Name: ex vivo oculomotor slice culture from embryonic gfp-expressing mice for time-lapse imaging of oculomotor nerve outgrowth
Uploaded: 2019-07-16T15:00:00
Description: Watch this Scientific Journal Video about Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth at JoVE.com

资金由国家眼科研究所 [5K08EY027850], 国家儿童健康与发展研究所 [U54HD090255], 哈佛-视觉临床科学家发展计划 [5K12EY016335], 圣殿骑士眼科基金会 [职业入门者]格兰特*和儿童医院眼科基金会[教师发现奖]。欧洲经委会是霍华德·休斯医学研究所的调查员。

此处描述的所有动物工作均符合波士顿儿童医院机构动物护理和使用委员会 (IACUC) 协议的批准和执行。
1. 分时配接
<ol><li>放置ISLMN:GFP (岛运动神经元绿色荧光蛋白;MGI: J:132726;Jax Tg (Isl-EGFP®)1Slp/J 库存号: 017952) 雄性小鼠和雌性小鼠一起过夜。在交配前称量雌性并记录重量。 注:ISL MN:GFP专门标记运动神经元与无细胞毒性?...

<ol>
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这种外生切片培养方案与传统的斧子导样测定23相比具有显著优势。每个颅骨运动核的大小不是一个限制因素,不需要任何困难的解剖。维持斧子传播的内源微环境,允许修改一个信令通路,同时保持其他信令通路。此外,可以在沿斧子轨迹的不同点评估效果。由于斧子制导需要多个线索和线索的组合,沿着路径29,这提供了一个显著的优势。与分离或外植培养不同,这?...

目视电机系统为研究斧子导向机构提供了一个优雅的系统。它相对简单,包括三个颅神经内侧六个移动眼睛的眼外肌肉(EOMs),以及抬起眼睑的悬浮肌(LPS)。oculo运动神经内枢LPS和四个EOMs - 下等斜和中,下等,和优越的直肠肌肉。其他两种神经, 三角和腹肌, 每个只有内瓦特一个肌肉, 优越的斜和侧直肠肌肉, 分别.眼动提供了一个简单的读出,显示内侧是否适当、缺失或异常。此外,还有由于神经元发育或斧突指导的不足而导致的人眼运动障碍,统称为先天性颅内疾病(CCDDs)1。
尽管有这些优点,目状电机系统很少用于斧子制导研究2,3,4,5,6,7,8, 9,10,由于技术缺陷。体外斧子制导测定有许多缺点11。共培养性测定,其中神经元外植与目标组织12或转染细胞13的外植一起培养,取决于外植的对称性和外植与目标组织之间的精确定位。条纹测定14,15,其中两个线索被放置在交替条纹和斧子被评估为优先增长一个条纹,只表明一个基板优于另一个,而不是说两个是有吸引力的或排斥,或生理相关。微流体室可以形成精确的化学梯度,但受试者生长的斧子会剪切应力16,17,18,这会影响它们的生长。此外,在每种方法中,收集外植或分离的细胞需要对生长的轴虫进行轴状化,因此这些测定实际上检查轴子再生,而不是初始轴子生长。最后,这些体外方法消除了影响斧子的微环境及其对不同点的线索的反应,传统上只单独测试一个线索。使这些缺点更加复杂的是,眼部运动系统中每个原子核的体积小,使得解剖在技术上对植物外或分离的培养物都具有挑战性。此外,眼动神经元的主要培养物通常是异构的,有显著的细胞死亡,并且密度依赖,需要从多个胚胎中汇集细胞(藤井裕久,个人交流)。然而,由于需要时间和费用,体内方法(包括敲除小鼠模型)不适合用于筛选。
开发培养整个胚胎的方法19允许标记迁移细胞20或阻断特定分子21,但整个胚胎培养需要在滚筒瓶中孵育,从而排除了标记的实时成像结构。允许操纵胚胎,然后在子宫或母亲腹部(维持胎盘连接)22的外科技术,允许操纵胚胎,然后进一步发育22,但这些也不允许延时成像。
为了克服体外检测的障碍,允许快速筛选信号通路,23日开发了一种体外胚胎切片培养技术,该技术改编自先前公布的外周神经外生长方案24。使用该协议,在沿其轨迹的许多周围结构(包括EOM目标)存在的情况下,可以随时间而对发育中的oculo运动神经进行成像。通过在培养介质中添加小分子抑制剂、生长因子或引导提示,我们可以评估沿斧子轨迹的多个点的制导扰动,从而能够更快速地评估潜在的生长和指导因素。

