这项工作是由德意志研究联合会（WE849/16 1 / 2，KD和RW）的支持。我们感谢托马斯欧拉（Töbingen德国）软件量子点的光刺激。

以下说明假设实验者有一个基本的了解视网膜结构，膜片钳记录和双光子显微镜。建立和运行一个设置膜片钳和双光子成像系统的有用信息，请参阅文献[7-12]。 1。动物和组织准备<ol><li>保持鼠标暗适应至少3小时。在此期间，准备1-2升胞组成的解决方案（毫米）125 2.5氯化钠，氯化钾，氯化钙，1.6 MgCl 2的，25日10碳酸氢钠3，D -葡萄糖和平衡pH值7.4 carbogen毒气（5％CO 2在O 2）在室温下。 </li>一<li> 二氧化碳过量颈椎脱位，安乐死在气密腔的鼠标。此过程，并应在黑暗中进行以下步骤。使用昏暗的长波长的光照（我们阻止短的波长由690纳米longpas的冷光源的过滤器），以支持个人愿景，同时保持动物的眼睛暗适应（小鼠只有在光谱的红色部分视力不佳）。在完全黑暗的使用红外线照明（&gt; 800 nm的）的工作，并佩戴夜视镜。 </li><li> Enucleate眼用一双弯曲的虹膜剪刀和他们转移到一碟放置在解剖显微镜下的细胞外液。 </li><li>开放与对春天的剪刀（我们第一次使用柳叶刀刺穿切割为起点）沿锯齿缘（视网膜及睫状体之间的边界）的眼球中取出的角膜和睫状体。取出镜头，仔细分离的视网膜色素上皮细胞。如果视网膜的方向是对你的学习至关重要，表明它像文献[12]。切断视神经和视网膜色素上皮之间，视网膜除去眼罩。注意视网膜表面的方向:卷曲的视网膜内的gangl离子电池的一面;外感光方。 </li><li>删除玻璃体视网膜内层表面轻轻拉出一个木制牙签援助。为了这个目的，使用了细胞外液的体积小，只是涵盖的视网膜。视网膜玻璃体支牙签，可以拖动离心关闭。然后，应用短切口，沿视网膜的周边，以方便扁平化的组织。 </li><li>转移到录音室视网膜感光的一面朝下，宣扬出去的玻璃底部（我们使用细刷）和固定的不锈钢尼龙串成帧。第二视网膜准备以同样的方式，并保持它在carboxygenated供日后使用外液暗适应。 </li></ol> 2。录音<ol><li>一个堂堂正正的激光扫描显微镜下安装在黑暗的录音室，并不断superfuse视网膜准备（不低于5毫升/公里n与细胞外液carboxygenated）加热到35 ° C。显微镜（还配备一个红外敏感CCD相机）坐落在一个吸震内的电子屏蔽法拉第笼的空气表。与非透明幕墙笼盖，继续在黑暗中准备。红色透明薄膜，我们还从电脑显示器屏幕关闭。 </li><li> 850-870 nm或更长的波长调谐红外激光，切换到锁模条件，并使用双光子激发可视化表达GFP的细胞。衰减激光的输出控制激光扫描软件在一定程度上的荧光细胞清楚地认识到，这只是足够使用中性密度过滤器</li><li>对于膜片钳记录，电流钳模式使用玻璃微量（我们用硼硅玻璃管材外径1.5毫米和0.225毫米壁厚）洋溢着细胞内组成的解决方案（毫米）125 K -葡萄糖酸，氯化钾10 0.5 EGT，10 HEPES，与氢氧化钾（给人一种吸管约5MΩ电阻）滴定至pH 7.4。请注意电压钳记录等其他实验条件下，需要不同的解决方案。如果需要注射染料，加入荧光探针（我们使用10毫米的Alexa Fluor 594）或示踪分子（3％Neurobiotin）。微管插入持有人，确保参比电极（氯化银线）与细胞外液在录音室接触。 </li><li>施加压力的微量和目标的GFP表达细胞（我们用40倍水浸泡的目的; NA 1.25）。坐落在神经节细胞层（直接在视网膜的电流方向表面之下）在内核层的近端部分（编制内深约55-75微米），以及无长突细胞组织。微量的靶细胞，内界膜（胶质endfeet鞘）在视网膜接触前表面有被击穿。是公认的成功渗透赶出从微管尖和分离从底层的视网膜组织内界膜红外线传输IR - CCD相机拍摄的图像上，可以观察细胞内的解决方案。 </li><li>办法从双光子图像进行比较SOMA的位置与图片的IR - CCD相机，记录微量的位置所需的细胞。请注意，这一步是不容易实现，需要一定的练习，因为绿色荧光蛋白表达细胞在确认以直接的微量红外线传输图像。