Name: visualization and live imaging of oligodendrocyte organelles in organotypic brain slices using adeno-associated virus and confocal microscopy
Uploaded: 2017-10-23T12:30:00.000+00:00
Duration: 8 min 38 s
Description: Watch this Scientific Journal Video about Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy at JoVE.com

我们感谢琳达 Hildegard 博格森和 Magnar Bjørås 获得细胞实验室和设备, Janelia 分子生物学共享资源人员的质粒和病毒生产和公园 Vervaeke, 以协助激光功率测量。这项工作由挪威卫生协会资助, 挪威研究理事会和显微镜设备由 Norbrain 资助。

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作者声明没有利益冲突。

此处描述的器官区域性的协议是由 De Simoni 和 Yu (2006)5所描述的协议的修改版本18 。下文概述了最重要的变化。培养基中添加了三个缓冲液, 在病毒转导和细胞培养基的变化过程中, 提高了孵化室外的切片成活率。纸屑的灭菌程序也发生了变化。虽然其他协议消毒五彩纸屑由灭菌, 我们不建议这样做, 因为它会导致几个五彩纸屑卷曲。避免使用卷曲的纸屑, 因为在这些片上生长的切片往往不太健康。我们的协议使用小鼠, 而不是老鼠, 并被修改, 以培养皮层而不是海马, 我们发现在更薄的切片 (230 µm vs 300 µm) 很好的工作。当鞘高峰在出生日附近 10-20 在老鼠和老鼠19, 培养由老鼠在 p7-9 (然后培养 11-14 天) 是优选的为研究形成突。在 11-14 天的体外, 文化中含有几个成熟的突与致密髓鞘13。在文化周期结束时, 由较年轻的老鼠组成的培养物将含有较少的成熟突, 而由较年长的老鼠所制作的文化更具挑战性, 以保持健康的20。
制作器官切片的过程涉及几个关键步骤。大脑的解剖和安装, 以及把切片放在五彩纸屑上, 必须做得快, 需要一些技巧。通常, 第一次尝试使器官切片的文化是不太成功的, 但2-3 轮的实践应该足以获得技能, 创造健康的文化所需要的。在整个过程中保持无菌环境是很重要的, 以避免对文化的污染。此外, 在使用活细胞时, 必须确保正确的 pH 值和渗透的解决方案, 以保持细胞健康。因此, 这是一个很好的建议, 以检查渗透 (应该是 270-310 mOsm/千克) 和 pH 的所有解决方案, 在切片上使用, 特别是在做实验的第一次。文化和解剖解答为器官文化包含 ph 指示 (酚红色), 但黄色血清在文化媒介能给印象比实际 ph 值低。培养基中含有青霉素、链霉素和制, 以尽量减少感染的几率。因此, 建议在实验室中建立器官切片培养技术时使用这些抗生素和药物, 但在应用专家无菌技术后, 可以避免这种方法。目前的协议使用细胞形态学来评估细胞的生存能力。对于更定量的检查, 切片可以加载碘碘化物或类似的染料在其他地方描述21。然而, 重要的是要知道, 碘碘的发射光谱重叠与 dsred, tdtomato 和其他红色指标。碘碘化物和类似试剂的另一个限制是, 它们不容易进入切片的更深层, 从而限制了对细胞的活性的评估到最上层的单元格5。
