我们感谢曼弗雷德HECKMANN的有益讨论建立方法的过程中。此外，我们感谢克劳迪娅·索默的技术援助和弗兰克·舒伯特支持制作视频。 01EO1002和耶拿大学医院的多学科临床研究中心（IZKF）：这项工作是由德国联邦教育与研究部（BMBF），德国，FKZ支持。

在下面的协议中提到的所有实验程序批准了图林根州当局（图林根森Landesamt献给Verbraucherschutz，Reg.-Nr. 02-044/12）。 1。准备实验<ol><li>吸电极的制作<ol><li>使用标准的高硼硅玻璃毛细管用一个微量拉马， 比如一个标准的贴片电极拔微量。 </li><li>制动用金刚石文件到尖端的0.5-1毫米（比背根的直径稍大）直径的电极。 </li><li>热打磨尖，所以它不会损害后根当它被吸入。标准实验室火炬将是合适的。 </li><li>装入玻璃长丝上连接，通过该负压可以应用注射器的电极保持器。 </li></ol></li><li>溶液的制备<ol><li>注射麻醉小鼠：混合0.75毫升氯胺酮10％，0.24毫升甲苯噻嗪2％第二5毫升0.9％的生理盐水。该溶液的10微升/克体重（BW）将腹膜内注射（IP）。 </li><li>人工脑脊液（ACSF）：稀释在双蒸水中下列盐（毫米）：134氯化钠，氯化钾3，KH 2 PO 4 1.25;硫酸镁4•H 2 O 2;的NaHCO 3 25， 氯化钙 2，D-葡萄糖10。加H 2 O 2至0.003％的最终浓度。始终使用新鲜配制的溶液。 </li></ol></li><li> （ 图2）录像设置的制备<ol><li>该放大器连接到PC接口，用于数字化数据。 </li><li>使用基于PC的程序或模拟定时器触发方波脉冲刺激器和数据采集在PC上的硬盘驱动器。 </li><li>使用连接到标准的玻璃电极持有人作为刺激和记录氯化silverwires。 </li><li>组装3操纵刺激，记录和参比电极周围分别立方米otactic帧为小鼠，以使所有的电极可使用所制备的脊髓以后。 </li><li>连接管及注射器电极持有人是苹果应用负压。 </li></ol></li></ol> 2。总评动物实验和动物准备录制程序<ol><li>利用老鼠根据各自的机构动物护理和使用委员会的指导方针进行的所有实验。深麻醉下确保动物的痛苦最小化执行所有的手术和录音。 </li><li>通过腹腔注射氯胺酮/甲苯​​噻嗪麻醉的动物（125毫克及每氯胺酮和甲苯噻嗪，克BW 8毫克分别与上述制备的溶液中加入10μl/ g体重）。如果在长期的录音需要，额外注资可制成IP或即时通讯 注意：重​​复肌肉注射0.05-0.1毫升在大腿上部的氯胺酮/甲苯​​噻嗪溶液已被证明是合适的以保持动物在深麻醉长达3小时。 </li><li>使用上的眼睛兽医药膏，以防止肌肤干燥，同时在麻醉下。 </li><li>固定在动物的头在立体定位框架，并使用加热垫与直肠探头和反射环路在实验过程中，以控制该动物的体温。在立体定位框架中的脊髓固定是没有必要的。 </li><li>开始前的准备，通过小鼠脚趾之间眨眼反射及抽搐检查麻醉深度。反射应予废除。 </li><li>打开沿从上胸椎水平降低腰椎区域用解剖刀将得到一个清晰的操作领域的脊髓上方中线皮肤。不要使用剪刀，使皮肤切口。小心松从下面的组织的皮肤上。在随后的步骤中保持了0.9％的生理盐水湿润的伤口。 </li><li>用手术刀和剪刀切筋，并从腰部椎骨到胸椎水平两侧的结缔组织。 </LI&gt; <li>取出棘突和结缔组织休息大约用小钳子椎骨。 </li><li>小心地从腰的水平（L4/L5）在解剖显微镜下钳板开始椎骨开裂。推钳板的尖端在椎骨和脊髓之间的空间，并解除骨片分开。不要伤害硬脑膜，避免压到脊髓。两者都是成功的关键。保持脊髓在整个过程中湿润。进入中旬胸椎水平。 </li></ol> 3。分离背根和DRP记录（图2） <ol><li>开启时小心使用细针（30克）与弯曲尖端硬膜。使用学联从现在开始，润燥。 </li><li>使用压力表分离背根尽可能切成两个相邻的根作为远端越好。剧烈分离期间拉动根部影响了实验的成功。 </li><li>降低吸电极下降到了吗RSAL根。添加尽可能多的脑脊液脊髓越好，因为液体有助于在背根吮吸。 </li><li>通过注射器施加负压吸一脊神经后根切断端插入一块玻璃移液管。如果需要的话，用细针将背根的电极开口的前面。 </li><li>如果背根位于干抽吸电极内，加入适量的脑脊液至其尖端而小心抽吸，直到吸液管被充分地充满脑脊液。 </li><li>后脊神经后根被吸入，从脊髓提高电​​极尖端。要小心，枪头和脊髓短路记录/刺激电极之间没有“水桥”。 </li><li>为同侧相邻根重复步骤3.3-3.6。 </li><li>定位参比电极尽可能接近的背根，并保持它通过应用学联湿润。 </li><li>通过建立该记录的设置，一个根已经选择了刺激，其他记录。提高电压，同时逐步从第二背根记录在电流钳模式下完成。有人可能会注意到一个短向下偏转后面是一个缓慢的，长期的向上偏转代表DRP。 </li><li>调整激励电压，以超强的水平，并记录多次扫描（至少20 - 30次扫描/背根在0.1赫兹，0.3赫兹，3 kHz滤波器）。 </li><li>记录三刺激（100赫兹）短列车，以获取有关DRP的时间依赖性求和信息。 </li><li>切换记录和刺激部位额外的对侧录音。 </li><li>动物并非旨在生存的过程。斩首最后一次录音后牺牲动物，而仍然在深度麻醉（氯胺酮/赛拉嗪，见上面，步骤2.2）。 </li></ol> 4。数据分析<ol><li>数据传送到分析程序（ 如适马图，伊戈尔Pro或MATLAB）。 </li><li>计算平均的痕迹FROM 20-30扫描。 </li><li>取的电压偏转（从基线; DRP峰值振幅）的最大振幅进行进一步的分析（ 图3）。 </li><li>后三个脉冲和单脉冲以获得后续的电位随时间的总和的量度计算的DRP峰值幅度之比。 </li></ol>

