我们还感谢加布里埃尔的手进行批判性阅读。

所有实验都是根据大阪大学动物福利委员会和日本神经科学学会制定的指导方针进行的。
1. 丘脑和大脑皮层的脊髓 cocultures
注: 有关详细过程, 请参阅原始出版物7,8,13。所有程序都应在无菌条件下进行。大 (SD) 大鼠用于神经元培养。
<ol><li>
试剂制剂 注意:以下试剂的容量是为一只老鼠脑子。<ol>
<li>
准备10x 修改后的 N2 补充13: 胰岛素 (50 微克/毫升), 黄体酮 (200 nm), 松 (200 纳米), 亚硒酸钠 (300 nm), 转铁蛋白 (1 毫克/毫升), 腐胺 (1 毫米), 葡萄糖 (60 毫克/毫升)</li>
		<li>
准备含有血清的培养基(10 毫升): 85% DMEM/F12, 10% 10x 改良 N2 补充, 5% 胎牛血清 (血清)</li>
		<li>
准备无血清培养基(20 毫升): 90% DMEM/F12, 10% 10x 改性 N2 补充剂</li>
		<li>准备鼠尾胶原蛋白溶液(0.5 毫升)。总之, 在无菌条件下, 从大鼠尾部取嫩纤维, 在醋酸溶液中孵化过夜。离心后, 收集上清 (几毫克/毫升) 和存储在冰箱13。</li>
	</ol>
</li>
	<li>
培养皿的制作
	<ol>
<li>将膜插入到35毫米培养皿中。</li>
		<li>添加 40-50 ul 的鼠尾胶原蛋白溶液的膜, 并传播胶原溶液围绕中心。</li>
		<li>空气在干净的长凳上完全干燥。</li>
		<li>将含血清培养基的 1.5-2 毫升放入盘中, 使膜与培养基一起浸泡。</li>
		<li>将培养基放在孵化器 (37 摄氏度, 湿润95% 空气, 5% CO2) 电镀前。</li>
	</ol>
</li>
	<li>
皮质切片的制备
	<ol>
<li>用70% 乙醇消毒所有手术器械10分钟或更长时间。</li>
		<li>从产后一天 (P) 2 大鼠在冰冷麻醉下快速解剖整个大脑 (浸泡在冰芯片中几分钟)。</li>
		<li>将大脑放入100毫米培养皿中, 其中含有100毫升的冷汉克斯平衡盐溶液。</li>
		<li>用显微外科剪刀从大脑中取出 pia, 从视觉和体感皮层切开皮质切片 (300-400 微米厚度) (详情请参阅图 1)。 注意:在双目显微镜下, 皮质切片厚度大致由网格尺度估计。 注意:另外, 皮质切片可以在无菌条件下 vibratome。</li>
		<li>用一次性塑料吸管将一些皮质切片转移到膜上。</li>
		<li>用火抛光的巴斯德吸管调整位于培养基中心附近的切片位置 (有关详细信息, 请参阅8、13) 并从插入中删除多余介质。 注意:调整介质的水平稍低于切片表面, 使切片能够得到足够的气体和介质供应。这对细胞活力至关重要。</li>
		<li>在湿润95% 空气和 5% CO2的环境中, 将含有血清的培养基中的切片保持在37摄氏度, 而仅在丘脑块被取走并放置在其旁边的某一天进行培养。</li>
	</ol>
</li>
	<li>
丘脑块的制备
	<ol>
<li>用70% 乙醇消毒所有手术器械10分钟或更长时间。</li>
		<li>麻醉一只怀孕的老鼠 (胚胎日 15), 腹腔注射戊巴比妥 (50 毫克/千克) 含有麻醉剂。</li>
		<li>解剖每个胚胎从子宫角, 并将胚胎放入一个100毫米培养皿中含有100毫升的冷汉克斯平衡盐溶液。</li>
		<li>在双目显微镜下, 从每个胚胎中取出大脑, 并切除主要包含 ventrobasal 复合体和侧膝状核的丘脑块 (大约0.5 毫米 x 3)7使用显微外科剪刀 (见图 1)。</li>
	</ol>
</li>
	<li>
文化与维护脊髓 cocultures
	<ol>
<li>使用巴斯德吸管, 将丘脑块放置在被放置在膜过滤器的皮质切片的心室表面旁。</li>