基因传递的方法多种多样。利用器官切片培养的 AAV 转导来可视化器的主要优点如下: AAV 转导与突转染方法相比, postmitotic 病毒的成功率更高.22。此外, 在器官文化中所有的脑细胞类型都存在, 而突有一个形态学类似于在体内, 包括致密髓鞘13。这是不同的突在单养, 缺乏与轴突的沟通, 从而不做致密髓鞘。成像在体外比较容易与在体内和提供更好的空间分辨率, 这是特别感兴趣的, 当成像小细胞器, 如线粒体。未来的研究将着眼于图像的在体内, 但面临着巨大的挑战, 由脂质丰富的髓鞘引起的光学畸变。此外, AAV 血清型取向和启动子特异性可能在体内和体外条件13,23之间有所不同。在这里提出的协议, AAV2/8 MBP_mito_dsred 和 AAV2/8 MBP_mito_GFP 是用来可视化胶质线粒体。AAV 血清型2/1 也测试了 MBP_mito_dsred, 但感染了较低数量的突在器官切片 (没有显示)。AAV2/8 CAG_GFP 和 AAV2/8 CAG_tdtomato 用于可视化胶质细胞质。尽管使用一般 CAG 启动子为此, AAV 2/8 CAG_GFP 和 CAG_tdtomato 选择性感染突, 只有少数的星形胶质细胞和神经元被感染 (这些可以很容易地识别其特征形态)。突的这种选择性可能是由于 AAV 2/8 型血清型24的取向。然而, 对于细胞蛋白靶向表达, 更高的特异性达到了与碱性髓鞘或其他胶质特定的促进剂推荐。其他由我们 CAG_GFP 的血清型 AAV 2/1, 2/2, 2/5, 2/7, 2/9, 其中大部分感染主要神经元和星形胶质细胞 (未显示)。
根据结构和实验条件, 需要对细胞转导进行修正。如果转细胞显得太暗淡, 尝试更长的时间在体外转导后增加表达。如果细胞出现明亮但不健康, 减少转导后的时间。如果细胞转很少, AAV 的浓度可以增加。然而, 也有可能是在体外转导时间过长, 转细胞已经死亡。对于这里使用的结构, 表达水平是最佳的 4-6 天后转导。在 7-8 天后转导, 细胞出现不健康, 与点的过程, 和10天后转导, 只有少数转细胞是可见的 (没有显示)。
本协议使用直立显微镜对器官切片进行实时成像。这是不同于大多数单一和 co-cultures, 通常使用倒置显微镜。这是不理想的使用倒置显微镜在这里, 因为切片必须然后用五彩纸屑面对向上, 这想必会导致切片是不太健康。直立显微镜的一个优点是有可能增加电生理的设置。此外, 使用泵改变介质意味着药物解决方案可以很容易地添加和删除在时间推移成像。水泵的缺点是在浴缸中保持振动和电流引起的焦点问题。在显微镜下, 细胞会逐渐变少健康.因此, 我们将切片放在显微镜下最多1小时。影像切片被丢弃 (特殊废料)。为了确保成像条件的最佳, 控制细胞的成像, 其中线粒体运动已经很好的特征, 应该并行运行。例如, 在轴突或树突中的线粒体运动可以通过换切片 AAV 2/1 Syn_mito_dsred (其中 dsred 表达由神经元特异的突启动子) 或 AAV 2/1 CAG_mito_dsred (随后免疫确认单元格标识)。线粒体运动的分析可以使用 ImageJ 中的多 kymograph 工具进行, 如别处所述25。
在这里, 我们描述核心的可视化不髓鞘在实时成像。与比分9, 使用三励磁波长, 核心只使用一 (488 毫微米) 并且因而是更加简单的实施。在分数, 三波长每揭示不同的 "片断" 髓鞘并且一起给鞘的一个充分的图象。此外, 评分可用于检测髓鞘结构, 如细胞质袋。随着单波长使用的核心, 一个较不详细的看法和鞘可能出现颗粒状或不连续 (图 3)。然而, 核心可视近100% 的髓鞘, 是横向的视野。因此, 核心是有用的检测髓鞘, 但不是分析髓鞘结构或完整性。