正确的定位是实现时，微管尖端导致细胞表面可以看到在双光子图像的凹陷。替代此过程中，使用荧光染料在细胞内的解决方案（例如的Alexa Fluor 594，100微米），以揭示在双光子图像的微量位置。红外双光子激发允许足够的这种染料的荧光微量明显。为此，调整激光扫描软件，还包括红色荧光的检测范围。 </li><li>从微量释放的压力，并获得一个全细胞膜片钳配置在电流钳模式。一个可用的录音应该给的-50至-55 mV的膜电位和持续至少20分钟。 </li><li>目前创建一个刺激软件（我们使用，德国蒂宾根大学的托马斯欧拉，量子点），在计算机显示器上11的视觉刺激。调整要记录的细胞刺激的空间位置是:马克显示器上显示的IR - CCD相机和中心到它的一个斑点状的刺激传输图像的细胞位置。中性密度​​滤光片插入光路中的调整刺激强度。校准显示器的频谱和强度，见参考文献[14]。选择一个prolongeð刺激间隔（如15秒），以避免适应。包括一个光电二极管保持在光束路径上的刺激定时记录。 </li><li>启动刺激的协议，并记录光响应（我们使用与非尖峰细胞过滤5 kHz时为10 kHz的采样率;穗录音建议像更高的采样率20千赫）。使用光刺激软件触发录音软件。 </li><li>染料充填让前仔细回缩荧光剂扩散到30-45分钟的记录细胞从细胞的微管。小心不拉细胞体。如果使用非荧光示踪Neurobiotin，它需要通过有约束力的可视化链霉亲和共轭一个荧光团（我们在夜间使用链霉亲和素- Cy3标记，1:400 4 ° C，经过20分钟的视网膜多聚甲醛固定;染料耦合研究链霉亲和孵化，可以延长至3天）。另外，注射剂也可以进行与尖锐的电极（&gt; 100MΩ）当染料驱动iontophoretically刺穿细胞（矩形脉冲:0.25-1 NA，100毫秒，间隔100毫秒，总时间3-6分钟）。 </li></ol> 3。代表性的成果: 以下结果来自对鼠标的研究表达绿色荧光蛋白（GFP）启动子下，儿茶酚胺合成的限速步骤（TH:GFP的小鼠）的酶催化酪氨酸羟化酶 13,14 。基于GFP信号的两个不同的细胞群的亮度分辨（图2A）。细胞中表达的GFP水平较高，拥有位于内核层（INL，图2A），或在神经节细胞层（保利协鑫，图2B）流离失所的胞体和内网状层中的分层（图2C，彩光） 。他们被确定为2型细胞14-16的形态（图3）可以系统地进行研究，并选举rical活动（图4），即使它的人口密度仅为250细胞/毫米2。 <img alt="图1" src="/files/ftp_upload/3457/3457fig1.jpg" /> 图1。实验装置示意图。双光子激发（红色点缀光路）荧光团，表达完整的视网膜细胞，使视觉由微管（绿色发射光路）的目标。通过冷凝器显微镜（黄色光路）预计的光刺激，视网膜细胞对光反应记录。 <img alt="图2" src="/files/ftp_upload/3457/3457fig2.jpg" /> 图2。绿色荧光蛋白表达细胞在视网膜的一个TH flatmount:GFP的鼠标。可以区分两类人群的GFP信号的亮度:1型细胞（DA细胞）位于显示弱荧光（A，见箭头）的INL和强烈的标记2型细胞INL（A，见箭头）或在保利协鑫（B）和树突状分层阶层的IPL S3（三）在流离失所的细胞体。比例尺，50微米。 <img alt="图3" src="/files/ftp_upload/3457/3457fig3.jpg" /> 图3 2型细胞注射示踪Neurobiotin的形态。示踪剂后来可视化链亲和素- Cy3标记的绑定（洋红色）。的显微照片，这也是描绘的GFP信号（绿色），是一个覆盖GCL和IPL的形象栈的投影。比例尺，50微米。 <img alt="图4" src="/files/ftp_upload/3457/3457fig4.jpg" /> 图4。位于保利协鑫2型细胞对光反应。在暗的范围内增加强度的照明白光满场的响应模式。刺激强度是每秒每杆（RH * /棒/ S）photoisomerizations。用于更好的不同的是一个3秒的长时间刺激离子刺激发病响应元件（ON响应）和偏移量（OFF响应）。