<ol>
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作者宣称没有竞争的财务权益。

体内的神经元活动和突触电位外和细胞内电生理记录是在调查中枢神经系统神经元功能和病理生理学的艺术手法状态。脊柱融合是运动功能， 如肢体运动和多式联运感官知觉的关键。突触前抑制是在这个计算过程，确保感官输入适当的反应的一个关键机制。在IA传入纤维的GABA能突触抑制运动神经元通过PAD的激励。背根电位记录在体内揭开了完整的动物直接测量垫的可能性。?...

突触前抑制是在脊髓中的最有力的抑制机制之一。它抑制兴奋性突触后电位（EPSPS）的单突触兴奋运动神经元而不改变突触后膜电位和运动神经元1-3的兴奋性。通过GABA能轴突轴突突触上诱导突触前感觉纤维初级传入去极化（PAD）是底层机制4-7（参见Figure1a）。这些突触含有GABA A和 GABA B-受体（GABA A R和γ-氨基丁酸乙 R）。 GABA A R活动导致的增加，氯电导而引发的PAD由于当地离子分布。这种去极化块的动作电位传播到轴突终末并降低其强度，导致降低的Ca 2 +内流和减少递质释放。 γ-氨基丁酸乙受体的激活并没吨有助于垫，但导致减少的Ca 2 +内流，从而提高突触前抑制的。而GABA A R的激活似乎涉及短期的抑制作用，γ-氨基丁酸乙 R的参与长期调制8-10。除了​​γ-氨基丁酸，占PAD和突触前抑制的主要部分，其它发射机系统也可能调节和促进这一机制11,12。 在突触前抑制病理变化似乎是至关重要的几种疾病状态，例如外周炎症和神经性疼痛13,14，以及异常的中枢性疼痛的处理15，脊髓损伤16，和中枢神经系统疾病与兴奋马达由有缺陷的GABA能传输17介导的18。因此，推定的突触前抑制是值得研究的实验病理条件下在体内对脊髓水平</em&gt;。垫产生了进行音量电位提供了一个直接测量突触前抑制脊髓。这些潜能被称为背根电位（DRP），可以从脊髓背根相邻背根7的刺激后进行测量。 DRP的第一测量已经报道在猫和青蛙19，并深入研究了猫被埃克尔斯，施密特等人在20世纪70年代初3,4,20,21。而DRP猫22和大鼠23 在体内的记录已被广泛使用，在小鼠中测量已经几乎完全执行在体外分离的脊髓制剂15,24。在这里，我们描述了一种方法， 在体内 ，允许直接测量突触前抑制在完整有机体的记录DRP麻醉小鼠。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass tubing (inner diameter 1.16 mm)</td>
			<td>Science Products (Hofheim, Germany)</td>
			<td>GB200F-10</td>
			<td>Other glass tubing might also be suitable</td>
		</tr>
		<tr>
			<td>Superfusion solution (sterile, 0,9% NaCl)</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>3570350</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Germany)</td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazine)</td>
			<td>Bayer Animal Health GmbH (Leverkusen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamin 10%</td>
			<td>Medistar GmbH (Ascheberg, Germany)</td>
			<td>KETAMIN 10%</td>
			<td></td>
		</tr>
		<tr>
			<td>30G micro needle/ Sterican</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>4656300</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Geramny)</td>
		</tr>
		<tr>
			<td>Salts for aCSF</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Diverse</td>
			<td></td>
		</tr>
		<tr>
			<td>S88 Dual Output Square Pulse</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>S88X</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
		</tr>
		<tr>
			<td>SIU5 RF Transformer Isolation Unit</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>SIU-V</td>
			<td></td>
		</tr>
		<tr>
			<td>InstruTECH LIH 8+8</td>
			<td>HEKA (Lambrecht, Deutschland)</td>
			<td>LIH 8+8 + Patchmaster software</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition&#160;</td>
		</tr>
		<tr>
			<td>Universal amplifier</td>
			<td>npi (Tamm, Deutschland)</td>
			<td>ELC-03X</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus (Tokyo, Japan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>MPC-200/MPC-325</td>
			<td>Mechanical micromanipulators also possible</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket System</td>
			<td>Stoelting (Wood Dale, USA)</td>
			<td>50300V</td>
			<td></td>
		</tr>
		<tr>
			<td>Intra-/extracellular recording electrode holder</td>
			<td>Harvard Apparatus (Holliston, USA)</td>
			<td>641227</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring spinal presynaptic inhibition in mice by dorsal root potential recording in vivo