大脑的白质是由髓鞘包裹的神经细胞轴突组成的, 这是由突所形成的一种专门的扩展等离子体膜。髓鞘是需要快速和可靠的动作电位传播和长期生存的髓轴突, 并失去髓鞘可能导致神经功能障碍。尽管它们的重要性, 与神经元和星形胶质细胞相比, 突的性质是不为人知的。因此, 为研究突而开发的工具越来越少。
活体成像的细胞器, 如线粒体, endoplasmatic 网 (ER) 或不同的水泡结构, 可以用来研究动态变化, 随着时间的推移。传统上, 生活突的成像已经在单一1,2中执行。然而, 突在单养不显示致密髓鞘, 因此, 器官或急性脑切片可能是一个更好的选择, 在研究的本地化和运动的细胞器。由于髓轴突与周围髓鞘之间的短距离, 髓鞘内的小细胞器和蛋白质的定位可能具有挑战性。因此, 光是显微免疫程序就没有空间分辨率来区分髓鞘内的细胞器和髓轴突的细胞。这可以通过病毒转导与细胞型特异性促进剂驱动的器官靶向荧光蛋白的基因来解决。其优点是细胞特异性和稀疏表达式, 能够准确地评估器官的定位和动态。转基因动物也可以用来实现这样一个以细胞为目标的特定表达式3。然而, 转基因动物的生产和维护费用昂贵, 通常不提供可通过病毒方法实现的稀疏表达。
本文所描述的方法使用的病毒转导突与线粒体靶向荧光蛋白 (dsred 或绿色荧光蛋白, gfp) 驱动的髓鞘碱性蛋白促进剂 (细胞内 dsred 或绿细胞-gfp), 以可视化器官脑切片中的胶质线粒体。此外, 另一种荧光蛋白在细胞质中的表达 (无论是 gfp 与 dsred 或 tdtomato 与美图-gfp 一起使用) 是用来使细胞形态学的可视化, 包括髓鞘鞘的细胞质。该协议包括制作器官脑切片的程序 (由 De Simoni 和 Yu 描述的修改后的协议版本, 20064,5)。然后我们描述了用于研究线粒体运动的延时成像程序。这个过程使用一个直立的共焦显微镜与连续交换成像介质, 设置, 使易于应用的药物或其他媒体的变化, 在成像。延时成像程序可以在任何共聚焦显微镜下进行, 有些额外的设备用于维持活体切片, 如下所述。该协议还包含一些优化成像和减少光的提示。
最后, 本文介绍了一种快速、简便的共焦反射显微镜 (CoRe) 可视化不髓鞘的方法。这可能是有用的, 以确定髓鞘鞘的实时成像。近年来, 一些技术已经发展到图像髓鞘不需要任何染色, 但其中大多数需要特定的设备和专长6,7,8。这里描述的过程使用髓鞘的反射的物产并且是一个简单的 single-excitation 波长版本光谱共焦反射显微镜 (比分, 几 laser 波长被结合形象化髓鞘)9. 核心可以在具有 488 nm 激光和 470-500 nm 带通发射过滤器或可调谐发射过滤器的任何共焦显微镜上进行。