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">24-Well Tissue Culture Plate</td>
			<td style="width:163px;">Genesee Scientific</td>
			<td style="width:344px;">25-107</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">6-Well Tissue Culture Plate</td>
			<td>Genesee Scientific</td>
			<td>25-105</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disposable Pasteur Pipet (Flint Glass)</td>
			<td>VWR</td>
			<td>14672-200</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11412-11</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fluorobrite DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1896701</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glucose (200 g/L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A2494001</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hank&#39;s Balanced Salt Solution (1X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14175-095</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heat Inactivated Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550H</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES Buffer Solution (1M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630106</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Glutamine (250 nM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Loctite Superglue</td>
			<td>Loctite</td>
			<td></td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Low Melting Point Agarose</td>
			<td>Thermo Fisher Scientific</td>
			<td>16520050</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Millicell Cell Culture Insert (30mm, hydrophilic PTFE, 0.4 um)</td>
			<td>Millipore Sigma</td>
			<td>PICM03050</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Moria Mini Perforated Spoon</td>
			<td>Fine Science Tools</td>
			<td>10370-19</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin/Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122&#160;</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Petri Dish (100 x 15mm)</td>
			<td>Genesee Scientific</td>
			<td>32-107G</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Phosphate Buffered Saline (1X, pH 7.4)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Razor Blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical Scissors - Blunt</td>
			<td>Fine Science Tools</td>
			<td>14000-12</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ti Eclipse Perfect Focus with TIRF</td>
			<td>Nikon</td>
			<td></td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome (VT 1200S)</td>
			<td>Leica</td>
			<td>1491200S001</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome Blades (Double Edge, Stainless Steel)</td>
			<td>Ted Pella, Inc.</td>
			<td>121-6</td>
			<td style="width:331px;"></td>
		</tr>
	</tbody>
</table>

ex vivo oculomotor slice culture from embryonic gfp-expressing mice for time-lapse imaging of oculomotor nerve outgrowth

精确的眼动对视力至关重要,但眼动系统的发展,特别是控制斧突引导的分子通路,尚未得到充分阐明。这部分是由于传统斧子制导测定的技术限制。为了识别影响奥库运动神经的其他斧子引导线索,开发了一种前体切片测定,以实时图像向眼睛生长的奥孔运动神经。E10.5 IslMN-GFP胚胎用于生成体外切片,将其嵌入到胶质中,在振动体上切片,然后在显微镜级顶培养箱中生长,用延时显微显微镜进行 24-72 h。从原子核到轨道的斧子生长的体内时间。小分子抑制剂或重组蛋白可以添加到培养培养物中,以评估不同斧头引导途径的作用。该方法的优点是维护轴子遍历的更多局部微环境,而不是对生长的轴子进行轴向,并沿其轨迹的多个点评估轴子。它还可以识别对斧子特定子集的影响。例如,对 CXCR4 的抑制会导致仍在中脑内的斧头生长,而不是口向生长,但已经退出的斧头不受影响。

正常结果:图1提供了实验的原理图。早在E9.5在小鼠,第一个斧头开始从oculmotor核26出现。通过E10.5,一种包含早期先驱神经元的迷法性神经,可以在中枢可见。E10.5的胚胎(即使在同一垃圾内)在神经向轨道前进的步数上存在显著差异,这可能是由于几个小时的发育差异。在正常子宫发育期间,第一个GFP阳性oculmotoraxons在接下来的18-24小时(E11.5)到达轨道/眼睛,然?...

