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度为 0.2% (w/v)。</li>
		<li>负载7.5 µL 的 CTB 溶液 (0.2% 瓦特/v) 在无菌 10-µL 微量泵与附加33口径针 (钝或短斜角) 为每个鼠标。</li>
	</ol>
</li>
	<li>经胸注射:<ol>
<li>将 CTB 在无菌水中的能量溶解到浓度为 0.1% (w/v)。</li>
		<li>负载20µL CTB 溶液 (0.1% 瓦特/v) 在一个无菌 500-µL 胰岛素注射器与斜面27口径针为每只老鼠。 注: 请务必使用蒸馏水和无菌水, 并适当溶解 CTB 粉。该解决方案可以保持一个月摄氏4摄氏度 (不要冻结)。沉淀可能在几天后形成。使用前用吸管将溶液带入室温, 并混合使用。</li>
	</ol>
</li>
</ol>2. 胸腔注射前的准备工作
<ol><li>使用高压釜或玻璃珠灭菌器, 在手术前消毒手术工具。准备一件干净的长凳做手术, 并处理手术工具。 注意: 在手术过程中使用的任何材料都应该是无菌的。不能灭菌的仪器, 如微量泵, 应用消毒剂 (必泰) 擦拭, 或者只应使用一次性。外科医生应该在手术开始前用消毒剂 (洗必泰擦洗) 洗手。外科医生应该戴上无菌手套、面罩和干净的长袍。</li>
	<li>称量动物, 并管理适当的麻醉剂量: 腹腔注射麻醉鸡尾酒 (如氯胺酮100毫克/千克和甲苯噻嗪5毫克/千克)。</li>
	<li>捏脚趾和/或检查眼睑反射的损失, 以确定是否正确麻醉鼠标。应用兽医软膏保护角膜。</li>
	<li>仔细剃须腹部皮肤 (为膈程序) 或右胸皮肤 (经胸手术), 使用电剪。剃得好, 足够宽, 以防止头发进入手术领域。</li>
	<li>通过局部应用10% 碘溶液在剃毛地区确保无菌条件。用碘溶液擦洗手术部位, 小心地从中心部位擦洗到周围。</li>
	<li>在整个手术中使用恒温垫保持动物的体温。</li>
</ol>3. 用膈方法进行胸腔注射
<ol><li>将麻醉鼠标放在加热垫上仰卧位。将卷纱垫放在动物的脖子下。对于成年鼠标, 请使用0.5 英寸厚度的滚动纱布垫。把这个垫子贴到手术板上, 以免发生运动。</li>
	<li>使用手术刀刀片, 切割腹侧皮肤沿中线: 做一个切口从剑突过程到脐区域, 而伸展皮肤侧向与另一只手, 使皮肤绷紧。不要对刀片施加太多的压力, 以免损害底层器官。</li>
	<li>使用小剪刀, 小心地把皮肤从腹部肌肉的切口周围剥离。这个程序将有助于缝合的肌肉和皮肤分开的手术结束。</li>
	<li>用小剪刀进行剖腹手术。通过在脐的水平上执行一个纽扣切口打开腹腔。切割腹部肌肉沿白色线 ("linea") 由剑突过程。</li>
	<li>为了形象化的腹部表面的隔膜, 收回腹部肌肉使用商业或自制拉钩。 注意: 这些拉钩可以由形状为 L 挂钩19的 midi 大小的回形针组成。</li>
	<li>伸展开腹的两侧, 并把拉钩到板凳大衣上。优化视野的光照, 使隔膜的腹面不再在半影中。最强大的照明设备为这个目的是一个 LED 灯与定向光束。</li>
	<li>一方面使用软镊子, 举起 xyphoid 阑尾, 拉下肝脏的侧面裂片 (图 2A)。另一方面使用小剪刀, 小心切断悬吊韧带, 不损害胆囊或隔膜 (图 2B)。</li>
	<li>一方面用软镊子, 举起 xyphoid 附录。另一方面抓住哈密尔顿注射器。目标三的注射地点在右 hemidiaphragm 分别覆盖胸骨, 内侧和脚区域 (图 3A, 插入图 3B)。</li>
	<li>由于膜片厚度约为0.37 毫米小鼠7, 插入针不超过1或2毫米超过隔膜片, 以防止肺部损害在呼吸期间。通过每个站点的右膜片 (图 4B) 提供2.5 µL 的 CTB 解决方案 (0.2% 瓦特/v)。不需要膜片的稳定。</li>
	<li>对三个同侧注射点 (图 1A) 重复此过程, 并为 PhMN 池的双向标记 contralaterally。</li>
	<li>闭合剖腹手术部位, 缝合腹部肌肉与可吸收4-0 缝合。使用被打断的针间距为3毫米, 主要皮肤闭合与9.0 毫米伤口剪辑。收紧主食, 以防止动物拔下主食。间隔约5毫米的空间钉。不要过度收紧伤口夹, 因为这会导致愈合受损。</li>
	<li>用蒸馏水清洁微型注射器, 防止堵塞。慢慢地起草和驱逐2-3 次。不要把空气抽进注射器。</li>
</ol>4. 胸腔内注射用经胸法
注意: 此方法灵感来自4中描述的过程, 并适合于鼠标。
<ol><li>在加热垫 (图 4A) 上, 将麻醉鼠标放在左侧压疮位置。</li>
	<li>用手动触诊 (图 4B) 识别肘部区域下的第六和第七根肋骨。</li>
	<li>当助手扩展右前-和后肢 (图 5A) 时, 插入注射器 cranially 定向和切下的第六或第七肋, 3 毫米深从皮肤, 与斜角向下 (图 1B和图 5B)。</li>
	<li>轻轻抬高针, 以确认, 通过解除肋骨, 它是很好地定位到胸腔 (图 5C, 插入图 1B)。</li>
	<li>用两个手指在胸壁上施加轻微的压力, 稳定胸部的呼吸运动。</li>
	<li>在拿着注射器的同时, 在单个注射中提供20µL 的 CTB 溶液 (0.1% 瓦特/v)。</li>
</ol>5. 术后护理
<ol><li>术后立即将鼠标放置在恒温垫上的右侧压疮位置, 以恢复。这确保了示踪剂传播在胸腔的右侧, 并允许监测动物的呼吸。</li>
	<li>管理1毫升无菌盐水溶液。提供后续注射, 如果动物出现脱水和/或无精打采。</li>
	<li>在手术后的头两天, 每天两次在术后0.1 毫克/公斤的丁丙诺啡, 以尽量减少任何潜在的疼痛。 注: 许多机构建议对侵入性手术进行多模镇痛, 如这里所描述的剖腹手术。这可能包括使用非甾体抗炎 (NSAID) 药物和/或局部麻醉剂在切口部位。这些药物通常是在第一个切口之前提供的, 以减少出现的疼痛。</li>
	<li>每天监视动物直至安乐死。</li>
</ol>

<ol>
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作者没有什么可透露的。

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。

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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 ...

retrograde-neuroanatomical-tracing-of-phrenic-motor-neurons-in-mice

Research

JoVE Journal

Neuroscience

12.6K 次观看.  Université de Namur. 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。

视频: Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

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

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the 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

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

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

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

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

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

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

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

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

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

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

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

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 ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

有条件的遗传跨突触标记在胚胎小鼠脑

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

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 ...

Laser-guided Neuronal Tracing In Brain Explants

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 through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

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

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

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

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

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

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 methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

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

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

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 ...

小鼠膈运动神经元逆行神经解剖学追踪的研究

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