<table><tbody><tr><td>Agarose&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>A9539</td><td></td></tr><tr><td>BD Microlance 19G</td><td>BD</td><td>301500</td><td>Needles used for in- and outlet of bath</td></tr><tr><td>Bioxide gas</td><td>AGA</td><td>105701</td><td></td></tr><tr><td>Brand pipette bulbs</td><td>Sigma-Aldrich</td><td>Z615927</td><td>Pipette bulbs</td></tr><tr><td>Bunsen burner (Liquid propane burner)</td><td>VWR</td><td>89038-530</td><td></td></tr><tr><td>Cable assembly for heater controllers</td><td>Warner Instruments</td><td>64-0106&amp;nbsp;</td><td>Temperature controller - thermometer part</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fluka</td><td>21100</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>AGA</td><td>100309</td><td>CO&lt;sub&gt;2&lt;/sub&gt; for incubator</td></tr><tr><td>Cover glass, square Corning</td><td>Thermo&amp;nbsp; Fischer Scientific</td><td>13206778</td><td>To attach under bath for live imaging. Seal with glue or petrolium jelly.</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7021</td><td></td></tr><tr><td>Delicate forceps</td><td>Finescience</td><td>11063-07</td><td>For dissection</td></tr><tr><td>Diamond scriber pen</td><td>Ted Pella Inc.</td><td>54463</td><td></td></tr><tr><td>Disposable Glass Pasteur Pipettes 230 mm</td><td>VWR</td><td>612-1702</td><td>Glass pipettes</td></tr><tr><td>Double edge stainless steel razor blade</td><td>Electron Microscopy Sciences</td><td>#7200</td><td>Razor blade for vibratome</td></tr><tr><td>Earle&#039;s Balanced Salt Solution (EBSS)</td><td>Gibco-Invitrogen</td><td>24010-043</td><td></td></tr><tr><td>Filter paper circles</td><td>Schleicher &amp;amp; Schuell</td><td>300,220</td><td>Filter paper used for filtration of PFA</td></tr><tr><td>Fun tack</td><td>Loctite</td><td>1270884</td><td>Use to connect/adjust position of in- and outlets in bath</td></tr><tr><td>Hand towel C-Fold 2</td><td>Katrin</td><td>344388</td><td></td></tr><tr><td>Harp, Flat for RC-41 Chamber,</td><td>Warner Instruments</td><td>&amp;nbsp;64-1418</td><td>Harp to hold down confetti in bath. Cut off strings before use with organotypic slices. 1.5 mm, 13mm, SHD-41/15</td></tr><tr><td>HEPES, FW: 260.3</td><td>Sigma</td><td>H-7006</td><td></td></tr><tr><td>Holten LaminAir, Model 1.2</td><td>Heto-Holten</td><td>96004000M</td><td>Laminar flow hood</td></tr><tr><td>Horse serum, heat inactivated</td><td>Gibco-Invitrogen</td><td>26050-088</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>LCR Membrane, PTFE,&amp;nbsp;</td><td>Millipore</td><td>FHLC0130</td><td>Confetti&amp;nbsp;</td></tr><tr><td>Leica VT1200</td><td>Leica</td><td>14048142065</td><td>Vibratome</td></tr><tr><td>MEM-Glutamax with HEPES</td><td>Thermo&amp;nbsp; Fischer Scientific</td><td>42360024</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>R.P. Normapur</td><td>25 108.295</td><td></td></tr><tr><td>Micro Spoon Heyman Type B</td><td>Electron Microscopy Sciences</td><td>62411-B</td><td>Small, rounded spatula with sharpened end for dissection</td></tr><tr><td>Millex-GP filter unit</td><td>Millipore</td><td>SLGPM33RA</td><td>Syringe filter unit</td></tr><tr><td>Millicell cell culture insert, 30 mm</td><td>Millipore</td><td>PICM03050</td><td>Cell culture inserts</td></tr><tr><td>Minipuls 