这项工作得到了国家耳聋与其他交流障碍研究所院内研究司。 NIDCD的5R01 DC007613提供了额外的支持。

1。囊解剖和文化<ol><li>安乐死的成年人（4周龄以上）鼠标使用批准的协议和斩首。 </li><li>剪掉头部两侧外耳道，并向前拉往鼻子的皮肤。开张头从后到前，消除双方的大脑揭示骨迷路。 </li><li>远离骨耳蜗和修剪颅骨骨迷路（包括泡）转移到组织文化引擎盖配备在解剖显微镜（注:腺病毒的工作应在II级生物安全柜中进行）。 </li><li>在引擎盖，每个耳蜗转移到一个35mm的组织培养皿含无菌夹层动脉瘤媒体（M199，GIBCO / Invitrogen公司＃12350）（ 图1A）。 </li><li>如果听泡仍然完好无损（这是可能的总清扫过程中消除的大疱，大疱和的顶点之间插入缩略图耳蜗），用两个镊子（一＃3和一个＃5）打破泡（ 图1B）。 </li><li>确定了骨耳蜗准备的标志:顶点，基地，听小骨，椭圆形和圆形的窗户，VIIIth神经根，半规管（ 图1中的C，D）。 </li><li>内侧的一面朝上，这样，椭圆形和圆形​​的窗户和APEX的菜和VIIIth的神经根底部朝上放置在盘子的内耳。 ＃3钳使用中的半规管（ 图1D）地区举行的筹备。 </li><li>使用手术刀配备＃11刀片，切断耳蜗的顶点正好顶到神经根（ 图1D）。 </li><li>关闭的准备，使切割部分朝上。作为手柄使用的前半规管，以稳定的准备（ 图1E）。 </li><li>使用＃5镊子，取出耳蜗的蜗轴下来该基地。在耳蜗钩地区变为向下的地步，使用细探针（一半断了＃55钳）解除骨骨螺旋叶片（ 图1F）最后货架，揭示了球囊。下方的球囊是镫骨足板，这是一个重要的里程碑。囊是紧邻的镫骨足板（ 图1G），它是由骨包围。使用探针芯片骨远足以显示，约1/3的囊。小心取出囊使用＃55钳（ 图1H）。此过程通常会导致去除色素屋顶上皮的囊骨的准备拉。但是，如果囊被拆除屋顶上皮完整，屋顶可以使用＃55钳去除。 </li><li>椭圆囊腺病毒感染（或实时成像实验）必须有夹层（感染前）拆除期间的耳石。在这种情况下，用鲁尔锁注射器，充满了解剖媒体和装有一个小口径的针（通常是26-28计）。持有，使用＃55钳（ 图1I）的边缘囊。使针尖接近囊针斜面朝下。用解剖媒体吹掉耳石（ 图1J）的流。另外，耳石可以去除刷牙他们使用睫毛工具（特德·佩拉，公司＃113）。椭圆囊，都不会接受腺病毒感染通常是在24孔组织培养板中培养的自由浮动的耳石完整（修复后耳石去除，这是比较容易见下文）。 </li><li>一旦所有的椭圆囊解剖，解剖媒体无菌培养基:DMEM培养基F12（Gibco公司＃11320）补充5％FBS和50 U / ml的青霉素G（Sigma公司）。 </li><li>在37°C和5％CO 2培养椭圆囊。如果文化是mainta独立非执行董事为超过48小时，一半的培养基应改为隔日。文化可以维持一个星期或更长的时间。 </li></ol> 2。支持细胞的腺病毒感染重要的是:工作与腺病毒需要生物安全等级2（BSL2）的程序和认证。与你的机构的生物安全主任检查的BSL2程序上的指导和培训。 <ol><li>解剖椭圆囊如上在夹层媒体（无血清）。如上取出耳石。 </li><li>当准备感染，1囊（毛细胞）转移到每个井1 NUNC迷你托盘（费舍尔＃12-565-68）含有15μL 无血清培养液F12（这是很重要的，因为血清灭活病毒） 。这种转移是容易与1.5毫米microcurette的（＃10082-1​​5从精细的科学工具）。 </li><li>我们的AD-GFP是5型与删除病毒E1和E3基因和人类CMV启动子驱动transg烯（GFP）。这种病毒取自，矢量BioLabs公司（费城，PA的<a href="http://www.vectorbiolabs.com/">vectorbiolabs.com</a> ）。股票病毒滴度在1.0-4.0×10 10 PFU / ml的。新增0.5-2股价病毒液，每口井含有囊（ 图1L）。实际使用的金额不等，取决于病毒和股票滴度。我们通常感染1.0-4.0×10 7 PFU每个囊。 </li><li>文化中的椭圆囊病毒的媒体在37°C / 5％CO 2小时2。 2小时后，转移的椭圆囊含血清培养基的24孔培养板。文化的椭圆囊在37°C / 5％CO 2过夜。 </li><li>可第二天椭圆囊实时成像实验，他们可以为毛细胞的免疫组织化学固定（见下文）。病毒基因表达增加，随着时间的推移，因此，如果转基因表达低，在感染后24小时有利于培养细胞在含血清媒体一个额外的24小时。如果你看到毛细胞毒性，减少使用量的病毒。 </li></ol> 3。囊固定，耳石去除，免疫组织化学<ol><li>在培养周期结束，固定在4％多聚甲醛的椭圆囊（在摇椅平台）是3小时室温或4℃过夜洗椭圆囊（3次，每次15 min）0.1米（1X）索伦森的磷酸盐缓冲液pH值7.4。 24孔培养板中执行这些步骤。 </li><li>耳石清除: 注:耳石腺病毒感染前必须拆除（见上面的步骤1.11）。然而，是不是被感染的椭圆囊，可以培养和固定与耳石仍完好无损。在这种情况下，使用这里描述的步骤去除固定后的耳石。检查任何剩余的色素屋顶上皮椭圆囊。小心取出任何剩余的屋顶上皮使用两个＃55 Forceps。要删除的耳石，取代索伦森的磷酸盐缓冲750-1000微升的Cal-，前decalcifier（费舍尔＃CS510-1D）。离开椭圆囊加州防爆为1分40秒（不超过2分钟）。取代0.1米索伦森的磷酸盐缓冲液洗（5次，每次5 min）CAL-EX。 </li><li>孵育10分钟的椭圆囊在硼氢化钠（西格玛＃452882，去离子水的1％）。在0.1米索伦森的磷酸盐缓冲液（5次，每次5 min）洗椭圆囊。 </li><li>椭圆囊封锁的解决方案的地方（PBS + 2％牛血清白蛋白+ 0.8％正常山羊血清+ 0.4％的Triton X-100）在室温下为3小时摇椅平台上。 </li><li>新增主要抗体孵育过夜4°C。抗肌球蛋白7A（要么变形虫Biosciences公司25-6790＃或＃MYO7A发展研究杂交瘤细胞银行138-1）1:100封闭液标签所有的毛细胞。分别标注striolar和非striolar的毛细胞，我们已经使用双标签protocol与单克隆抗钙调素（西格玛＃C 3545; 1:150）和抗钙结合蛋白（Chemicon公司＃AB1778，特曼库拉，美国加利福尼亚州; 1:200）。 （通常的Alexa Fluor从Invitrogen公司，卡尔斯巴德，美国加利福尼亚州的共轭抗体）二级抗体封闭液1:500稀释，并在室温摇椅平台上4个小时在黑暗中培养。 </li><li>山椭圆囊上使用Fluoromount-G的（南方生物技术，伯明翰，AL，美国）的玻璃幻灯片。 </li></ol> 4。代表结果使用这种方法保留完整的椭圆囊培养补充的毛细胞和支持细胞（ 图2）。健康文化的毛细胞，四周薄，肌球蛋白7A阳性细胞质地区的全面核型材（ 图2，顶部面板）。支持细胞（反SOX2标记）是较小的和较稠密的毛细胞（ 图 2，下部面板）的包装。阿德novirus感染囊支持细胞的25-50％，无毛细胞被感染（ 图2，AD-GFP板）。应当指出，每个腺病毒滴度的最佳工作必须凭经验确定，因为毛细胞死亡是可能的，如果病毒滴度过高。此外，机械损伤（期间造成的夹层）的地区，将占用大量的病毒。这些地区的机械损伤的细胞GFP的表达和缺失的毛细胞非常高的水平不断线很容易区分。相反，这是一个完好的文化支持细胞（ 图2）分散的绿色荧光蛋白表达。 <img src="/files/ftp_upload/3734/3734fig1.jpg" alt="图1"/> 图1。囊解剖。答:听泡。（AB）乙:使用两个坚固的镊子，泡坏了:骨耳蜗（RW =圆窗，下=镫骨动脉，耳蜗，ST =镫骨）D:手术刀刀片（SB），用来切断的VIIIth颅神经（8 日 N）的升序=前半规管 é耳蜗的顶端的顶点。用一把叉子插入前半规管（ASC）和最外层骨（箭头）＃3钳牢牢准备。使用镊子去除膜迷路楼:删除膜迷路，骨螺旋叶片的基础又是附近的钩地区可见。罚款探针插入下面这骨货架解除货架和删除（箭头）G:拆除后的球囊，椭圆囊（U）是立即可见相邻的镫骨足板（SF）的H:椭圆囊（美）被删除＃55钳（箭头）SF =镫踏板 我:。椭圆囊耳石（O）的部分删除记者:耳石（海外），D感觉上皮（SE）可见下从囊的使用装用26号针头注射器提供的流媒体。针（）的影子是在图像的底部可见K:椭圆囊耳石删除和感觉上皮（SE）可见L:腺病毒感染与每个囊（U）是放置在一个小型的个人托盘。支持细胞吹打成井（P值吸取尖）病毒与腺病毒感染。 <img src="/files/ftp_upload/3734/3734fig2.jpg" alt="图2"/> 图2。推动绿色荧光蛋白表达的腺病毒（AD-GFP）感染腺病毒介导的支持细胞的感染。椭圆囊。所示的毛细胞层和支持细胞层在同一地区的一个囊的共聚焦图像。毛细胞标记抗体对肌球蛋白7A（洋红色）。支持细胞标记抗体对SOX2（红色）。示意图显示囊感觉上皮细胞和毛细胞的位置（HC）和支持细胞（SC）的结构。聚焦（光）在上（U）和较低的显示部分的位置（L），板在原理图中虚线表示。上面板:在水平头发细胞核采取的共聚焦图像，AD-GFP信号出现在毛细胞之间的空隙，不重叠的毛细胞标记肌球蛋白7A。较低的面板:聚焦部分在支持细胞核的水平，AD-GFP信号与支持细胞标记SOX-2共定位。 AD-GFP感染的结果，在GFP的表达仅在支持细胞，无毛细胞被感染。 <img src="/files/ftp_upload/3734/3734fig3.jpg" alt="图3"/> 图3。腺病毒感染不会导致 UT 毛细胞或支持细胞的死亡。在4×10 8 PFU / ml的AD-GFP感染ricles。椭圆囊标记与抗肌球蛋白7A（毛细胞标记）和Sox2（支持细胞标记）抗体。控制椭圆囊和AD-GFP感染的椭圆囊毛细胞和支持细胞计数平等。

