我们感谢他们在帮助建立器官切片培养体系和沃尔夫冈夜蛾和批判性阅读的手稿Liviu加布里埃尔Bodea马汀娜Emond和伊莎贝尔Brachmann。我们要感谢弗兰克Costantini的R26记者小鼠和SHH CreER小鼠的克里夫塔宾。这项研究是从科学和北莱茵 - 威斯特法伦州（Programm楚Förderung DER Rückkehr DES wissenschaftlichen Spitzennachwuchses AUS DEM Ausland）的研究部资助的一个研究奖。

本协议修改Daza 等 ，2007年7月 。 1。筹备工作<ol><li>可以准备提前一天<ol><li>准备1X克雷布斯缓冲液（1.5升）: 126 mM氯化钠，氯化钾2.5毫米，1.2毫米的NaH 2 PO 4:H 2 O MgCl 2的 1.2毫米，2.5毫米氯化钙 ，葡萄糖11毫米，25毫米碳酸氢钠3，调整pH至7.4。过滤消毒（0.22微米孔径），并储存在4 ° C </li><li>准备培养液（20毫升）:5毫升的HBSS，9 mL的DMEM高糖，30％葡萄糖液850，5毫升马血清（25％）。添加200μL100X Penicllin /链霉素。保存在4 ° C。 </li></ol></li><li>开始清扫前的准备<ol><li>准备100毫升4％低熔点琼脂糖（LMP琼脂糖）在1X克雷布斯缓冲区:微波解决方案，直到琼脂糖完全溶解，然后置于45℃水浴琼脂糖。 </li><li>填充的振动圣多美与冰冷1X克雷布斯缓冲区的缓冲区托盘和开始冷却元件（保持在4 ° C）。修正在刀架和设置的手术刀，精细的画笔和一个小型的穿孔勺子拿起切片vibratome面积刀片。收集切片，准备与1X克雷布斯缓冲区的无菌培养皿中（35 × 10毫米）。置于冰上。 </li><li>设置无菌培养皿（100 × 15毫米），清扫，并嵌入较小的无菌培养皿中（35 × 10毫米），小剪刀，两个钳（杜蒙5）的罚款，迷你穿孔勺，玻璃夹层面积巴斯德吸管火抛光圆封闭尖冰和1升1X克雷布斯缓冲区。用70％乙醇擦拭所有的清扫工具。 </li><li>培养基中添加一个6孔板（1.5毫升/孔）的井，并放置在37℃培养箱。 </li><li>准备用1.5毫升/以及无菌1X克雷布斯缓冲区和15μL青霉素/链霉素（100X）/六孔板。在无菌条件下，地方米尔licell细胞插入入井文化。将六孔板的vibratome使大脑切片可以转移到滤膜后切片即可。 </li></ol></li></ol> 2。胚胎的大脑解剖和嵌入<ol><li>使用异氟醚麻醉怀孕母鼠和牺牲颈椎脱位（胚胎阶段E12.5）的鼠标。解剖出鼠标的子宫，子宫与产钳拉起。使用其他钳分开从子宫mesometrium。放置在冰冷1X克雷布斯缓冲区的子宫。使用细镊子从胚胎中分离出来的肌壁的子宫，赖克特的膜和卵黄囊。从子宫中取出胚胎。解剖胚胎放入一个单独的无菌1X克雷布斯缓冲区的Petri菜。 </li><li>解剖立体显微镜下的大脑。解剖大脑，先切断胚胎的头部。固定头刺入的头（眼平）细钳。使用另一镊子小心取出皮肤和颅骨。使用镊子小心地抬起大脑，并转移到一个培养皿用无菌1X克雷布斯缓冲区。这是非常重要的，整个大脑的完整性保持在清扫过程中，由于脑组织的损害问题（如组织切碎）在切片的vibratome创建。 </li><li>洗一次的大脑在4％低熔点（LMP）的琼脂糖。嵌入在新鲜的4％琼脂糖LMP 2-3大脑。尽量均匀放置在冰上的嵌入菜。使用用火抛光的圆尖，解除大脑，直到底部是凝固的琼脂糖的巴斯德吸管。在一个平面位置的水平琼脂糖块底部的大脑应该解决。 </li><li>在琼脂糖完全凝固（约3分钟后），修剪周围的大脑琼脂糖胶琼脂糖块标本舞台上vibratome。当刷胶块，确保大脑的腹侧方是平行的平台，因为大脑应该是在一个横截面切。 </li></ol> 3。 