3 Speed Control Module</td><td>GILSON</td><td>F155001</td><td>Peristaltic pump for live imaging - Control module part (connect to two-cannel head)</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S7907</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S8282</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fluka</td><td>71628</td><td></td></tr><tr><td>Nunclon Delta Surface</td><td>Thermo&amp;nbsp; Fischer Scientific</td><td>140675</td><td>Culture plate</td></tr><tr><td>Nystatin Suspension</td><td>Sigma-Aldrich</td><td>N1638</td><td></td></tr><tr><td>Objective W &quot;Plan-Apochromat&quot; 40x/1.0 DIC&amp;nbsp;</td><td>Zeiss</td><td>441452-9900-000&amp;nbsp;</td><td>Water immersion objective used for live imaging. (WD=2.5mm), VIS-IR</td></tr><tr><td>Parafilm</td><td>VWR</td><td>291-1211</td><td></td></tr><tr><td>Paraformaldehyde, granular</td><td>Electron Microscopy Sciences</td><td>#19208</td><td></td></tr><tr><td>PC-R perfusion chamber</td><td>SiSkiYou</td><td>&amp;nbsp;15280000E</td><td>Bath for live imaging</td></tr><tr><td>Penicillin-Streptomycin, liquid</td><td>Invitrogen</td><td>15070-063</td><td></td></tr><tr><td>Petri dish 140 mm&amp;nbsp;</td><td>Heger</td><td>1075</td><td>Large Petri dish&amp;nbsp;</td></tr><tr><td>Petri dish 92x16 mm&amp;nbsp;</td><td>Sarstedt&amp;nbsp;</td><td>82.1473</td><td>Medium Petri dish&amp;nbsp;</td></tr><tr><td>Petridish 55x14,2 mm</td><td>VWR</td><td>391-0868</td><td>Small Petri dish</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma</td><td>P4417</td><td>PBS tablets</td></tr><tr><td>R2 Two Channel Head&amp;nbsp;</td><td>GILSON</td><td>F117800</td><td>Peristaltic pump for live imaging - Two channel head part (requires control module)</td></tr><tr><td>Round/Flat Spatulas, Stainless Steel</td><td>VWR</td><td>82027-528</td><td>Large spatula for dissection</td></tr><tr><td>Sand paper</td><td>VWR</td><td>MMMA63119</td><td>Optional, for smoothing broken glass pipettes</td></tr><tr><td>Scissors,&amp;nbsp; 17,5 cm</td><td>Finescience</td><td>14130-17</td><td>Large scissors for dissection</td></tr><tr><td>Scissors, 8,5&amp;nbsp;</td><td>Finescience</td><td>14084-08</td><td>Small, sharp scissors for&amp;nbsp; dissection</td></tr><tr><td>Single edge, gem blade</td><td>Electron Microscopy Sciences</td><td>#71972</td><td>Single edge razor blade</td></tr><tr><td>Single inline solution heater SH-27B</td><td>Warner Instruments</td><td>64-0102</td><td>Temperature controller - heater part</td></tr><tr><td>Steritop-GP Filter unit, 500 ml , 45mm</td><td>Millipore</td><td>SCGPT05RE</td><td>Filter to sterilize solutions</td></tr><tr><td>Super glue precision</td><td>Loctite</td><td>1577386</td><td></td></tr><tr><td>Surgical scalpel blade no. 22</td><td>Swann Morton Ltd.</td><td>209</td><td>Rounded scalpel blade</td></tr><tr><td>Temperature controller TC324B</td><td>Warner Instruments</td><td>64-0100</td><td>Temperature controller for live imaging (requires solution heater and cable assembly)</td></tr><tr><td>Trizma base</td><td>Sigma&amp;nbsp;</td><td>T1503</td><td></td></tr><tr><td>Trizma HCl</td><td>Sigma</td><td>T3253</td><td></td></tr><tr><td>Water jacketed incubator series II</td><td>Forma Scientific</td><td>78653-2882</td><td>Incubator</td></tr></tbody></table>