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小鼠进行处理，并按照指引，对动物福利机构，由德国政府颁发的动物实验的法律安乐死。

这种方法提供了可能性研究的完整的视网膜视觉引导下的具体神经元的电性能，不影响视网膜adaptational条件。它特别适合于细胞，目前不佳而由于人口密度低，像大多数人口的无长突细胞研究的表征。双光子激发允许高分辨率和高对比度的影像，甚至较深部位的组织17，一个准确的定位的前提和成功的膜片钳细胞，特别是在其细胞体的高密度的INL。 孤立的小鼠视网膜是可行的实验条件下3-4小时。如果第二个视网膜下连续carbogen毒气完全黑暗中，它保留紫色的原色感光色素，可用于后整理与第一视网膜的实验。在开始的时候，针对选择与在视网膜wholemount微量细胞是一个挑战，需要一些练习，尤其是当在黑暗中工作。包括在微荧光染料，可以简化程序，因为细胞和微管是在同一时间的双光子激发下可见。然而，将组件添加到细胞内的解决方案可能阻碍gigaseal形成或导致录音的质量遭受。一旦成功地实现了，一个好的记录可以持续约1小时而红外激发光本身仅仅是视网膜感光细胞吸收不足，兴奋表达GFP的细胞发出的光在可见光谱部分。然而，荧光团很高兴，只有在一个小焦量，是不会改变视网膜adaptational条件。所有的多，激发只需要为目标，录制的光反应时，可以切换。 该usefulnESS是已经证明这种方法通过研究特定人群的无长突细胞14,18从电生理记录，否则会一直只能由事故12。最后，这个强大的技术，可以进一步扩展，包括药理学方法 14， 钙离子成像 19或使用注射的细胞免疫研究或电子显微镜。通过这种方式，一个特定类型的细胞在视网膜的电路的功能定位可以揭开。

<table border="1"><tbody><tr><th>试剂/设备</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>夜视镜</td><td> Gutzeit有限公司，Warthausen，德国</td><td> Xtron的F1 </td><td>包括一个800纳米光源</td></tr><tr><td>光学过滤器</td><td>肖特，美因茨，德国</td><td> RG9 </td><td>在690纳米，长通与截止过滤</td></tr><tr><td>虹膜剪刀</td><td>精细科学工具</td><td> 14061-09 </td><td>与22毫米刀片弯曲</td></tr><tr><td>春季剪刀</td><td>精细科学工具</td><td> 15000-00 </td><td>直3 mm的螺丝刀</td></tr><tr><td>录音室</td><td> Luigs诺依曼有限公司，德国拉廷， </td><td> 200-100 500 0180 A型（TC） </td><td>底部安装玻璃盖玻片聚四氟乙烯室（约0.15毫米; 200-100 500 0182号） </td></tr><tr><td>流量热水器</td><td>多声道系统，德国罗伊特林根， </td><td> PH01，TC01 </td><td>与温度小号可加热灌注导管恩索尔和温度控制器</td></tr><tr><td>激光扫描显微镜</td><td>徕卡显微系统</td><td>马克的LFS </td><td>徕卡共聚焦软件控制</td></tr><tr><td>空气球台</td><td>新港</td><td> VH 3036W - OPT </td><td></td></tr><tr><td>激光</td><td>光谱物理</td><td></td><td>海啸锁模钛蓝宝石激光</td></tr><tr><td> CCD相机</td><td> PCO公司，德国，Kelheim </td><td> PixelFly QE </td><td>包括控制软件</td></tr><tr><td>微管玻璃毛细管</td><td> Hilgenberg，Malsfeld，德国</td><td> 1408411 </td><td></td></tr><tr><td>微管拉拔</td><td>萨特仪器</td><td> P - 97 </td><td></td></tr><tr><td>的Alexa Fluor 594 </td><td> Invitrogen公司</td><td> A - 20004 </td><td>荧光染料</td></tr><tr><td> Neurobiotin </td><td> Axxora </td><td> VC - SP - 1120 - M050 </td><td>非荧光示踪</td></tr><tr><td>链霉菌- Cy3标记</td><td> Dianova </td><td> 016-160-084 </td><td></td></tr><tr><td>麦克风romanipulator </td><td> Luigs诺依曼有限公司，德国拉廷， </td><td> 210-100 000 0010 </td><td>电动Mini25操盘单元与显示SM - 5 </td></tr><tr><td>膜片钳放大器</td><td> NPI电子有限公司，德国，塔姆</td><td> SEC - 05LX NPI </td><td></td></tr><tr><td>数字化仪</td><td>美国国家仪器公司</td><td> BNC - 2090 </td><td></td></tr><tr><td>数据采集​​软件WinWCP </td><td>约翰登普斯特，苏格兰，英国格拉斯哥大学</td><td></td><td> http://spider.science.strath。 ac.uk / sipbs / software.htm </td></tr><tr><td>视觉刺激发生器量子点</td><td>托马斯欧拉，德国蒂宾根大学</td><td></td><td>要在一个单独的控制2台显示器（用户界面，刺激显示器）的计算机操作</td></tr><tr><td>中性密度​​滤光片</td><td> ITOS，美因茨，德国</td><td></td><td></td></tr></tbody></table>

