Name: retrograde neuroanatomical tracing of phrenic motor neurons in mice
Uploaded: 2018-02-22T14:00:00
Description: Watch this Scientific Journal Video about Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice at JoVE.com

我们感谢罗伯特 Graffin 和宝琳 Duhant 的技术支持。

实验性议定书是按照欧洲共同体理事会动物实验指令 (2010/63/欧盟、86/609/欧洲经济共同体和 87-848/欧洲经济共同体) 进行的, 并得到了纳尔大学动物伦理委员会 (伦理项目 n°17-284) 的批准。).图 1描述了两种方法: 膈或经胸注射。在研究中使用雄性 C57bl/6J 小鼠 (n=18), 年龄从3个月到4月。
1. CTB 溶液的制备
<ol><li>膈注射:<ol>
<li>将 CTB 在无菌水中的能量溶解到浓?...

<ol>
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本文所描述的协议可以适用于任何成年小鼠的应变或任何实验范式, 其中的 PhMN 电路的完整性应评估。例如, 肌萎缩侧索硬化 (ALS) 和颈脊髓损伤 (cSCI) 是与 PhMN 丢失、膈轴突顺变性和随后的呼吸折衷有关的条件。动物模型的 ALS 或 cSCI 模拟病理学和功能性呼吸衰竭观察到人类疾病。在这些模型中, 组织学技术经常被用来评估 PhMN 生存后, 针对 PhMN 神经保护18,20</...

本研究的目的是提出一种可靠的方法来识别膈运动神经元 (PhMN) 在小鼠脊髓部分。在胸腔内注射荧光神经解剖学示踪剂作为传递方法, 以达到膈肌神经的投射到膜片上, 并使用逆行运输沿膈轴突标记膈细胞体。本文介绍两种胸腔内分娩技术: 膈与胸肺。
膈运动神经元是脊髓中继细胞, 其轴突汇聚成膈神经, 最终支配膈肌。这些是较低的马达神经元接受吸气驱动从球呼吸中心和中继到隔膜神经肌肉连接 (NMJ)。PhMN 结构成两个马达柱, 一个为每个 hemicord, 运行沿中颈椎脊柱。在包括人类在内的大多数哺乳动物物种中, 膈电机列从 C3 级别扩展到 C61,2,3。我们和其他人已经证实, PhMN 集中在大鼠和小鼠脊髓的 C3-C5 水平4,5,6,7,8。膈细胞的地形图分布不随机;在膈肌运动池 (C3) 的颅骨部分, 支配的运动神经元分布较密, 而支配脚部的运动神经元则更是尾鳍 (C5)9。此外, PhMN 在腹角灰质中有不同的聚集。在 C3 水平, 膈细胞的簇在侧向, 然后他们在一个延髓方向转移并且被发现 ventromedially 在最尾部水平10,11。
考虑到他们在灵感中的重要作用, 准确地识别 PhMN 在健康的脊髓中是至关重要的, 但在病理条件下, 如退行性疾病或脊髓外伤损伤, 也应遵循他们的命运。由于 PhMN 与其他颈椎运动神经元的形态学不同, PhMN 的识别依赖于在初级呼吸系统中心 (8) 的水平, 或在隔膜 NMJ7或在膈神经4。追踪器由神经纤维占去并且运载到膈肌细胞身体在颈椎, 在那里它可以被视觉化使用直接或间接检测系统。逆行或顺示踪剂在商业上有广泛的共轭。值得注意的是, 每个追踪器都被赋予无、低或高的跨突触追踪能力。
在目前的研究中, 我们选择了霍乱毒素的β亚基 (CTB) 功能性与 Alexa 555 (从今以后称为 CTB-荧光) 作为荧光标签, 允许直接可视化 PhMN 的冰冻脊髓部分。CTB 通常被描述为 monosynaptic 示踪器, 尽管实验数据倾向于显示 transneuronal 通道12。CTB 有能力将苷 GM1 在神经末端的等离子膜上。CTB 是通过网格蛋白依赖或独立的机制内化, 并通过反式高尔基网络进入内质网, 在逆行的时尚13, 14.内部化和逆行传输似乎依赖于肌动蛋白骨架15、16以及微管网络17。
为了证明 CTB 作为逆行神经解剖学示踪贴标膜片-PhMN 电路的用处, CTB 荧光被交付 intrapleurally。CTB 使用两种技术进行管理: 第一个包括剖腹手术和多膈注射;第二个, 较少侵入, 使用了独特的经胸注射。四天后, 荧光标记的 PhMNs 从健康和 spinally 受伤 (C4) 动物的颈椎脊髓中定量。