visualization and live imaging of oligodendrocyte organelles in organotypic brain slices using adeno-associated virus and confocal microscopy

神经元依赖于形成突的电绝缘和营养支持。尽管突的重要性, 目前用于研究神经元的先进工具, 只是部分被胶质研究人员所接受。细胞类型特异性染色的病毒转导是一个有用的方法来研究活体细胞动力学。本文介绍了一种在转录控制下利用腺相关病毒 (AAV) 携带线粒体靶向荧光蛋白的基因对器官脑切片中胶质线粒体进行可视化的协议髓鞘碱性蛋白启动子。包括制作器官冠状小鼠脑切片的协议。一个过程中的线粒体的延时成像, 然后跟随。这些方法可以转移到其他的细胞器, 可能是特别有用的研究细胞在髓鞘。最后, 我们描述了一个现成的技术, 可视化的活体切片不髓鞘的共聚焦反射显微镜 (核心)。核心不需要额外的设备, 并可以用于识别髓鞘鞘在现场成像。

器官脑切片的培养和转如上所述, 显示了稀疏分布的皮质突表达 mito_dsred 和 GFP。免疫与抗体抗 Olig2 和碱性髓鞘证实, 表达特定于突 (图 1)。
对于活体成像, 转突是公认的几个平行运行的髓鞘鞘的特征形态学 (图 1和图 2A)。通过在转突上执行延时成像, 可以监测主要过程和髓鞘 (图 2B-D) 中的线粒体运动。
在低 GFP 表达的细胞中, 髓鞘可以在活成像过程中通过照亮样品在 488 nm 和捕获发光 470-500 nm (图 3A) 中识别。这种共焦反射显微镜 (核心) 技术也工作在粉煤灰固定和 immunostained 组织, 虽然髓鞘出现变暗 (图 3B-C)。通过与抗髓鞘碱性蛋白抗体免疫, 我们发现接近100% 的髓鞘水平到成像平面 (由箭头指示,图 3B-C) 是可见的与核心, 而髓鞘垂直于视图的字段 (由星号表示,图 3B-C) 的位置更少或根本不可见。虽然几乎所有节间都是可视化的, 但许多节间距出现不连续, 鞘的小部分不可见 (图 3A插入)。髓鞘中的这些小的 "间隙" 可以通过三互补波长的成像来填充, so-called 分数为9。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56237/56237fig1.jpg"> 
图 1:免疫组织化学证实, dsred 在突有选择性表达, 对 Olig2 和碱性髓鞘均具有免疫力。 图像来自培养的小鼠脑切片转与 (AAV2/8) dsred (红色, 线粒体标记) 和 CAG-GFP (绿色, 胞)。切片为髓鞘标记髓鞘碱性蛋白 (immunolabeled, 蓝) 和 Olig2 (胶质谱系细胞核标记, 品红)。(从引用13中修改的图)<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56237/56237fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56237/56237fig2.jpg"> 
图 2: 在突中跟踪线粒体运动的时间推移成像.(A) 投影 z 堆叠 (光学部分10.35 µm) 的胶质转与 MBP_mito_dsred (红色线粒体) 和 CAG_GFP (绿色荧光)。(B) 同一个单元格的单个共焦图像 (光学截面1.04 µm)。正方形表示为时移成像 (C) 选择的区域, 并指示在 (D) 中绘制 kymographs (许姆) 的线。(C) 图像来自在不同时间点拍摄的 a 和 B 中的单元格的延时记录。移动的线粒体被表明 (红色箭头)。(D) 从 (B) 中所示的轨迹 Kymographs。固定的线粒体被视为直线垂直线, 移动的线粒体被视为对角线。正如可以看到在 kymographs, 并在时效图像 (C), 只有线粒体在主要过程中标记 Kymograph 1 (许姆 1) 正在移动。其他三 kymographs 的线粒体在延时记录期间是静止的。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56237/56237fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56237/56237fig3.jpg"> 
图 3: 不髓鞘的共聚焦反射显微镜 (核心) 的可视化.(a) 从活体器官脑切片的大脑皮层投射 z 堆叠 (光学切片10.35 µm)。在这里显示的胶质中的 GFP 表达 (绿色细胞体) 太暗淡, 无法识别主要过程和髓鞘。相反, 髓鞘被激发在 488 nm, 捕捉发光光在 470-500 nm (以洋红显示)。一些富含细胞质的 paranodes 的 GFP + 胶质可以看到在髓鞘节间 (箭头) 的提示。插入: 髓鞘的放大部分显示髓鞘形象化与核心出现 "参差不齐" 或不连续 (参见主要文本为细节)。(B-C)共聚焦图像从纹状体 (B) 和海马 CA1 (C) 的层 radiatum 的一个急性小鼠脑切片, 已固定在4% 粉煤灰和 immunostained 髓鞘碱性蛋白 (碱性)。Horisontal 髓鞘 (箭头) 是可见的与核心, 而鞘是垂直于视野 (星号) 是最经常不可见的。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56237/56237fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
试剂 "表:<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56237/REAGENTS_table_Kennedy_and_Rinholm_april.xlsx" target="_blank">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy at JoVE.com

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

形成突促进了快速动作电位的传播和神经元的存活。这里描述的是一个协议的胶质特异表达荧光蛋白在器官脑切片与后续的延时成像。此外, 一个简单的程序, 可视化不髓鞘提出。

腺相关病毒和共聚焦显微镜对器官脑切片胶质细胞器的可视化和实时成像

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

visualization-live-imaging-oligodendrocyte-organelles-organotypic

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

10.8K 次观看.  University of Oslo. 形成突促进了快速动作电位的传播和神经元的存活。这里描述的是一个协议的胶质特异表达荧光蛋白在器官脑切片与后续的延时成像。此外, 一个简单的程序, 可视化不髓鞘提出。

