这项工作是由SFB 604，东风集团MO 1421/2-1和Krebshilfe 107089（陛下）的支持。 AS是从儿童肿瘤基金会（美国纽约）收件人的青年研究者奖。

本研究根据保护的德意志联邦共和国（Tierschutzgesetz DER德意志联邦共和国）的动物法进行，并批准了图林根州厅食品安全和消费者保护（图林根森Landesamt献给Lebensmittelsicherheit UND Verbraucherschutz）。 1，设置的测量<ol><li>麻醉小鼠通过异氟烷/ O 2吸入-对麻醉3％的起始，在100％的氧气维持2％异氟醚（ 图1）。通过测试简单的反射，如运动反射和低档疼痛的敏感性测试确认足够的麻醉。使用软膏对眼睛，以防止麻醉期间干涩建议，但没有必要的，因为该过程一般只需30分钟/动物的总和。如遇生存实验，及时管理长效止痛剂用于管理术​​后疼痛。 </li><li>刮胡子的毛皮覆盖后肢用电动剃须刀，并与市售的脱毛霜脱毛执行，而动物已经在进行镇痛。为了在整个过程维持无病原状态，穿无菌手套，总是用70％乙醇仔细清洗工具。 </li><li>通过反馈控制的加热板和热直肠探头（ 图2）控制身体的温度稳定性。如果需要的话，给使用的无菌披盖以覆盖动物之间的加热板，以保持无菌的实验环境。此外，它是推荐使用的加热板，这是通过一个集成的传感器的电子控制，限制加热温度为≤40℃，以避免组织损伤。 <img alt="图1" fo:content-width="4in" fo:src="/files/ftp_upload/51181/51181fig1highres.jpg" src="/files/ftp_upload/51181/51181fig1.jpg" /> 图1。地契erimental设置显示剃光后肢麻醉鼠标。 </li><li>采取心电图（ECG）记录监视心脏率作为一个重要的参数。对于心电图记录安装三个电极如下：在颈部区域中的皮肤下的每个前肢和一个电极的皮肤下的一个电极。 </li><li>采用接触凝胶的最佳电导率/电阻转移地方环形电极。所述感测电极（标示为黑色）被放置在那里的腓肠肌具有其最大直径位置。参比电极（以红色表示）置于正好在传感电极的下方。 注：请参阅“材料表”的设备的详细信息。 </li><li>标记测量与近端和远端坐骨神经刺激和引线电极之间的预定但一致的距离上的正确位置上。实验方案：在从所述感测电极4毫米的距离，远端刺激就会发生。在16毫米到感测电极的距离，近端刺激将进行（ 图2）。 </li></ol><img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/51181/51181fig2highres.jpg" src="/files/ftp_upload/51181/51181fig2.jpg" /> 图2代表图像显示之前，为了测量开始时的实验情况。的白色箭头表示传感（黑色）和参考（红色）在左后肢的腓肠肌电极的位置。通过针形电极的刺激，便会在定义的位置相对于黑色的感应电极来进行。远端刺激的点（黑线标记为“DS”在左后肢）具有从所述感测电极4 mm的距离;近端刺激（黑标有“PS”）的地方为16毫米远。在右后肢红线表示的约<html> iMate的坐骨神经的解剖过程。此外，相关的后肢肌肉的粗略位置显示为地标。星号表示直肠温度探头。 2，测量<ol><li>原理：执行一系列的神经刺激与0.1毫秒的持续时间由单极支配28号针头电极重复生成的单个方波脉冲（重复频率200毫秒;参见图3A）。离线数据分析，建议以同时获得刺激信号连同神经肌肉功能的响应曲线（ 图3B）由于神经刺激。平均一系列的最大反应（“代表神经肌肉功能反应”），为后续的数据分析。