这项工作是由美国国立卫生研究院授予安永012031（ET），FM柯比基金会（ET），研究奖，以防止失明，公司（MAZ），新泽西州狮子会眼研究基金会（MAZ）的支持，新泽西州（MAZ），约瑟夫J和玛格丽特DiSepio视网膜病变的研究基金（MAZ），贾尼斯米切尔瓦萨和Ashby米切尔奖学金（AMK）的眼科研究所。

1。一个细胞培养插入定制的立场 5毫升枪头（ISC BioExpress，＃P - 3250 - 19）的基础内径13毫米，这完全匹配​​的底部尺寸（12.6毫米，外径）10毫米（内径）NUNC细胞文化插入（聚碳酸酯膜，热，8微米，＃137443）。要的立场，吸液管被切断基地附近产生一个长6毫米的空心圆柱。然后，件从气缸侧面，采用火焰加热的刀片下创建一个环（2毫米高度），这引起了插入的高度约5毫米的3腿（4毫米高度）。 2。猪视网膜的依恋和文化的无菌滤纸的制备 4M的Whatman滤纸（猫号1004）移动到一个玻璃管的杀菌或一旦视网膜重视滤纸具有体积小，三角形的处理（如被切断成圆弧状（直径6.3毫米）在视频显示）。 3。视网膜外植体的制备从5眼 - 9个月的老，150-230英镑，美国约克夏猪屠宰场（在冰上运输），在三个小时眼球摘除内从一个地方。抵达后，眼睛是多余的组织清理，蘸优碘的解决方案（碘伏10％，在普渡大学弗德瑞克公司，斯坦福，CT）和贝科的修改鹰中等（DMEM培养液1克/大号葡萄糖，L -谷氨酰胺洗2次，补充2.5微克/毫升两性霉素B（GIBCO Invitrogen公司，卡尔斯巴德，加利福尼亚州）和丙酮酸钠，Cellgro科技股份有限公司，Manasses，弗吉尼亚州）。眼前段被拆除，露出视网膜神经。六毫米的环钻刀片（Storz成为眼科博士伦，曼​​彻斯特，MO），用来切割赤道，后段全层植。随后视网膜剥落一块干燥无菌滤纸轻轻地应用到神经节细胞层，解除神经视网膜，并放置到文化插入附加视网膜滤纸，光感受器朝上。插入，然后放入培养液，确保全面覆盖的视网膜，在12孔培养皿（费舍尔）。多植获得常规，并从单一的眼睛培养。 4。组织培养鹰的最低限度的基本中培养的视网膜组织，Invitrogen公司GIBCO -生命科技公司，马里兰州洛克维尔（MEM），pH值7.4，辅以谷氨酰胺为0.2毫克/毫升，10微克/毫升猪胰岛素，丙酮酸1毫米，0.1毫米L -抗坏血酸，100 U / ml青霉素，100μg/ mL链霉素，和250 ng / ml的两性霉素B（抗生素Antimycotic：Sigma公司，圣路易斯，密苏里州），加湿5％的CO 2，平衡空气曝气，在37 ° C交换液每两天。 组织可使用多种试剂或探针治疗，或向左，为后续的生化或形态学分析未处理。以下部分介绍了程序，以创建一致的全层视网膜截面。 5。切片<ol><li>使用一个低温恒温器<ol><li>一夜之间修复视网膜组织在4％parafomaldehyde 4℃; </li><li>简单地冲洗组织与磷酸盐缓冲液（PBS）; </li><li>小心取出滤纸的组织，而它在PBS是在解剖镜下使用的35毫米菜; </li><li>组织转移30％的蔗糖，并保持在4℃过夜℃; </li><li>周围组织吸走多余的溶液，添加足够的组织TEK培养基（华侨城复方4583），以支付组织，并让它浸泡2小时，在室温下渗透到组织; </li><li>建立一个与牙科用蜡的方形镜框（10毫米× 10毫米，5毫米的高度）和周围的组织，它添加更多的组织TEK介质填充帧（确保组织样本是平）和在冷冻（ - 20 ° C）的至少1个小时; </li><li>冷冻样品块传输到样品台上（确保样本块垂直平台）; </li><li>预冷却的低温恒温器安装在样品台上（-20 ° C，莱卡，CM1900），并确保该块刀片垂直; </li><li>删除前所需厚度的组织切片的蜡和帧块; </li><li>明胶处理过的幻灯片上放置的部分。 </li></ol></li><li>使用一个Vibratome <ol><li>上面所述的低温恒温器的组织修复和冲洗; </li><li>组织放入预先加热，融化的琼脂在一个小容器4％，并保持在4 ° C，直到琼脂凝结完全; </li><li>使用锋利的刀片，修剪成小块状的组织样本里的琼脂; </li><li>胶vibratome与Krazy胶样品架（确保组织样本中的一块是垂直的TH平​​台的样品块发送样品架，即，垂直的刀片），并装上vibratome切片系统完全淹没在冰冷的海水样品块（莱卡，1000系列）的持有人; </li><li>切成50微米的部分样品块和明胶处理过的幻灯片，用软毛刷。 </li></ol></li></ol> 6。免疫组织化学使用任何标准的协议。 Immunolabeled厚的部分可以被看作共聚焦显微镜。 7。代表性的成果： 保存在七天内的文化形态。前和后培养的视网膜图像，视频。