视频: Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

颅内注射腺相关病毒载体

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

科研

神经科学

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

一个<em&gt;体外</em&gt;急性脑片的少突胶质细胞定向T细胞攻击模式

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

免疫与感染

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 ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

封闭系统中脑切片长期培养中精确定位和重复间歇成像的改进辊筒法

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A large share of ATP is needed to re-install ion gradients across plasma membranes degraded by electrical signaling of neurons. There is evidence that astrocytes - while not generating fast electrical signals themselves - undergo increased production of ATP in response to neuronal activity and support active neurons by providing energy metabolites to them. The recent development of genetically encoded sensors for different metabolites now enables the study of such metabolic interactions between neurons and astrocytes. Here, we describe a protocol for cell-type specific expression of the ATP-sensitive Fluorescence Resonance Energy Transfer- (FRET-) sensor ATeam1.03YEMK in organotypic tissue slice cultures of the mouse hippocampus and cortex using adeno-associated viral vectors (AAV). Furthermore, we demonstrate how this sensor can be employed for dynamic measurement of changes in cellular ATP levels in neurons and astrocytes upon increases in extracellular potassium and following induction of chemical ischemia (i.e., an inhibition of cellular energy metabolism).

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

We describe a protocol for cell-type specific expression of the genetically encoded FRET-based sensor ATeam1.03YEMK in organotypic slice cultures of the mouse forebrain. Furthermore, we show how to use this sensor for dynamic imaging of cellular ATP levels in neurons and astrocytes.

使用基于 FRET 的传感器 ATeam1.03YEMK在小鼠大脑的组织切片中进行细胞内 ATP 成像

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

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 ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding and treatment of ocular diseases. This study introduces an optimized adeno-associated virus (AAV) approach, utilizing specific serotypes and promoters to achieve optimal transfection efficiency in targeted retinal cells, including retinal ganglion cells (RGCs) and M&#252;ller glia. Leveraging the precision of confocal scanning laser ophthalmoscopy (CSLO), this work presents a non-invasive method for in vivo imaging that captures the longitudinal expression of AAV-mediated green fluorescent protein (GFP). This approach eliminates the need for terminal procedures, preserving the continuity of observation and the well-being of the subject. Furthermore, the GFP signal can be traced in AAV-infected RGCs along the visual pathway to the superior colliculus (SC) and lateral geniculate nucleus (LGN), enabling the potential for direct visual pathway mapping. These findings provide a detailed protocol and demonstrate the application of this powerful tool for real-time studies of retinal cell behavior, disease pathogenesis, and the efficacy of gene therapy interventions, offering valuable insights into the living retina and its connections.

This protocol describes the use of adeno-associated virus (AAV) vectors for cell-specific labeling and in vivo imaging using a confocal scanning laser ophthalmoscope (CSLO). This method enables the investigation of different retinal cell types and their contributions to retinal function and disease.

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding ...

腺相关病毒和共聚焦显微镜对器官脑切片胶质细胞器的可视化和实时成像

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颅内注射腺相关病毒载体

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

一个

器官切片文化研究少突胶质细胞动力学和髓鞘

封闭系统中脑切片长期培养中精确定位和重复间歇成像的改进辊筒法

使用基于 FRET 的传感器 ATeam1.03^YEMK在小鼠大脑的组织切片中进行细胞内 ATP 成像

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

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

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

腺相关病毒和共聚焦显微镜对器官脑切片胶质细胞器的可视化和实时成像

摘要

探索更多视频

此视频中的章节

颅内注射腺相关病毒载体

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

一个

器官切片文化研究少突胶质细胞动力学和髓鞘

封闭系统中脑切片长期培养中精确定位和重复间歇成像的改进辊筒法

使用基于 FRET 的传感器 ATeam1.03YEMK在小鼠大脑的组织切片中进行细胞内 ATP 成像

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

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

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

使用基于 FRET 的传感器 ATeam1.03^YEMK在小鼠大脑的组织切片中进行细胞内 ATP 成像

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