为了产生可靠的数据，记录和以后平均至少3个独立的，最佳响应每刺激位点和动物的曲线。 p_upload/51181/51181fig3highres.jpg“SRC =”/ files/ftp_upload/51181/51181fig3.jpg“/&gt; 图3的程序进行数据采集和分析（概略表示）。重复地产生脉冲，通过针形电极（在图3A中上排）施加到坐骨神经。同时，若干个相应的神经肌肉反应曲线被记录（在图3A中下排）。当平均并放大，神经肌肉响应曲线由于刺激脉冲（ 图3B中上排）显示如下的特性（在图3B中下排）：信号响应的延时以及持续时间和信号的振幅的数目，而可以为后续计算和统计获得。不规​​则的信号传导和/或次优的记录通常会导致更多的各种信号的变形不是只有一个正极和一个负极偏转或变形的信号（双峰形状）具有降低的幅度（ 图3C）。 </li><li>与在确定的位置针电极（ 图2“PS”）进行近端刺激。 <ol><li>为了达到最佳的拍摄条件，最大振幅，可视化的实际响应曲线，同时对刺激的过程。通过这样做，实验者能够立即评估的响应曲线的形状以及振幅的大小。 </li><li>如果需要，轻微和仔细操作的位置和/或刺激针相对于坐骨神经的角度。刺激条件下，这种温和的优化使得达到恒定的幅度尽可能大的价值和典型的双相形状的响应曲线（ 图4）。 </li></ol></li><li>用在确定的位置（ 图2中“DS”）的针电极进行远端刺激。 </li><li> completio后n次测量，测试鼠标转移到一个单独的笼子里，直到它已经恢复了足够的意识，以保持胸骨斜卧。不要让动物无人值守和其他动物的公司，直到它完全从麻醉中恢复。及时管理长效止痛剂用于管理术​​后疼痛。的非甾体抗炎药（NSAID）和阿片类药物全身给药被推荐为1-3天。 </li><li>或者，牺牲还是麻醉鼠标在一个快速和人性化的时尚没有任何进一步的疼痛的动物， 例如通过颈部脱位。 </li></ol><img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/51181/51181fig4highres.jpg" src="/files/ftp_upload/51181/51181fig4.jpg" /> 图4插图，以确定记录CMAP最大振幅。一个完整系列的注册提出。 （ 一 </st荣&gt;）插入点以最小的CMAP响应。 （二）轻微刺激针运动的结果CMAP录音与最大振幅。 （ 三）在针放置额外产生的变化记录CMAP振幅不同，包括接近最大振幅。 （ 四）刺激针替换为近最大振幅串行CMAP录音。注：典型的递减在CMAP振幅可以在最佳刺激位点12，13发生在重复刺激。星号表示CMAP记录与描绘的平均最大振幅。 3。分析<ol><li>提取的基础上使用适当的软件（ 如 AtisaPro）代表神经肌肉功能的响应数据集神经传导参数。 注：请处理与时间相关的数据测定，特别小心，因为复合运动动作锅拐点确定差分（CMAP）开始和终止是很困难的。与重复性验证的过程是在图3B，其中是用在发病后和结束前的信号偏差切线。 </li><li>计算&#39;的信号延时“和”CMAP的价值观。 <ol><li>延迟“表示刺激和CMAP发作之间的时间延迟，而CAMP发病的初始负偏转之间的时间跨度初始回归到基线被称为&#39;CMAP持续时间&#39;。使用远端和近端的延迟之间的差来计算传导速度与远侧和近侧的刺激部位之间的距离一起。 刺激部位之间传导速度=延迟/距离 </li><li> CMAP&#39;（复合肌肉动作电位）的幅度代表了CMAP信号（毫伏给出）的最大正，负转机点之间的幅度。 CMAP =值“积极转变点” -价值“负婆转亏为盈INT&#39; </li></ol></li></ol>