Vibratome切片<ol><li>使用切割刀片。要获得完整的切片，这是非常重要切片的过程中温度保持在4 ° C。 </li><li>第300微米厚的水平切片在频率为50赫兹，刀片幅度为1.1毫米和25毫米/秒的速度。 </li><li>使用精细的画笔，以推动在一个小穿孔勺的切片收集脑片，并转移到一盘菜用无菌冰冷1X克雷布斯缓冲区。选择包含腹侧中脑组织切片（ 见图1）。在一个E12.5小鼠大脑中有一个300微米的水平切片包含腹侧中脑组织，包括多巴胺能神经元。 </li></ol> 4。切片文化步骤4.2-4.5应在无菌条件下进行。 <ol><li>转移到一个Millicell小细胞培养在6孔板1X克雷布斯缓冲区（见1.2.5）的膜插入大脑切片。为了转移切片，使用迷你穿孔勺子（精细科学的工具）和罚款的画笔。一膜，可放置最多3片。 </li><li>转移细胞膜片与培养基的6孔板（见1.2.5）。不应该由中等覆盖膜的顶部。脑片接收从下面，从上面的空气介质。 </li><li>广场在孵化器的6孔板， 用 5％的CO 2在37 ° C。这是非常重要的切片放置在孵化器后，在2小时内解剖的初步步骤。准备时间更加延长，可能会导致穷人的生存切片。 </li><li>片可维持在体外培养3天。更改第二天50％的培养基。 </li></ol><ol><li>让我们为4-5小时开始前的时间推移成像片在孵化器中恢复。 </li><li>对于时间推移成像，保持膜插入切片，并转移到35毫米的Ibidiμ-的碟（盘底部的高光学质量的材料包括）插入。 </li><li>添加1毫升培养液中加1.5μL抗坏血酸（200毫米）的菜。抗坏血酸防止光毒性片。 </li><li>环境室切片在37 ° C，5％CO2，在时间推移成像。 </li></ol> 6。代表性的成果 图1说明了从E12.5小鼠大脑器官切片文化的准备。图 2显示水平的器官切片（急性和文化几天后）获得形式E12.5小鼠大脑。为了便于比较，在同等发展阶段的冷冻脑切片所示。脑多巴胺能神经元酪氨酸羟化酶（TH）免疫组化显示。中脑多巴胺能神经元的项目，前脑的目标。这些预测在E12.5开始形成，并在随后的几天延长对前脑。我们认为，发展文化中的多巴胺能神经元的正常发展的一个很好的迹象的前脑预测。水平切片中脑多巴胺能神经元对他们适当的前脑的目标区域扩展的预测。 3格（体外天）或前脑的目标地区被破坏后，异常预测延长对背脑。 E12.5脑冠状切片的例子如图3所示。可视化与免疫TH延长异常预测背脑多巴胺能神经元。 图4显示了增殖，坏死和细胞凋亡在器官切片文化的分析。 BrdU IMmunostaining可视化增殖细胞示范养殖条件下，细胞增殖后，1格。扩散减少4格后。 3格后，许多在腹脑细胞发生坏死（碘化丙啶染色）和凋亡（免疫裂解的caspase - 3）。图5显示了YFP标记的神经元在急性切片成像实验时间推移监测的迁徙路径。 <img alt="图1" src="/files/ftp_upload/3350/3350fig1.jpg" /> 图1。原理说明编写的器官切片文化。 300微米水平的大脑切片切片E12.5大脑使用一个vibratome准备。 （一）原理的E12.5小鼠大脑矢状观点。部分级别表示。含有多巴胺能神经元的面积是在粉红色描绘的，预测在蓝色表示。 （二）三个可以从背获得片示意图腹侧和Tha吨包含两个前脑（FB）和脑（MB）。请注意，只有一个切片包含多巴胺能神经元（片二）。基于心室的位置和脑和前脑皮层组织（箭头，B节与节A和C）的连续性，可确定相应的切片。含有多巴胺能神经元的面积是在粉红色表示的，含有多巴胺的前体的面积是描绘绿色，蓝色箭头表示发展预测。 （三）含有多巴胺能神经元的片膜插入培养。缩写:VMB，中脑腹侧; DMB，脑背;羟脯氨酸，下丘脑;血红蛋白，后脑。 <img alt="图2" src="/files/ftp_upload/3350/3350fig2.jpg" /> 图2。