<table>
	<tbody>
		<tr>
			<td>Glass-bead sterilizer Steri 250</td>
			<td>Keller</td>
			<td>31-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Small scissors</td>
			<td>F.S.T.</td>
			<td>14058-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Soft tweezers</td>
			<td>F.S.T.</td>
			<td>11042-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Swann Morton</td>
			<td>No.11 or 15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin subunit beta conjugated to Alexa Fluor 555</td>
			<td>Life Technologies</td>
			<td>C22843</td>
			<td>Bring at room temperature before use&#160;</td>
		</tr>
		<tr>
			<td>10ul Hamilton syringue, removable needle</td>
			<td>Sigma-Aldrich</td>
			<td>701RN</td>
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		</tr>
		<tr>
			<td>33-gauge needle for Hamilton syringue, 20mm length, point style 4</td>
			<td>Filter Service</td>
			<td>7803-05</td>
			<td></td>
		</tr>
		<tr>
			<td>500ul insulin syringue MyJector, 27-gauge</td>
			<td>Terumo</td>
			<td>BS05M2713</td>
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			<td>Orientable LED lamp</td>
			<td>V.W.R.</td>
			<td>631-0995</td>
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			<td>Resorbable 4/0 sutures</td>
			<td>S.M.I. AG</td>
			<td>15151519</td>
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			<td>Needle holder</td>
			<td>F.S.T.</td>
			<td>12002-14</td>
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			<td>9mm autoclips</td>
			<td>Bioseb</td>
			<td>205016</td>
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			<td>Autoclip 9mm applier</td>
			<td>Bioseb</td>
			<td>MikRon 9mm</td>
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</table>

retrograde neuroanatomical tracing of phrenic motor neurons in mice

膈运动神经元是由 C3 到 C6 水平的颈椎运动神经元。轴突投射到膈神经支配呼吸隔膜。在脊髓切片中, 膈运动神经元不能从其他运动神经元中识别出形态学或生物化学标准。我们提供描述的程序, 以可视化膈运动神经元细胞体的小鼠, 后胸腔注射霍乱毒素亚基β (CTB) 共轭荧光。该荧光神经解剖学示踪剂有能力被抓住在隔膜神经肌肉交界, 被携带 retrogradely 沿膈轴突和到达膈细胞体。比较了胸腔 CTB 分娩的两种方法: 膈与经胸注射。这两种方法都是成功的, 并导致了类似数量的 CTB 标记的膈运动神经元。总之, 这些技术可用于可视化或量化的膈运动神经元的各种实验研究, 如那些关注膈膈电路。

研究中包括雄性 C57bl/6J 小鼠 (n=18), 年龄从3个月到4月。在实验的0天, 8 只小鼠接受了单边 C4 挫伤, 右侧, 根据发布的协议7,18。作为假手术, 10 只小鼠在 C4 顶部进行了椎板切除术, 无挫伤。3天, 根据上述两种不同的程序, 对 CTB 荧光胸腔注射的小鼠进行了准备。7天, 所有小鼠在麻醉后被安乐死 (氯胺酮100毫克/千克和甲苯噻嗪5毫克/千?...

Watch this Scientific Journal Video about Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice at JoVE.com

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

本文描述了荧光共轭霍乱毒素亚单位β胸腔内分娩后小鼠膈运动神经元识别的一种协议。比较两种方法注射胸腔: 膈与经胸的方法。

小鼠膈运动神经元逆行神经解剖学追踪的研究

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Research

JoVE Journal

Neuroscience

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

在小鼠体内的汽车平衡和协调的评估使用的平衡木

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

科研

神经科学

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Measuring Motor Coordination in Mice

测量小鼠运动协调

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

运动神经元细胞外识别的肌肉运动游泳池<em&gt;海兔夜蛾</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

隔膜的神经支配膈运动神经元的功能和形态评估

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

肌肉注射沿运动终板：微创方法来接送示踪剂直接为运动神经元

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Laser-guided Neuronal Tracing In Brain Explants

激光制导神经跟踪在脑外植体

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...