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作者什么都没有透露。

所描述的协议提供了一个简单而可靠的方法，而不需要暴露感兴趣的神经，以确定麻醉小鼠坐骨神经传导性能。然而，这一实验过程由穿刺针引起组织损伤。因此，这是一个合理的选择来完成录音后牺牲动物。然而，相对于其他更具侵入性的程序，这需要神经前录音曝光，组织损伤相对较小3,14。因此，重复的测量是可能的，并且取决于相应的研究设计。然而，某些点具有以保证一致的结?...

电生理测量是必不可少的工具在临床和实验室环境调查周围神经功能的完整性。在人类中，有大量神经肌肉疾病和神经病变的诊断依赖于电生理学测量。通过测量神经性质传导速度或信号的电势的振幅，能够表征外周神经疾病的大致原点。 神经传导速度是高度依赖于由髓鞘启用快速信号传播。因此，脱髓鞘过程一般表现出下降传导速度4。复合肌肉动作电位（CMAP） -与功能性轴突的数量关联-是轴索损害时显著减少5的指标。 因此，通过电生理学方法手段的外周神经损伤的病因能够被辨别，如遗传性神经病6,7，糖尿病性神经病8,9，慢性炎症性脱髓鞘性多神经病（CIDP）10，或代谢性神经病11。 通常，在人类应用上的腓肠或尺神经的非侵入性的录音是优选的。在小鼠中，它是直接分析坐骨神经的神经性，同时含有较大的外周神经系统（PNS）中最大的神经 - 及肌肉运动和感觉系统的小口径轴突。 因为这里展示的过程是一个快速，简单和可靠的方法来测量相关的电生理学上的完整的小鼠外周神经的所有标准值。通过采取录音从保存生物，神经环境的生理条件有保证。

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Concentric Needle Electrodes (Stimulation)</td>
			<td style="width:311px;">Natus Medical Incorporated 
			San Carlos, CA 94070, USA</td>
			<td style="width:119px;">9013S0901</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital Ring Electrodes (Recording)</td>
			<td style="width:311px;">Natus Medical Incorporated 
			San Carlos, CA 94070, USA</td>
			<td>9013S0302</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ToM - Tower of Measurement (A/D converter)</td>
			<td>GJB Datentechnik GmbH, Langewiesen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AtisaPro, Data acquisition &#38; analysis software</td>
			<td>GJB Datentechnik GmbH, Langewiesen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HSE-Stimulator T</td>
			<td colspan="2">Hugo Sachs Elektronik, Hugstetten, Germany</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo electrophysiological measurements on mouse sciatic nerves

电生理学研究允许各种神经肌肉疾病的合理的分类，并帮助连同神经病理学技术，在病理生理学1的理解。在这里，我们描述了在体内进行对小鼠坐骨神经的神经电生理研究的方法。 将动物用异氟烷麻醉，以确保镇痛试验小鼠并在此期间，约需30分钟/动物的测量不受干扰的工作环境。 37℃恒定的体温是由加热板维护和直肠温探头2连续测量。另外，心电图（ECG）中，以便测量被定期地记录连续监视所研究的动物的生理状态。 电生理记录均于坐骨神经，日最大神经进行ë外周神经系统（PNS），与两个肌肉运动和感觉纤维束供给鼠标后肢。在我们的协议，坐骨神经留在原位 ，并因而不具有要被提取或暴露，允许测量无任何不良神经刺激随着实际录音。使用适当的针电极3正进行近端和远端的神经刺激，登记所发送的电位与感测电极在腓肠肌。经过数据处理的，可靠的和对神经传导速度（NCV）和化合物肌肉动作电位（CMAP）高度一致的值，关键参数为外周神经机能总值的量化，可以实现的。

<html>我们进行了一系列体内对12只小鼠的坐骨神经电生理测量的总本研究：每组6只性别。的测量进行了与所提出的协议，并提供了以下的结果： 雄性和雌性小鼠显示约20米/秒，平均坐骨神经传导速度（ 图5）。这与文献中的其他测量是一致的。此外，它表明，根据我们的数据是男性和女性之间没有相关的差异神经传导速度。 <p class="jove_content" fo:keep-togeth...

Watch this Scientific Journal Video about In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves at JoVE.com

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

体内神经传导特性的测量体现了强大的工具来表征神经肌肉疾病的多种动物模型。在这里，我们提出了一个简单而可靠的协议，其中对麻醉小鼠坐骨神经电生理分析可以执行。

在对小鼠坐骨神经体内的电测量

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

in-vivo-electrophysiological-measurements-on-mouse-sciatic-nerves

Research

JoVE Journal

Neuroscience

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

33.0K 次观看.  Fritz Lipmann Institute.  体内神经传导特性的测量体现了强大的工具来表征神经肌肉疾病的多种动物模型。在这里，我们提出了一个简单而可靠的协议，其中对麻醉小鼠坐骨神经电生理分析可以执行。 

视频: In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

从蛾的嗅觉系统的电生理测量

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

科研

神经科学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

在人类婴儿的伤害性电生理测量和分析

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

体内运动神经元变性小鼠前肢肌作用电位的电生理测量

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Methods Article

在对小鼠坐骨神经体内的电测量