器官切片培养的中脑多巴胺能神经元的预测都依赖于前脑的完整性。为标签的多巴胺能神经元的酪氨酸羟化酶（TH）免疫组化。 （一）急性片（0格）显示在腹侧中脑多巴胺能神经元的正常位置。白色箭头指示，在更高的放大倍率在插图所示的领域。尚未开发前脑的预测。 1格后，前脑的预测开始形成。预测延长到2-3格的前脑。白色箭头指示的insets高放大倍率（白框）中的细胞体的位置。黄色箭头突出，在更高的放大倍率的insets（黄框）中的完整的片正常的预测。异常预测开发与损坏前脑切片。红色箭头指示向背侧中脑的insets高放大倍率（红色框）中的异常预测。损害是用红色星号表示。 3格后，大多数片（N = 5 / 7）对背脑异常的预测。 （二）次水平在不同发育阶段的冷冻脑切片免疫组化显示的发展在体内的多巴胺能预测。白色箭头指示的insets高放大倍率（白框）中的细胞体的位置。黄色箭头突出位置在更高的放大插图（黄框）所示的预测。冰冻切片的水平选择密切匹配的器官切片的文化水平。需要注意的是一个单一的冰冻切片（12微米），并不代表整个器官片（300微米）。因此，在E13.5和E14.5所示的预测观察到120微米以上腹包含细胞体（黄色星号）的部分路段上。 <img alt="图3" src="/files/ftp_upload/3350/3350fig3.jpg" /> 图3。冠状切片中脑多巴胺能神经元的标志物酪氨酸羟化酶（TH）染色的文化。 （AC）切片后，1，2或3格。多巴胺能神经元对脑背侧（R发展异常预测编箭头）。箭头显示多巴胺的细胞体的位置。 <img alt="图4" src="/files/ftp_upload/3350/3350fig4.jpg" /> 图4。脑器官切片文化细胞增殖和细胞活力。 （一）增殖细胞标记的BrdU（50毫微克/毫升，Sigma公司）除了到培养液中18小时随后免疫染色切片BrdU。 1格后，BrdU标记细胞位于脑室区（红色箭头）。 4格后，增殖细胞较为分散，不再保持鲜明的脑室区。 （二）坏死细胞被打成除了碘化丙啶（1μg/μLSigma公司），以2小时的培养液，荧光显微镜下观察。 1格后，腹侧中脑（VMB）是不坏死，但许多碘化丙啶标记细胞可在背侧中脑和前脑。 3格后的细胞活力降低腹midbra英寸比例尺:500微米（三）Immunostaing裂解的caspase - 3可视化细胞凋亡。在1或2格，只有少数（TH）的多巴胺能神经元凋亡。 3格后，多巴胺能神经元开始发生凋亡。在中间和右边的面板，在左侧面板盒装面积的放大倍率越高，。 14-16帧栈Z -放大倍率越高，图像的最大强度的预测。图像与蔡司Apotome设置每0.5微米。 <img alt="图5" src="/files/ftp_upload/3350/3350fig5.jpg" /> 图5。YFP标记的神经元在急性切片的迁徙路线。 （一） YFP标记的神经元失效成像水平切片用于。切片孵育5小时前成像。细胞标记使用诱导的Cre / loxP系统6。 嘘CreER小鼠8和ROSA的loxP位一站式loxP位EYFP记者小鼠9我们重新使用。罗莎记者等位基因（EYFP的表达）的重组是在细胞诱导表达CreER（SHH -表达细胞），但只有在他莫昔芬（Sigma公司）的管理。在这个例子中，他莫昔芬（3 mg/40 g体重）给怀孕小鼠在E8.5。这个实验装置，主要是在多巴胺能神经元前体和他们的后裔在腹脑 10,11标签的结果。比例尺:500微米。 （二）在急性切片追踪YFP标记的神经元的迁徙路线。 YFP标记的命运映射神经元的延时图像被收购每蔡司显微镜Axio的观察员（客观EC PlnN 10倍/ 0.3）30分钟的总时间为5小时30分钟。切片孵育在37 ° C的环境室（孵化器XLS1 Pecon），并提供5％的CO 2在成像。标有红色箭头的细胞的初始位置;迁移位置用红色星号标记。

