我们感谢他在帮助准备手稿博士迈克尔·伍滕。这项工作是由NIH 2RO1NS033661（MWW）支持。

1。设置收获前<ol><li>为了产生神经元的收获产前幼仔，安排成年鼠的繁殖19天前神经隔离的日子。 （只C57BL / 6小鼠年龄2-8个月内被用来发展这个协议的目的交配）。在怀孕的女性，心悸或视觉确认，确认成功的交配可以通过检测阴道塞。 </li><li>前一天神经隔离： <ol class="ualpha"><li>对于免疫的应用，在24孔板以3:1的胶原蛋白，鼠尾光涂层的外衣盖玻片：聚-D-赖氨酸的解决方案。 </li><li>用于细胞培养的应用，大衣适当大小的组织文化以3:1的胶原蛋白，鼠尾光涂层塑料制品：聚-D-赖氨酸的解决方案。 </li></ol></li><li>在组织文化引擎盖发现板UV光下休息过夜。 </li><li>洗板前用无菌汉克的平衡盐溶液（HBSS中）使用。涂层板，可以填补与HBSS中，并储存在4℃黑暗中一个星期。 </li></ol> 2。组织收获<ol><li>不育性始终是一个日益增​​长的初级细胞培养等，最大的，应谨慎行事，以确保在无菌的环境中最可能的因素。仔细注意无菌技术，初步解剖和神经组织的收获，这个协议可以完成的层流罩外，污染最小的风险。初步收获后，所有后续步骤应最大的无菌条件下进行细胞培养额定罩内。 </li><li>斩首怀孕鼠标安乐死在受精后约19天。使用麻醉安乐死的怀孕女性不建议作为麻醉已知会导致脑细胞死亡（Stratmann，等，2010）。使用无菌解剖剪刀和镊子，在中腹侧面创建一个开放鼠标完全揭示体腔。仪器可以消毒用酒精和明火。 </li><li>对小鼠的体腔后，将位于产前幼仔，应该很容易在子宫内可见。与蒸压消毒钳，打开子宫，取出幼仔。杀头与新鲜无菌剪刀和消毒纱布的地方在解剖显微镜下取出头幼仔。无菌，灭菌的仪器，可以使用酒精和明火之前火焰清理和使用过程中。 </li><li>用无菌剪刀或手术刀打开颅骨，从脖子后面的小狗的鼻子。此过程通常可以完成插入一剪刀尖椎孔，然后进行前方。用钳子小心地取出整个大脑。将无菌纱布上的大脑。用消毒的手术刀，删除的小脑和大脑中线切开分成两半球（ 图1）<html> 。 </li><li>把握脑膜周围，用无菌镊子海马的一小部分，并把它轻轻地走。虽然这不是绝对必要之前删除隔离海马脑膜，脑膜的存在可以使夹层海马更加困难，由于膜的韧性。在任何情况下，海马将更加清晰可见脑膜后已被删除。海马是一个弧形的结构，在半球和腹侧弯曲（ 图2）远端部分开始。由于内，凹侧（尾鳍）正面临着一个心室，这已经是免费的。因此，分离海马，需要削减沿凸外侧。剥离后，轻轻提起每海马组织成一个小温暖（37℃）下的细胞培养罩的HBSS培养皿用无菌组织钳和转让。脑组织可结合多个幼仔。 </li></ol>“&gt; 3。组织离解<ol><li> 3毫升100毫米组织培养皿无菌HBSS中使用无菌手术刀，轻轻百果脑组织。 </li><li>碎组织和HBSS中转移到15毫升锥形管。新增的HBSS 1.5毫升和0.5毫升到5毫升的总量0.25％胰蛋白酶溶液。 </li><li>帽和轻轻颠倒离心管4-5次混合。尽量避免产生气泡释放的DNA消化组织将坚持气泡，导致碎组织，而不是解决试管底部（ 图3）浮动。 </li><li>在37℃孵育15分钟后，海马组织相管，每5分钟以上。 </li><li>允许组织，定居在试管底部。 </li><li>仔细去除多余的解决方案，使用无菌吸管，留在试管底部组织原状。 </li><li>洗颗粒组织在37°C 5分钟，5毫升的HBSS。重复共3次。允许组织完全解决管底部的每一次前出发到下一个清洗步骤。 </li><li>从组织颗粒最终清洗和更换2毫升新鲜的HBSS。 </li></ol> 4。神经元研磨<ol><li>开始研磨步骤之前，你需要准备火抛光的巴斯德吸液管。使用Bunson燃烧器，保持无菌9英寸的巴斯德吸管中的火焰尖端（ 图4a），直到吸管开幕直径约0.5毫米大小和边缘的吸管开幕的已略圆（ 图4b）。