<ol>
	<li>Johnson, T. V., Martin, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+an+adult+retinal+explant+organotypic+tissue+culture+system+as+an+in+vitro+intraocular+stem+cell+transplantation+model.">Development and characterization of an adult retinal explant organotypic tissue culture system as an in vitro intraocular stem cell transplantation model.</a> Invest Ophthalmol Vis Sci. 49, 3503-3512 (2008).</li><li>Kretz, A., Marticke, J. K., Happold, C. J., Schmeer, C., Isenmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primary+culture+technique+of+adult+retina+for+regeneration+studies+on+adult+CNS+neurons.">A primary culture technique of adult retina for regeneration studies on adult CNS neurons.</a> Nat Protoc. 2, 131-140 (2007).</li><li>Donovan, S. L., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+square+wave+electroporation+of+retinal+explant+cultures.">Preparation and square wave electroporation of retinal explant cultures.</a> Nat Protoc. 1, 2710-2718 (2006).</li><li>Koizumi, A., Zeck, G., Ben, Y., Masland, R. H., Jakobs, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+physiologically+functional+adult+mammalian+retinas.">Organotypic culture of physiologically functional adult mammalian retinas.</a> PLoS One. 2, e221-e221 (2007).</li><li>Lye, M. H., Jakobs, T. C., Masland, R. H., Koizumi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+adult+rabbit+retina.">Organotypic culture of adult rabbit retina.</a> J Vis Exp. , (2007).</li><li>Ames, A., Li, Y. Y., Heher, E. C., Kimble, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+rabbit+retina+as+related+to+function:+high+cost+of+Na++transport.">Energy metabolism of rabbit retina as related to function: high cost of Na+ transport.</a> J Neurosci. 12, 840-853 (1992).</li><li>Kobuch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+adult+porcine+retina+and+retinal+pigment+epithelium+in+perfusion+culture:+characterisation+of+an+organotypic+in+vitro+model.">Maintenance of adult porcine retina and retinal pigment epithelium in perfusion culture: characterisation of an organotypic in vitro model.</a> Exp Eye Res. 86, 661-668 (2008).</li><li>Kaempf, S., Walter, P., Salz, A. K., Thumann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+organotypic+culture+model+of+adult+mammalian+neurosensory+retina+in+co-culture+with+retinal+pigment+epithelium.">Novel organotypic culture model of adult mammalian neurosensory retina in co-culture with retinal pigment epithelium.</a> J Neurosci Methods. 173, 47-58 (2008).</li><li>Guduric-Fuchs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+study+of+pig+retinal+development.">Immunohistochemical study of pig retinal development.</a> Mol Vis. 15, 1915-1928 (2009).</li><li>Chandler, M. J., Smith, P. J., Samuelson, D. A., MacKay, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+density+of+the+domestic+pig+retina.">Photoreceptor density of the domestic pig retina.</a> Vet Ophthalmol. 2, 179-184 (1999).</li></ol>

视觉研究人员已经建立了各种视网膜文化系统，包括器官系统，研究各种问题，如视网膜干细胞移植，视网膜再生 2，外源基因表达 3-5 。然而，成年哺乳动物视网膜是难以维持在体外 ，主要是由于高能源需求的光感受器6。小泉纯一郎等人描述了兔子的视网膜器官文化系统中，光感受器的氧气供应，提高文化插入的高度和搅拌培养基减轻。他们成功地维持长?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>纪念</td><td> Invitrogen公司，Gibco公司</td><td> 10370-021 </td><td></td></tr><tr><td> 5毫升枪头</td><td> ISC的BioExpress </td><td> P - 3250 - 19 </td><td></td></tr><tr><td> NUNC细胞文化插入</td><td>热</td><td> 137443 </td><td> 8微米，聚碳酸酯</td></tr><tr><td>滤纸4M滤纸</td><td>瓦特曼</td><td> 1004 </td><td></td></tr><tr><td> DMEM培养液</td><td> Cellgro - mediatech公司</td><td> 10 - 013 - CV </td><td></td></tr><tr><td>洋菜</td><td> Fluka公司</td><td> 05040 </td><td></td></tr><tr><td> Fungizone两性霉素B </td><td> Invitrogen公司，Gibco公司</td><td> 15290018 </td><td></td></tr><tr><td>环钻刀片</td><td>博士伦</td><td> T3096 </td><td> 6.00毫米</td></tr><tr><td>华侨城复合</td><td>电子显微镜科学</td><td> 62550-01 </td><td> 4583 </td></tr><tr><td></td><td>科学</td><td></td><td></td></tr><tr><td>低温恒温器</td><td>徕卡</td><td> CM1900 </td><td></td></tr><tr><td> Vibratome </td><td>徕卡</td><td> 1000系列</td><td></td></tr><tr><td>共聚焦显微镜; </td><td>卡尔蔡司</td><td> LSM510 </td><td></td></tr><tr><td>反突触囊泡蛋白2（SV2）鼠单克隆抗体</td><td> DSHB </td><td> N / A </td><td></td></tr><tr><td>抗神经胶质纤维酸性蛋白（GFAP）多克隆兔抗体</td><td> DAKO公司</td><td> DAKO公司</td><td></td></tr><tr><td>碘化丙啶（PI） </td><td>西格玛</td><td> P4170 </td><td></td></tr></tbody></table>