		<li>在湿润95% 空气和 5% CO2的环境中, 保持37摄氏度的含血清培养基中的 cocultures。</li>
		<li>在培养两天后, 用无血清培养基交换一半培养基。此后, 每 2-3 天交换媒介。 注意:媒介的水平应该在媒介交换以后检查, 因为它是重要的对细胞生存。</li>
	</ol>
</li>
</ol>2. 电穿孔
所有程序都应在无菌条件下进行。
注意:在准备丘脑块后的某一天进行电穿孔, 以防止块体脱离。
<ol><li>
粒
	<ol>
<li>使质粒的混合物如下: pCAGGS-DsRed 2 微克/ul;pCAG-SYP-EGFP 3.5 微克/ul。 注意:用无内毒素 Maxiprep 试剂盒纯化两种质粒, 并在汉克斯平衡盐溶液中溶解。</li>
	</ol>
</li>
	<li>
电穿孔设备安装 诸如刺激器和隔离器之类的电子仪器应该连接起来, 如图 2所示。<ol>
<li>将刺激器连接到两相隔离器。</li>
		<li>将电极 (铜线直径0.2 毫米) 连接到隔离器的负端。</li>
		<li>将电极 (银线1毫米直径) 串联到100Ω电阻器, 并将该隔振的正极端子连接起来。</li>
		<li>将放大器连接到示波器, 通过测量放大器的电压来监测电流是否通过电阻。</li>
	</ol>
</li>
	<li>
玻璃吸管的制备 注: 两种玻璃吸管用于质粒溶液的喷射和电脉冲的应用。<ol>
<li>用电极拉拔器将吸管从玻璃毛细血管中制成。</li>
		<li>打破喷射吸管的尖端 (尖端直径应该是 20-50 微米)。</li>
		<li>用砂纸研磨和抛光电极吸管的尖端, 然后进行火抛光 (内径应为 50-200 微米)。 注意:电极尖端应该是平坦和平滑的, 因为它直接接触组织。</li>
	</ol>
</li>
	<li>
电穿孔程序
	<ol>
<li>将弹射和电极吸管连接到机械手。</li>
		<li>将银线 (0.2 毫米直径) 插入电极吸管。</li>
		<li>用塑料管将弹射吸管连接到1毫升的注射器上。</li>
		<li>采用注射器吸入法, 将5质粒溶液的 ul 引入到弹射吸管中。</li>
		<li>将共培养在电穿孔阶段, 并将1毫米直径电极插入培养基中。</li>
		<li>将弹射吸管放在丘脑外植体上。</li>
		<li>在提示触及丘脑块的表面后, 手动推注射器。 注意:大约0.5 质粒溶液的 ul 通常使用每一个丘脑块。</li>
		<li>将电极吸管放在丘脑块上, 在弹射吸管缩回后立即放置。</li>
		<li>提供电脉冲 (50-400 μA, 3 到5列车200平方脉冲1毫秒的持续时间在200赫兹)。监控示波器上列车的振幅和数量。 注意:电流的振幅取决于电极吸管的内径13。</li>
		<li>收回电极吸管, 并返回到孵化器的盘子。</li>
	</ol>
</li>
	<li>
显微观察
	<ol>
<li>将培养皿放在直立共焦显微镜的微观阶段, 配备两组 EGFP (励磁、488 nm、发射、515 nm) 和 DsRed (励磁、514 nm、发射、565 nm)。</li>
		<li>采用20X 长工作距离物镜, 以 1-7 µm 级尺寸拍摄 10-25 光学剖面图。选择单独区分标记的轴突, 表达 DsRed 和 SYP-EGFP。 注意:观测到的面积约为 0.7 x 0.7 毫米 (对应于 1024 x 1024 像素, 即0.68 微米/像素)。</li>
		<li>每24小时捕获图像以进行每日成像。在室温下10分钟内完成, 并将培养物返回到孵化器。对于短间隔的时间推移成像, 保持文化在一个文化室, 维持环境的湿润95% 空气和 5% CO2在37摄氏度。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