允许吸管完全冷却，然后再开始研磨过程。 </li><li>使用正常的无菌9英寸巴斯德吸管，轻轻磨碎共组织了7次。较大的组织块，在这一点上是正常的，应允许移动到下一个步骤之前，解决管底​​部。 </li><li>将上清转移到一个新的无菌50毫升锥形管。 </li><li&gt;其余组织，加2毫升无菌HBSS中，磨碎共5次使用火抛光的巴斯德吸管。 </li><li>允许所有剩余较大的组织块，以解决管的底部，并结合以前共4毫升分离神经细胞上清上清。 </li></ol> 5。细胞接种<ol><li>使用血球计数游离细胞。 </li><li>作为一般规则，一旦细胞的数量已经确定，从这个最后的数字减去20％，占电镀后可能发生的任何细胞死亡。 </li><li>细胞可以镀使用以下建议： 在24孔板盖玻片- 6×10 4细胞/ 0.5毫升 对于60毫米的组织培养板- 4×10 5细胞/板，3毫升 对于100 mm组织培养皿- 6×10 6细胞/ 6毫升板</li><li>混合适当的细胞数量的Neurobasal电镀媒体的指示量（的Neurobasal媒体含有B27的补充[1毫升/ 50毫升，，谷氨酰胺解决方案0.5毫米，25μM的谷氨酸（先生147.13克/摩尔），青霉素（10,000单位/毫升）/链霉素（10,000微克/毫升）[250μL/ 50毫升] ，1mm的肝素钠（先生238.3克/摩尔），10％热灭活供体马血清），并添加细胞板。旋流板轻轻细胞均匀分布。您好捐助者马血清加电镀媒体，以丰富的增长在第一个24小时的细胞。细胞随后断奶从血清和血清串行减少在每个媒体更换无血清的环境。还应当指出的是，而在较高浓度的谷氨酸是神经细胞培养的毒性，在这里添加浓度，它会抑制非神经细胞的附件11。然而，它应该只被添加到培养的第一个24小时的电镀媒体，随后离开任何饲养媒体，以防止细胞的神经毒性。 </li><li> P级花边神经元在37℃，5％CO2培养箱过夜。 </li><li>从细胞中取出一半的媒体数量和相同体积的Neurobasal饲养媒体（含B27添加剂的Neurobasal媒体取代[1毫升/ 50毫升，0.5毫米谷氨酰胺​​溶液，青霉素（10,000单位/毫升）/链霉素（10,000微克/毫升）250μL/ 50毫升，1毫米肝素钠（先生238.3克/摩尔）。 </li><li>通过消除旧媒体的一半，并用相同体积的新鲜的Neurobasal饲养媒体的神经元应投喂，每4天。神经过程应开始第1天（ 图5a）可见，成为流行的10天（ 图5b）。 </li></ol> 6。代表结果增长和文化的主要神经细胞的能力，已成为神经科学的一个不可缺少的一部分。小学文化的允许研究者分析特定的细胞通路，化学修饰和处理，目标禄在受控环境中alization和增长模式。许多这些程序利用先进的方法，可视化细胞的反应中的具体变化。在这种情况下，海马神经元用于研究特定的神经通路，将被证明是困难的，如果不是不可能的完整的大脑在分析。从大脑的特定区域的神经元附近的同质人口的制备是研究脑功能的关键。在单个神经元的分子作用，可以划定高阶通路，如内存或学习。由于该协议得到较纯的文化，而不需要的神经胶质细胞的饲养层，海马神经元，这些神经元很容易利用免疫研究。然而，小学文化的所有器官，含有多种细胞类型，一些污染少所需的细胞可以发生。在隔离神经细胞，神经胶质细胞污染是一个普遍的问题。神经胶质细胞c可以很容易被发现后微观可视化的文化形态目标神经元（ 图6）显着不同。胶质细胞污染的影响将取决于计划使用的文化。如果正在使用免疫荧光检查细胞，神经胶质污染可以尝试拍摄单个神经元时，是什么比一个多不便。然而，如果被用于生化分析的神经细胞是由神经胶质细胞的任何重大污染可能会导致结果的重大变化。概述了进一步讨论的方法来解决胶质细胞污染。 一旦神经已经成功地分离和培养，一个典型的应用是检查细胞免疫荧光技术的进程。 如图7所示，如线粒体的细胞器，可以使用添加到培养基修复前的重要染料染色ATION。内源性细胞的蛋白质，可以可视化固定细胞，使用标准的免疫荧光技术（ 图8）。