electrophysiological characterization of gfp-expressing cell populations in the intact retina

学习的生理特性和完整的组织中的特定神经元的突触连接，这些细胞缺乏突出的形态特征，或显示一个人口密度低，是一种挑战。这尤其适用于视网膜无长突细胞，interneurons，包括大约30亚型在哺乳动物1分外多种形式类。虽然被塑造视网膜输出2的视觉处理的重要组成部分，这些亚型没有研究到现在功能方面，因为遇到这些细胞与记录电极是一种罕见的事件。 最近，众多的转基因小鼠线是可用的表达像促销员的控制下的绿色荧光蛋白（GFP）的膜受体或酶，是具体 ​​的，只有在一个给定的组织3,4神经元的一个子集的荧光标记。这些预标记细胞，因此accessiblE定向微电极定位的微观控制下，允许他们在原地的生理特性的系统的研究。然而，激发的荧光标记物是伴随着风险的光毒性的活组织。在视网膜上，这种做法是另外的问题阻碍，激发光引起的光感受器适当的刺激，从而造成感光色素，漂白和光适应条件转移到视网膜电路。克服了这些缺点是使用一个锁模飞秒短脉冲激光红外激发交付。双光子激发提供的能量足够激发荧光基团，并在同一时间限制激发到一个小的组织体积减少光损伤5危害。此外，叶以来未能photopigments 6吸收红外光（850纳米）的视觉刺激反应的视网膜。 达到原位录音电从绿色荧光蛋白表达细胞在视觉上有针对性的双光子激发。视网膜是在黑暗中准备和维护，并可以通过显微镜的冷凝器（图1）预计的光刺激。膜片钳记录的光反应，可与染料填充揭示的形态和检查耦合到邻近的细胞间隙连接介导的染料，使靶细胞可以通过不同的实验水平上研究。

Watch this Scientific Journal Video about Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina at JoVE.com

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

本文描绘出单个细胞荧光标记完好的小鼠视网膜神经人口的记录。通过使用双光子红外激发transgenetically标记的细胞是有针对性的膜片钳记录，研究他们的光反应，感受野特性和形态。

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

electrophysiological-characterization-gfp-expressing-cell-populations

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

13.4K 次观看.  University of Oldenburg. 本文描绘出单个细胞荧光标记完好的小鼠视网膜神经人口的记录。通过使用双光子红外激发transgenetically标记的细胞是有针对性的膜片钳记录，研究他们的光反应，感受野特性和形态。

视频: Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

科研

神经科学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

量化的活动顺</em在小鼠视网膜>调控元件外植体电穿孔

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

发育生物学

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

生物学

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

在OVO在鸡胚视网膜电穿孔

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

评估视网膜神经节细胞层中的钙活性

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

作者聚焦:一种使用钙成像进行神经电刺激的创新方法 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

电刺激平装视网膜中的钙成像

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...