目前的协议也是一个强大的工具来研究发展方面的增长轴突以外的 TC 投影11。例如, 皮质切片培养和电穿孔技术的结合使皮层神经元的单个轴突形态学可视化, 长期观察9,18。
利用目前的协议, 在轴突分支和突触形成中, 有趣基因的作用也可以通过荧光蛋白的共表达和兴趣基因来分析。通常, 当质粒溶液的摩尔比率调?...

哺乳动物大脑中的丘脑-皮质 (TC) 投射是研究轴突导引和靶向机制的合适系统。在发育过程中, 感觉 TC 轴突生长在皮质板块, 并形成分支和突触在大脑皮层的主要感官区域 IV.1,2中优先。即使在基础连接建立后, 轴突和突触端子也会根据环境变化而重建3,4。然而, 如何动态改变 TC 轴突形态是很不清楚的。其中一个主要原因是缺乏足够的技术来观察单个细胞水平的结构变化。虽然最近的显微学, 如双光子显微学的发展, 允许直接观察活体皮层神经元在体内, 但仍有技术上的限制, 捕获的总体 TC 轨道5,6. 因此,体外方法对 TC 轴突进行实时成像, 将为轴突分支和突触形成的结构分析提供有力的工具。
我们的小组首次建立了具有渗透性膜的静态切片培养方法7。使用这种方法, 鼠皮质切片被共培养的感觉丘脑块, 和叶片特定的 TC 连接概括在这个脊髓 cocultures 7, 8.稀疏标记与荧光蛋白进一步允许我们观察 TC 轴突生长和分支形成9,10,11。最近, 我们开发了一种新的方法, 同时成像分支和突触形成的个别 TC 轴突在脊髓 cocultures12。为了同时可视化 TC 轴突和突触前的地点, DsRed 和 EGFP 标记突触素 (SYP EGFP) 通过电穿孔脊髓共培养与小数目的丘脑神经元联合转染。目前的方法有利于 TC 轴突的形态学分析和长期观察, 可用于显示轴突分支与突触形成之间的因果关系。

<table><tbody><tr><td>DMEM/F12</td><td>GIBCO</td><td>11320-033</td><td></td></tr><tr><td>Hanks&amp;rsquo; balanced salt solution (HBSS)</td><td>Nissui</td><td>5905</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Scientific</td><td>SH30396-03</td><td>Hyclone</td></tr><tr><td>Insulin</td><td>Sigma</td><td>I6634</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma&amp;nbsp;</td><td>H0888</td><td></td></tr><tr><td>Sodium selenite</td><td>Wako Pure&lt;br/&gt; Chemical Industries</td><td>192-10843</td><td></td></tr><tr><td>Transferrin&amp;nbsp;</td><td>Sigma</td><td>T1147</td><td></td></tr><tr><td>Putrescine&amp;nbsp;</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>Glucose</td><td>Wako&lt;br/&gt; Pure Chemical Industries</td><td>16806-25</td><td></td></tr><tr><td>35 mm petri dishes</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>Millicell-CM insert</td><td>Millipore</td><td>PICMORG50</td><td></td></tr><tr><td>100 mm petri dishes</td><td>BIO-BIK</td><td>I-90-20</td><td>petri dish sterrile</td></tr><tr><td>HiPure Plasmid Maxiprep Kit</td><td>Invitrogen</td><td>K210006</td><td></td></tr><tr><td>Disposable sterile plastic pipettes</td><td></td><td>202-IS</td><td>transfer pipets sterile</td></tr><tr><td>Glass capillary: OD 1.2 mm</td><td>Narishige</td><td>&amp;nbsp;G-1.2</td><td>inner diameter, 1.2 mm</td></tr><tr><td>Silver wire: 0.2 and 1 mm&amp;nbsp;</td><td>Nilaco</td><td>AG-401265 (diameter, 0.2 mm), AG-401485 (diameter, 1.0 mm)</td><td></td></tr><tr><td>1 mL syringe</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Stimulator&amp;nbsp;</td><td>A.M.P.I</td><td>Master 8</td><td></td></tr><tr><td>Biphasic isolator&amp;nbsp;</td><td>BAK ELECTRONICS</td><td>BSI-2</td><td></td></tr><tr><td>Amplifier&amp;nbsp;</td><td>A-M Systems</td><td>Model 1800</td><td></td></tr><tr><td>Oscilloscope</td><td>Hitachi</td><td>VC-6723</td><td></td></tr><tr><td>Manipulator</td><td>Narishige</td><td>SM-15</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>MO-10</td><td></td></tr><tr><td>Stereomicroscope&amp;nbsp;</td><td>Olympus</td><td>SZ40</td><td></td></tr><tr><td>Universal stand&amp;nbsp;</td><td>Olympus</td><td>SZ-STU2</td><td></td></tr><tr><td>Light illumination system&amp;nbsp;</td><td>Olympus</td><td>LG-PS2, LG-DI, HLL301</td><td></td></tr><tr><td>Electrode puller&amp;nbsp;</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>Digital eclipse C1 laser</td><td></td></tr><tr><td>x20 objective</td><td>Nikon</td><td>ELWD 20x/0.45</td><td></td></tr><tr><td>Culture chamber</td><td>Tokai Hit</td><td>UK A16-U</td><td></td></tr><tr><td>Sprague-Dawley (SD) rat</td><td>Japan SLC and Nihon-Dobutsu</td><td></td><td></td></tr><tr><td>Microsurgery scissors</td><td>Natsume&amp;nbsp;</td><td>MB-54-1</td><td></td></tr></tbody></table>

visualization of thalamocortical axon branching and synapse formation in organotypic cocultures