一旦神经细胞是固定的，可以将感兴趣的蛋白质的特异性抗体的细胞，这些蛋白质可以用荧光显微镜可视化。培养的神经元，还提供与手段的研究人员检查个别蛋白影响神经功能。使用多种技术，包括DNA转染，电穿孔或病毒转导，蛋白质可在神经细胞（ 图9）过度。神经细胞如何回应过表达蛋白的影响，可以对大脑的反应可能有直接的推论，并提供细胞的目标确定为药物治疗的可能性。这些类型的实验细节超出了本文的范围，但他们没有说明这种技术制备的文化是适合广泛的为羽绒-Stream应用。然而，这个协议的整体简洁，以及为短的时间内，需要准备这些神经细胞，使这是一个在今日的神经科学实验室使用的理想方法。 <img alt="图1" src="/files/ftp_upload/3634/3634fig1.jpg" /> 图1。剖析产前鼠脑。第一个切口是大脑分成两个半球的中线。 <img alt="图2" src="/files/ftp_upload/3634/3634fig2.jpg" /> 图2。在产前鼠脑海马区的位置。纹状体一边移动到海马可视化，并注意到在每个半球的远端地区弯曲的“芸豆”型结构。 <img alt="图3" src="/files/ftp_upload/3634/3634fig3.jpg" /> 图3。海马组织中的胰蛋白酶溶液分解。 <p类“jove_content”&gt; <img alt="图4" src="/files/ftp_upload/3634/3634fig4.jpg" /> 图4。巴斯德枪头用于海马组织研磨。 （一）普通巴斯德枪头。 （二）消防抛光的巴斯德枪头。注意的圆边和吸管开口尺寸减少约50％。 <img alt="图5" src="/files/ftp_upload/3634/3634fig5.jpg" /> 图5。海马神经元分离使用此程序，并镀在NB媒体。 （一）细胞生长1日后电镀。神经过程的开始，是在第1天可见。 （二）细胞生长10天电镀后，突起是分支和重叠。 <img alt="图6" src="/files/ftp_upload/3634/3634fig6.jpg" /> 图6。污染7天生长的神经胶质细胞和细胞器标记MitoTracker红，CM-H2XRos（Invitrogen公司＃M7515）和transfe染色的海马神经元反恐执行局与GFP-LC3的使用脂质体2000（Invitrogen公司＃11668019）。线粒体是可见的，但是只有一个单一的神经元，在所有细胞中成功转染荧光构造。与胶质细胞的污染，使GFP-LC3的表达很难想象，在神经元突起的分析。 <img alt="图7" src="/files/ftp_upload/3634/3634fig7.jpg" /> 图7。海马神经元增长为7天，细胞器标记MitoTracker红CM-H的2 XRos（Invitrogen公司＃M7513）染色。这一重要的染料来染色积极组织培养细胞中的线粒体。这些细胞被固定在4％多聚甲醛/ PBS和荧光显微镜观察。染料本身无荧光，直到在线粒体氧化。活跃​​的线粒体中可以看出整个神经过程。 <img alt="图8" src="/files/ftp_upload/3634/3634fig8.jpg" /> 图8。 </strONG&gt;海马神经生长7天，4％多聚甲醛/ PBS及免疫染色与单克隆抗-微管蛋白β抗体（适马＃T0198）固定。初级抗体，俄勒冈州的绿色标记的羊抗鼠二抗（Invitrogen公司＃O11033）加入荧光显微镜观察。 <img alt="图9" src="/files/ftp_upload/3634/3634fig9.jpg" /> 图9。海马神经细胞生长5天，并与GFP-LC3βDNA转建设用脂质体2000（Invitrogen公司＃11668019）。第7天，细胞固定用绿色荧光蛋白标记纳入其外膜LC3β4％多聚甲醛/ PBS和aggresomes用荧光显微镜进行可视化。 aggresomes位于整个细胞体和突起，并用箭头表示。