organotypic culture of full-thickness adult porcine retina

是公认的体外模型的需求，可以取代或减少动物实验。猪视网膜有一个类似于人类视网膜的神经元结构，因此，为研究人类的视网膜损伤及退行性疾病的机制的珍贵物种。在这里，我们描述了从屠宰场取得的眼睛分离成年猪视网膜器官文化的成本效益的技术。取出后眼前段，环钻刀片是用来创建多个神经视网膜布鲁赫的成年猪眼后段膜的RPE -脉络膜巩膜植。一块无菌滤纸是用来解除每个外植体视网膜神经。滤纸视网膜复杂的培养（感光面朝上）之上插入一个定制的立场，这是从培养皿的底部。使培养基中的良好循环，视网膜双方的立场。总体而言，这个过程很简单，重现性好，并允许保存至少7天的本地视网膜结构，使其成为一个有用的模型的各种哺乳动物视网膜的形态，药理，生化研究。

Watch this Scientific Journal Video about Organotypic Culture of Full-thickness Adult Porcine Retina at JoVE.com

Organotypic Culture of Full-thickness Adult Porcine Retina

在这里，我们描述了一个成本效益的技术，七天为成年猪视网膜器官文化。简单地说，无菌滤纸用于解除神经视网膜视网膜色素上皮和地方感光一面向上插入一个定制的立场提出。

器官型培养的全厚成年猪视网膜

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

organotypic-culture-of-full-thickness-adult-porcine-retina

Research

JoVE Journal

Neuroscience

14.9K 次观看.  University of Medicine and Dentistry of New Jersey - UMDNJ. 在这里，我们描述了一个成本效益的技术，七天为成年猪视网膜器官文化。简单地说，无菌滤纸用于解除神经视网膜视网膜色素上皮和地方感光一面向上插入一个定制的立场提出。

视频: Organotypic Culture of Full-thickness Adult Porcine Retina

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

海马脑片的器官型检查神经元损伤的文化模式

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

科研

神经科学

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

成年小鼠视网膜电生理研究水平切片的制备

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

来自成人大鼠尿膀胱的原发神经元和胶质的隔离与培养

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Culture of Neurospheres Derived from the Neurogenic Niches in Adult Prairie Voles

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the differentiation and proliferation potential of neural stem cells, as well as to generate cell lines than can be assayed over time. Also, neurospheres can create a niche (in vitro) that allows the modeling of the dynamic changing environment, such as varying growth factors, hormones, neurotransmitters, among others. Microtus ochrogaster (prairie vole) is a unique model for understanding the neurobiological basis of socio-sexual behaviors and social cognition. However, the cellular mechanisms involved in these behaviors are not well known. The protocol aims to obtain neural progenitor cells from the neurogenic niches of the adult prairie vole, which are cultured under non-adherent conditions, to generate neurospheres. The size and number of neurospheres depend on the region (subventricular zone or dentate gyrus) and sex of the prairie vole. This method is a remarkable tool to study sex-dependent differences in neurogenic niches in vitro and the neuroplasticity changes associated with social behaviors such as pair bonding and biparental care. Also, cognitive conditions that entail deficits in social interactions (autism spectrum disorders and schizophrenia) could be examined.

We established the conditions to culture neural progenitor cells from the subventricular zone and dentate gyrus of the adult brain of prairie voles, as a complementary in vitro study, to analyze the sex-dependent differences between neurogenic niches that could be part of functional plastic changes associated with social behaviors.

从成人草原卷的神经性尼奇衍生的神经球培养

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

小鼠河马兴奋和类特异性抑制神经元的不同组织型切片培养的单细胞电位

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...