焦显微镜</td><td>蔡司</td><td></td><td></td></tr><tr><td>学生杜蒙＃5镊子</td><td>精细科学的工具</td><td> 91150-20 </td><td></td></tr><tr><td>超纯低熔点琼脂糖</td><td> Invitrogen公司</td><td> 16520-050 </td><td></td></tr><tr><td>剥离一个双向嵌入模具</td><td> Polysciences </td><td> 18986或18646A </td><td></td></tr><tr><td>牛血清白蛋白</td><td>杰克逊ImmunoResearch </td><td> 001-000-162 </td><td></td></tr><tr><td>山羊血清</td><td> Invitrogen公司</td><td> 16210-064 </td><td></td></tr><tr><td> S100 </td><td> Dako公司</td><td> Z0311 </td><td></td></tr><tr><td>的Alexa Fluor 568山羊抗兔IgG </td><td> Invitrogen公司</td><td> A - 11036 </td><td> 1:1000稀释</td></tr><tr><td> Vectashield </td><td>媒介实验室</td><td> H - 1000 </td><td></td></tr><tr><td> YO - PRO - 1 </td><td> Invitrogen公司</td><td> Y3603 </td><td></td></tr><tr><td>真空润滑脂</td><td>费舍尔</td><td> S41718 </td><td></td></tr><tr><td>阿菲 - 凝胶蓝胶</td><td> Bio - Rad公司</td><td> 153-7301 </td><td></td></tr></tbody></table>

vibratome sectioning for enhanced preservation of the cytoarchitecture of the mammalian organ of corti

尔蒂哺乳动物器官是一个高度有序的mechanosensory头发和nonsensory支持细胞的细胞镶嵌 （在1,2审查）。可视化的这种细胞的马赛克往往需要Corti器的横截面。特别是nonsensory支柱和Deiters细胞，其细胞核位于毛细胞基部，不能无交叉切片Co​​rti器的可视化。然而，细腻尔蒂哺乳动物器官的细胞结构，包括罚款的支柱和Deiters细胞的胞质突起，是难以保持常规病理过程，如石蜡及冷冻切片，这是与标准的免疫组织化学染色技术兼容。 在这里，我描述了一个简单而强大的程序，包括耳蜗，在整个装载这些vibratome部分的免疫组织化学染色，共聚焦显微镜，切片vibratome。这个程序已经广泛用于多个器官，包括鼠标肢芽，斑马鱼肠道，肝，胰，心（见3-6选定的例子）的immunhistochemical分析。此外，此过程成像和quantitificaton支柱细胞在胚胎和成年小鼠7尔蒂突变和控制机关数sucessful。然而，这种方法是目前没有被广泛用来研究哺乳动物器官的尔蒂。在此过程中的潜在的罚款尔蒂成人器官的细胞结构都提供了增强的保存和允许各种细胞类型的量化描述。

Watch this Scientific Journal Video about Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti at JoVE.com

Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti

一个简单的程序，切片，免疫组织化学和激光共聚焦显微镜Corti器的vibratome描述。此过程允许的罚款尔蒂哺乳动物器官的细胞结构的改善保存，因此可以准确定量的细胞类型。

Vibratome切片增强保尔蒂哺乳动物器官的细胞构筑

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of ...

vibratome-sectioning-for-enhanced-preservation-cytoarchitecture

Research

JoVE Journal

Neuroscience

24.2K 次观看.  Medical College of Wisconsin. 一个简单的程序，切片，免疫组织化学和激光共聚焦显微镜Corti器的vibratome描述。此过程允许的罚款尔蒂哺乳动物器官的细胞结构的改善保存，因此可以准确定量的细胞类型。

视频: Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

在哺乳动物的中枢神经系统视神经横断后的实验操作方法

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

科研

神经科学

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

在犁鼻器官的切片准备工作表示一个基因编码的钙传感器成像神经元的反应

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

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 ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

的vibratome切片小鼠视网膜准备感光文化

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

扁平哺乳动物皮层皮质模块的可视化研究

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial companies have devised complex section transfer systems using fluid water. To simplify this technology, we created a simple method using homemade equipment that combines cutting and floating within a simple thermostatic chamber; therefore, the sections automatically enter the water bath on the water surface. The hippocampus from adult mouse brains, adult mouse kidneys, embryonic mouse brains, and adult zebrafish eyes were cut using both conventional paraffin sectioning and the presented method for comparison. Statistical analysis shows that our improved method saved time and produced higher quality sections. In addition, paraffin sectioning of a whole specimen in a short time is easy for junior operators.

Here, we present a protocol to improve paraffin sectioning. This method combines cutting and floating using a simple thermostatic chamber to avoid the transfer process required by the conventional method. As a result, the efficiency and the number of intact paraffin sections were greatly improved.

切片中石蜡嵌入组织的切割和漂浮方法

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...