轴突分支和突触形成是建立精确神经回路的关键过程。在发育过程中, 感觉丘脑-皮质 (TC) 轴突形成分支和突触在特定层的大脑皮层。尽管轴突分支与突触形成之间存在明显的空间相关性, 但它们之间的因果关系却不甚明了。为了解决这个问题, 我们最近开发了一种方法, 同时成像的分支和突触形成的个别 TC 轴突在脊髓 cocultures。
本协议描述了一种由脊髓共培养和电穿孔组合组成的方法。丘脑和大脑皮层的脊髓 cocultures 促进基因操纵和轴突过程的观察, 保留特征结构如层流结构。用电穿孔技术将两种不同的质粒编码 DsRed 和 EGFP 标记的突触素 (SYP EGFP) 与小数目的丘脑神经元共转染。这种方法使我们能够同时可视化 TC 神经元和它们的突触前点的单个轴突形态。该方法还能够长期观察, 揭示轴突分支与突触形成之间的因果关系。

本文的实验目的是揭示 TC 轴突分支与突触形成之间的关系。为了同时可视化突触前的轴突轨迹和位置, 脊髓 cocultures 中的单个或几个丘脑细胞被转染为 SYP EGFP 和 DsRed 使用电穿孔的两种质粒。在文化的第二周中, DsRed (图 3) 清楚地标记了单独区分的 TC 轴突。只有显示 DsRed 和 SYP-EGFP 的轴突被选择观察。标记 TC 轴突侵入皮层切片和形成分支主要在上层, ?...

Watch this Scientific Journal Video about Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures at JoVE.com

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

本协议描述了丘脑和大脑皮层脊髓 cocultures 中丘脑-皮质轴突分支和突触形成同时成像的方法。单个丘脑-皮质轴突及其突触前终端的可视化细胞电穿孔技术与 DsRed 和 GFP 标记突触素。

脊髓 Cocultures 丘脑-皮质轴突分支和突触形成的可视化研究

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

visualization-thalamocortical-axon-branching-synapse-formation

Research

JoVE Journal

Neuroscience

6.4K 次观看.  Kanazawa University. 本协议描述了丘脑和大脑皮层脊髓 cocultures 中丘脑-皮质轴突分支和突触形成同时成像的方法。单个丘脑-皮质轴突及其突触前终端的可视化细胞电穿孔技术与 DsRed 和 GFP 标记突触素。

视频: Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

与器官型脑片和癌细胞的3D球体共培养系统

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

科研

医学

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

一个多室中枢神经系统的神经胶质细胞共培养的微流体平台

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

生物学

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

发育生物学

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

生物工程

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

基于2D和3D人诱导多能干细胞的模型，用于剖析新皮质发育过程中原发性纤毛受累

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

修改一个Colliculo  - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

神经科学

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

扁平哺乳动物皮层皮质模块的可视化研究

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

莱纳尔皮质-希波坎普斯有机切片文化中的癫痫发生模型

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

<meta charset="utf8"></head><body>小鼠感觉皮层发育中的神经元活动映射

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following the discovery of numerous genes contributing to these developmental changes, it is now known that neuronal activity also substantially modulates these processes. In the developing cerebral cortex, neurons exhibit synchronized activity patterns that are specialized to each primary sensory area. These patterns markedly differ from those observed in the mature cortex, emphasizing their role in regulating area-specific developmental processes. Deficiencies in neuronal activity during development can lead to various brain diseases. These findings highlight the need to examine the regulatory mechanisms underlying activity patterns in neuronal development. This paper summarizes a series of protocols to visualize primary sensory areas and neuronal activity in neonatal mice, to image the activity of individual neurons within the cortical subfields using two-photon microscopy in vivo, and to analyze subfield-related activity correlations. We show representative results of patchwork-like synchronous activity within individual barrels in the somatosensory cortex. We also discuss various potential applications and some limitations of this protocol.

<meta charset="utf8"></head><body>作者聚焦：从双光子显微镜和单神经元成像破译神经回路形成

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

在单神经元分辨率下新生小鼠感觉皮层自发活动的体内可视化

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...

脊髓 Cocultures 丘脑-皮质轴突分支和突触形成的可视化研究

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在单神经元分辨率下新生小鼠感觉皮层自发活动的体内可视化

脊髓 Cocultures 丘脑-皮质轴突分支和突触形成的可视化研究

摘要

探索更多视频

此视频中的章节

与器官型脑片和癌细胞的3D球体共培养系统

一个多室中枢神经系统的神经胶质细胞共培养的微流体平台

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

基于2D和3D人诱导多能干细胞的模型，用于剖析新皮质发育过程中原发性纤毛受累

修改一个Colliculo - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

扁平哺乳动物皮层皮质模块的可视化研究

莱纳尔皮质-希波坎普斯有机切片文化中的癫痫发生模型

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

在单神经元分辨率下新生小鼠感觉皮层自发活动的体内可视化

原位急性离体胚胎脑切片培养物中轴突生长和生长锥动力学的可视化