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我们什么都没有透露。

这里介绍的器官切片培养方法在体外分析发展中国家在胚胎腹侧中脑多巴胺能神经元和它们的迁徙和投影路线系统提供了一个短期的。我们发现，有一些在协议的重要步骤应仔细出席了会议，以获得片，使腹侧中脑多巴胺能神经元的正常发展。最关键的一步是胚胎脑，快速和精确的解剖。相比之下一代成人的大脑切片，这是关键部分的大脑上配有一个冷却系统，使用频率低，与高速结合，?...

特定的试剂和设备表<table border="1"><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> DMEM培养液</td><td> Sigma - Aldrich公司</td><td> D6429 </td><td></td></tr><tr><td>葡萄糖30％ </td><td> Sigma - Aldrich公司</td><td> G7528 - 250 </td><td></td></tr><tr><td>马血清</td><td> Invitrogen公司</td><td> 26050-088 </td><td></td></tr><tr><td> DMEM（4,5克/ L，GLC，与L -谷氨酰胺，娜PYR，碳酸氢钠） </td><td> Sigma - Aldrich公司</td><td> D6429 - 500 </td><td></td></tr><tr><td>青霉素/链霉素100X </td><td> Sigma - Aldrich公司</td><td> P4333 - 20 </td><td></td></tr><tr><td> L -抗坏血酸</td><td> Sigma - Aldrich公司</td><td> A4403 </td><td> 200mm的股票和存储准备在-20 ° C </td></tr><tr><td>超纯LMP琼脂糖</td><td> Invitrogen公司</td><td> 15517-022 </td><td></td></tr><tr><td> Millicel插入</td><td> Millipore公司</td><td> PICMORG50 </td><td></td></tr><tr><td> μ盘35毫米，低</td><td> Ibidi </td><td> 80136 </td><td></td></tr><tr><td> Vibratome </td><td> MICROM </td><td>航模650V </td><td></td></tr><tr><td>刀片</td><td>普莱诺有限公司</td><td> 121-6 </td><td></td></tr><tr><td> Histoacryl胶</td><td> BRAU9381104 </td><td>布劳恩蛇牌</td><td></td></tr><tr><td>穿孔勺直径 直径15毫米</td><td>精细科学工具</td><td> 10370 -18 </td><td></td></tr><tr><td>钳5 Dumoxel </td><td>精细科学工具</td><td> 11252 - 30 </td><td></td></tr></table> immunostainings使用的抗体: <table border="1"><tr><tD&gt; 抗体的名称 </td><td> 公司 </td><td > 目录编号 </td><td> 评论（可选） </td></tr><tr><td>兔抗酪氨酸羟化酶</td><td> Millipore公司</td><td> AB152 </td><td>稀释1:500 </td></tr><tr><td>小鼠抗酪氨酸羟化酶</td><td> Millipore公司</td><td> MAB318 </td><td>稀释1:500 </td></tr><tr><td>小鼠抗BrdU </td><td> BD Pharmingen公司</td><td> 555627 </td><td>稀释1:200 </td></tr><tr><td>兔抗裂解的caspase 3 </td><td>细胞信号技术</td><td> 9661 </td><td>稀释1:200 </td></tr><tr><td>驴抗兔IgG - Alexa的488 </td><td> Invitrogen公司</td><td> A21206 </td><td>稀释1:500 </td></tr><tr><td>驴抗鼠IgG - Cy3标记</td><td>杰克逊ImmunoResearch </td><td> 715-165-150 </td><td>稀释1:200 </td></tr></table>