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supporting+cells+eliminate+dying+sensory+hair+cells+to+maintain+epithelial+integrity+in+the+avian+inner+ear.">Supporting cells eliminate dying sensory hair cells to maintain epithelial integrity in the avian inner ear.</a> J. Neurosci. 30, 12545-12556 (2010).</li><li>Sato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+fractalkine+receptor+CX3CR1+on+cochlear+macrophages+influences+survival+of+hair+cells+following+ototoxic+injury.">Expression of fractalkine receptor CX3CR1 on cochlear macrophages influences survival of hair cells following ototoxic injury.</a> J. Assoc. Res. Otolaryngol. 11, 223-234 (2010).</li></ol>

没有利益冲突的声明。

感觉毛细胞受到的各种压力，包括老龄化，噪声外伤，和接触，包括氨基糖甙类抗生素和抗癌药顺铂的耳毒性药物引起的死亡。在哺乳动物毛细胞，导致永久性听力丧失和/或平衡紊乱导致死亡。 在体外模型系统研究旨在确定的细胞和分子机制的毛细胞死亡，以及那些旨在防止或扭转毛细胞死亡的关键工具。不同于成年哺乳动物耳蜗毛细胞，毛细胞的囊良好的生存文化。囊毛细胞是敏感的?...

A correction was made to <a href="http://www.jove.com/video/3734">Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection</a>. There was an error with a figure, the authors middle names were omitted, and the acknowledgements were updated.