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没有利益冲突的声明。

海马文化已被应用于分子生物学，为20年以上。虽然原则上，神经细胞可以从大脑的任何部分，海马文化已被证明是最流行 ​​，由于在7海马神经细胞的人口相对简单的架构。海马文化通常是由后期的胚胎组织。这个组织是容易分解和包含不少于成熟的脑组织的神经胶质细胞。从胚胎组织的海马神经元的分离，也减少了由于更少的黏附接触3的轴突和树突的剪切破坏。虽然海马的?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 供应商 </td><td> 目录编号 </td></tr><tr><td>鼠尾胶原1 </td><td> BD公司</td><td> 354236 </td></tr><tr><td>聚-D-赖氨酸的解决方案</td><td> Chemicon公司</td><td> -003-E的</td></tr><tr><td>汉克斯平衡盐溶液</td><td> Invitrogen公司</td><td> 14175-095 </td></tr><tr><td>胰蛋白酶溶液（1X）0.25％，液体</td><td> Invitrogen公司</td><td> 15050-065 </td></tr><tr><td>的Neurobasal中等液（1X） </td><td> Invitrogen公司</td><td> 21103-049 </td></tr><tr><td> B27的补充（50X）液体</td><td> Invitrogen公司</td><td> 17504-044 </td></tr><tr><td> L-谷氨酰胺200毫米（100X）液体</td><td> Invitrogen公司</td><td> 25030-149 </td></tr><tr><td>青霉素（10,000单位/毫升）/链霉素（10,000微克/毫升） </td><td> Invitrogen公司</td><td> 15140-148 </td></tr><tr><td> HI捐助者马血清</td><td>亚特兰大生物</td><td> S12150H </td></tr></tbody></table>

isolation and culture of hippocampal neurons from prenatal mice

被广泛用于揭示小学文化的大鼠和小鼠海马神经元细胞在神经生物学机制。通过隔离和不断增长的单个神经元，研究人员能够分析贩运细胞，细胞结构和个别蛋白质本地化，使用各种生化技术相关的属性。从这些实验的结果是测试解决学习和记忆功能的神经基础理论的关键。然而，从这些形式的实验结果明确的增长能力，神经细胞与其他脑细胞类型的最低污染的前提。在这个协议中，我们使用特定的神经细胞生长和胚胎海马组织小心剥离，旨在优化健康的神经细胞的生长，同时最大限度地降低污染类型的细胞（即星形胶质细胞）的媒体。胚胎小鼠海马组织能更加困难比DI大小的样品，由于类似的灭鼠组织隔离开来ssection。我们发现从胚胎19天（E19）小鼠幼仔海马的详细解剖技术。一旦海马组织是孤立的，温柔的神经细胞的分离，实现稀浓度的胰蛋白酶和机械设计单独从结缔组织细胞的破坏，同时提供单个细胞的损害最小。包括一个详细说明如何准备用于在中断移液器。免疫荧光协议，以最大限度地提高成功的细胞培养提供最佳的电镀密度。该协议提供了一个快速（约2小时）和有效的技术从小鼠海马组织神经细胞文化。

Watch this Scientific Journal Video about Isolation and Culture of Hippocampal Neurons from Prenatal Mice at JoVE.com

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

我们没有从产前的老鼠大脑海马神经元的神经胶质细胞饲养层的使用高度纯化的文化提供了一个协议。

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

isolation-and-culture-of-hippocampal-neurons-from-prenatal-mice

Research

JoVE Journal

Neuroscience

70.0K 次观看.  Auburn University. 我们没有从产前的老鼠大脑海马神经元的神经胶质细胞饲养层的使用高度纯化的文化提供了一个协议。

视频: Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

原代海马神经元的磷酸钙转染

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

分离和游离感觉神经元的文化活动从鸡胚

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

从产前Islmn：GFP转基因小鼠的奥库罗运动、特罗赫利尔和脊柱运动神经元的分离与培养

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...

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 ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

从成人和老化小鼠的河马切片中尽量减少低氧

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

小鸡西里神经神经元的分离与培养

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...