Watch this Scientific Journal Video about Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth at JoVE.com

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

前体切片测定允许实时成像奥库运动神经外生。切片通过嵌入E10.5 Isl MN:GFP胚胎在生长中,在振动体上切片,并在一个顶的培养箱中生长。斧子引导通路的作用是通过向培养培养物添加抑制剂来评估的。

来自胚胎GFP-表达小鼠的Ex Vivo Oculo运动切片培养,用于对Oculo运动神经外生长进行时移成像

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

该协议使我们能够识别在奥库洛运动神经中活跃的轴顿制导通路，并实时评估它们在神经轨迹的不同点的作用。此技术保留了轴子旅行的本地环境及其最终目标。生长的轴子不切割，因此可以评估初始轴子的生成，而不是再生。该方法提供了对眼动系统中轴孔制导的见解，但可以适应其他神经轴翁制导的研究。这种技术需要实践来掌握，快速工作是非常重要的。第一次尝试时，只使用几个胚胎，而不是整个垃圾。在开始手术之前，首先在胚胎10.5的胚胎日通过超声波确认妊娠的时分交配。收获胚胎，用70%乙醇喷洒怀孕小鼠的腹部，用剪刀打开腹腔提取子宫。在将器官放入新鲜冰冷HSS的第二个培养皿中之前，将子宫用冰冷的HS培养皿中清洗子宫。在解剖范围内，从子宫角及其单个羊膜囊中去除胚胎，将每个胚胎放在12井板盖的底面，在收获时放在冰上。使用滤纸去除每个胚胎周围的任何液体，而不接触胚胎本身，将胚胎淹没在液体中4%的低熔化糖。将板盖放在冰上，以凝固加糖。当阿加罗斯变硬时，翻转胚胎，用额外的阿加罗斯覆盖每个样品的另一侧。当第二卷阿加罗斯凝固后，使用荧光解剖显微镜修剪每个胚胎周围的阿加罗斯，使每个胚胎在振动体阶段正确定向。胶体运动核和早期轴龙生长应该是荧光的。对齐每个胚胎，使核，外生长的轴子，和眼睛形成一条线，并使用剃刀刀片修剪与这条线平行的红糖。接下来，用冰冷的切片缓冲液填充振动室，将第一个胚胎超粘到振动体阶段，使叶片与眼核和眼睛平行。当超级胶水干燥时，将振动体阶段淹没，使胚胎朝向远离刀片，并使用新的振动体刀片获得 400 到 450 微米切片。使用无菌转移移液器将每片在获得时转移到冷切片缓冲液中，并使用荧光解剖显微镜选择含有眼细胞核和眼睛的切片。使用无菌转移移液器，将切片转移到六井板中的细胞培养物中，每井含有1.5毫升培养介质，并将该切片放在37摄氏度的培养箱中。从振动体阶段去除残留的阿加罗斯，将下一个胚胎超粘到舞台上，继续收集切片，直到所有胚胎被切开并镀上。然后将感兴趣的抑制剂或重组分子的适当浓度添加到每个井的介质中，用适当的溶剂稀释。创建剂量-响应曲线。每 30 分钟通过相位对比度和荧光显微镜对切片进行一次图像，长达 72 小时。在子宫发育的正常情况下，第一个绿色荧光蛋白阳性的细胞运动轴子在胚胎第11.5天到达轨道，然后分支到其最终目标，如在这个代表性的切片培养中观察到的。振动器舞台上切片的方向至关重要，因为如果切片方向不正确，则无法解释。例如，如果胚胎向其侧倾斜，则切片内只能观察到一个细胞运动核。如果嵌入的胚胎向背部倾斜太远，则眼睛将不在切片内，相反，可能会包括上肢、后脑或脊髓。同样重要的是，在将切片放置在培养膜上时，组织不会折叠。溶解有机溶剂中的抑制剂和生长因子时小心。例如，在这个实验中，该片在将乙醇添加到介质中后死亡。请记住，应将所有内容放在冰上，并尽量减少提取胚胎和将切片放入培养箱之间的时间。利用这项技术，我们已经开始识别在眼动系统中起作用的其他轴孔制导机制。使用剃须刀刀片时，请小心谨慎，某些抑制剂可能很危险，应根据其安全配置文件小心处理。

Research

JoVE Journal

Neuroscience

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

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

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

科研

神经科学

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

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 ...

VisualEyes: A Modular Software System for Oculomotor Experimentation

VisualEyes：一个模块化的软件系统动眼神经实验

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and ...

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

VisioTracker，创新的自动化到动眼神经分析方法

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

电机神经切断和时间推移成像胶质细胞行为在现场斑马鱼

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

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

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

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

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

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

从成年斑马鱼中制备新鲜视网膜切片用于前体成像实验

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...