organotypic slice cultures of embryonic ventral midbrain: a system to study dopaminergic neuronal development in vitro

鼠标是一个很好的的模式生物研究哺乳动物大脑的发展，由于大量的分子和基因数据。然而，发展中国家的小鼠大脑是不容易操纵和体内成像合适，因为小鼠胚胎是交通不便，不透明。器官型胚胎的大脑切片文化，因此被广泛用于研究在体外培养的小鼠大脑发育 。体外操纵或允许使用的转基因小鼠基因表达的修改，所以可以用荧光蛋白标记的神经元或神经胶质细胞的亚群。标记细胞的行为，然后可以使用时间推移成像观察。已定时成像研究细胞行为背后晚1-2萌芽阶段大脑皮层的发展，特别是成功。胚胎器官的大脑区域以外的前脑切片在文化系统较差establisHED。因此，时移成像数据描述了神经细胞迁移的财富，是仅限于前脑3,4。它仍然是不知道，是否背的大脑中发现的原则腹脑区的真实。在腹侧的大脑，神经元是神经元集群，而不是层组织，他们往往要经过复杂的迁徙轨迹，以达到他们的的最终位置。腹脑不仅是一个腹侧大脑发育良好的模型系统，也包含如多巴胺能神经元疾病过程中的有关神经元的人口。虽然功能和多巴胺能神经元变性一直在成人和脑老化的非常详细的调查，很少有人知道这些神经元的行为在其分化和迁移阶段5。我们在这里描述从胚胎一天（五）12.5鼠腹侧中脑切片培养的一代。这些切片邪教URES有可能适合监测在体外几天的多巴胺能神经元发展。我们强调在胚胎发育的早期阶段产生的脑切片的关键步骤， 并讨论保持在体外培养的多巴胺能神经元的正常发展的必要条件。我们也可以从时间的推移成像实验结果。在这些实验中，被打成腹侧中脑前体（包括多巴胺的前体）和他们的后裔在镶嵌的方式，使用一个基础的Cre / loxP位诱导的命运映射系统 6 。

Watch this Scientific Journal Video about Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro at JoVE.com

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

描述一个方法来生成从E12.5小鼠胚胎脑器官切片。可用于器官切片文化观察多巴胺能神经元或其他腹侧中脑的神经元的行为。

胚胎腹侧中脑器官型片文化:一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

organotypic-slice-cultures-embryonic-ventral-midbrain-system-to-study

Research

JoVE Journal

Neuroscience

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

17.9K 次观看.  University of Bonn. 描述一个方法来生成从E12.5小鼠胚胎脑器官切片。可用于器官切片文化观察多巴胺能神经元或其他腹侧中脑的神经元的行为。

视频: Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

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 Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

在微RNA在腹鸡中脑大毛表达

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

接口3D工程神经细胞微电极阵列:一种创新<em&gt;体外</em&gt;实验模型

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

生活多巴胺能神经元可靠的鉴定中脑培养使用RNA测序和TH-启动子驱动的EGFP表达

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

胚胎腹侧中脑器官型片文化:一个系统来研究多巴胺能神经元发展在体外

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