Erratum: Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;试剂</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品目录号</strong</td&gt;</tr&gt;<tr&gt;<td&gt;中等199</td&gt;<td&gt; Gibco公司/ Invitrogen公司</td&gt;<td&gt; 12350</td&gt;</tr&gt;<tr&gt;<td&gt;的DMEM/F12</td&gt;<td&gt; Gibco公司/ Invitrogen公司</td&gt;<td&gt; 11320</td&gt;</tr&gt;<tr&gt;<td&gt;精细镊子（＃55）</td&gt;<td&gt;精细的科学工具</td&gt;<td&gt; 11255-20</td&gt;</tr&gt;<tr&gt;<td&gt;精细镊子（＃5）</td&gt;<td&gt;精细的科学工具</td&gt;<td&gt; 11252-30</td&gt;</tr&gt;<tr&gt;<td&gt;钳（＃3）</td&gt;<td&gt;精细的科学工具</td&gt;<td&gt; 11231-30</td&gt;</tr&gt;<tr&gt;<td&gt;青霉素G</td&gt;<td&gt;西格玛</td&gt;<td&gt; P3032</td&gt;</tr&gt;<tr&gt;<td&gt;胎牛血清</td&gt;<td&gt; Invitrogen公司</td&gt;<td&gt; 10082-1​​39</td&gt;</tr&gt;<tr&gt;<td&gt; NUNC小托盘</td&gt;<td&gt;费舍尔</td&gt;<td&gt; 12-565-68</td&gt;</tr&gt;<tr&gt;<td&gt;腺病毒GFP</td&gt;<td&gt;矢量Biolabs公司</td&gt;<td&gt; 1060 *</td&gt;</tr&gt;<tr&gt;<td&gt;加州前decalcifier</td&gt;<td&gt;费舍尔</td&gt;<td&gt; CS510-1D</td&gt;</tr&gt;<tr&gt;<td&gt;硼氢化钠</td&gt;<td&gt;西格玛</td&gt;<td&gt; 452882</td&gt;</tr&gt;<tr&gt;<td&gt;抗肌球蛋白7A（多克隆）</td&gt;<td&gt;变形Biosciences公司</td&gt;<td&gt; 25-6790</td&gt;</tr&gt;<tr&gt;<td&gt;抗肌球蛋白7A（单克隆）</td&gt;<td&gt;发展研究杂交瘤细胞银行</td&gt;<td&gt; MYO7A 138-1</td&gt;</tr&gt;<tr&gt;<td&gt;抗-钙调蛋白</td&gt;<td&gt;西格玛</td&gt;<td&gt;ç3545</td&gt;</tr&gt;<tr&gt;<td&gt;反，钙结合蛋白</td&gt;<td&gt; Chemicon公司</td&gt;<td&gt; AB1778</td&gt;</tr&gt;<tr&gt;<td&gt; FluoromountĞ</td&gt;<td南方生物技术公司</td&gt;<td&gt; 0100-01</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"<strong&gt;表1</strong&gt;。在囊文化和腺病毒感染的试剂和使用的工具。</p&gt;<p class="jove_content"&gt; *注:通常是由AD-GFP载体Biolabs公司提供股票滴度在有1x10<sup&gt; 10</sup&gt; PFU / ml的。我们要求定制，以提供股票病毒滴度为1.2×10放大<sup&gt; 11</sup&gt; PFU / ml的。</p&gt;

dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection

听力损失和平衡紊乱往往造成mechanosensory毛细胞是内耳的受体细胞死亡。由于没有细胞行圆满代表了哺乳动物的毛细胞，毛细胞的研究依赖于基层机关文化。最好具有成熟的哺乳动物毛细胞在体外模型系统，利用从成年鼠（ 图1）1-6椭圆囊器官培养。囊是前庭器官，以及椭圆囊毛细胞的听觉器官，Corti器毛细胞结构和功能相似。成年小鼠的囊准备成熟的感觉上皮细胞的生存，动态平衡，这些细胞死亡的分子信号调节的研究。 哺乳动物耳蜗毛细胞的终末分化，当他们失去了，不能再生。在非-MAM其次是强大的再生，恢复听力和平衡功能7，8，马里脊椎动物，听觉或前庭毛细胞死亡。再生毛细胞介导的胶质状的支持细胞，毛细胞的基底表面的接触中感觉上皮9，10。支持细胞是重要的调停毛细胞生存和死亡的11。我们最近开发出一种技术，支持在使用培养椭圆囊腺病毒的细胞感染。利用腺病毒5型（dE1/E3）提供含有CMV启动子控制下的绿色荧光蛋白的转基因，我们发现，腺病毒特别有效地支持细胞的感染。支持细胞的感染效率约为25-50％，并没有感染毛细胞（ 图2）。重要的是，我们发现，不支持细胞的腺病毒感染导致毛细胞或支持细胞的毒性，因为在AD-GFP INF细胞计数ected椭圆囊相当于在非疫区的椭圆囊（ 图3）。因此，在支持细胞培养椭圆囊腺病毒介导的基因表达提供了一个功能强大的工具，研究支持细胞作为调解人毛细胞生存，死亡和再生的作用。

Watch this Scientific Journal Video about Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection at JoVE.com

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

mechanosensory毛细胞是内耳的受体细胞。最好的特点<em&gt;在体外</em成熟的哺乳动物毛细胞模型系统利用从成年鼠的椭圆囊器官培养。我们目前的成年小鼠的囊的剥离，我们证明腺病毒介导的支持细胞在体外培养椭圆囊的感染。

解剖成年小鼠的椭圆囊和腺病毒介导的支撑细胞的感染

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear.  Since there ...

dissection-adult-mouse-utricle-adenovirus-mediated-supporting-cell

Research

JoVE Journal

Neuroscience

23.6K 次观看.  Medical University of South Carolina. mechanosensory毛细胞是内耳的受体细胞。最好的特点在体外

视频: Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

科研

神经科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

成年鼠脑铸造三维成像和形态分析

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

小鼠多巴胺和三维胶原基质检测纹状体外植体的解剖和文化

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

幼虫，蛹和成人的解剖和免疫组化<em&gt;果蝇</em&gt;视网膜

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

成年小鼠背根节外植体和分离细胞模型调查可塑性和对包括病毒感染在内的环境侮辱的反应

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

成年小鼠中的科利耳表面制备

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...