突触前抑制是在脊髓中的最有力的抑制机制之一。底层的生理机制是通过GABA能轴突 - 轴突突触（初级传入去极化）介导的初级传入纤维的去极化。初级传入去极化强度可以通过录音的音量进行电位在背根（背根电位，DRP）进行测量。突触前抑制的病理改变是至关重要的某些疼痛疾病的中央异常处理和电机兴奋的有些失常。在这里，我们描述了记录的DRP的方法体内对小鼠。在麻醉动物，并用吸电极记录过程脊髓背根的编制进行了说明。这种方法允许测量GABA能DRP和由此估计在活小鼠脊髓突触前抑制。与转基因小鼠模型的组合，DRP记录可能本身相比于体外分离脊髓的准备工作， 如同时记录或操纵棘上网络和感应DRP由外周神经刺激的可能性RVE作为一种强大的工具，调查疾病相关的脊髓病理生理学。 在体内录音有几个优点。

典型的DRP迹线示于图3。突出刺激伪迹通常后跟一个短的向下偏转。此后缓慢，持久向上偏转，占本DRP是清晰可辨。在录音的一个子集，背根反射是因为在DRP的顶部小幅冲高可见。在正常野生型小鼠，背根反射最常出现时，刺激电压过高。作为背根反射，不能以高的重现性在此制剂引起，它们不用于分析和注意采取减少刺激电压至最低，但仍然超最大强度。为DRP的峰值振幅分析，平均2...

Watch this Scientific Journal Video about Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo at JoVE.com

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABA能突触前抑制是在脊髓电机和脊髓网络感官信号集成的重要功能强大的抑制机制。底层的初级传入去极化可以通过记录背根电位（DRP）来测量。在这里，我们证明在体内录音DRP小鼠的方法。

测量脊髓突触前抑制小鼠背根电位记录<em&gt;在体内</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

measuring-spinal-presynaptic-inhibition-mice-dorsal-root-potential

Research

JoVE Journal

Neuroscience

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

13.9K 次观看.  Jena University Hospital, Jena, Germany.  GABA能突触前抑制是在脊髓电机和脊髓网络感官信号集成的重要功能强大的抑制机制。底层的初级传入去极化可以通过记录背根电位（DRP）来测量。在这里，我们证明在体内录音DRP小鼠的方法。 

视频: Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

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 ...

科研

神经科学

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Polygraphic Recording Procedure for Measuring Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

印刷业记录程序在小鼠体内测量睡眠

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

背根神经节的啮齿动物的脊髓和隔离的液压挤压

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

背根神经节内注射和背根挤压伤为感官轴突再生模型

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Laminotomy 对猪腰背根神经节通路和注射的治疗

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

使用 Vivo 单纤维记录和带附骨神经的固定背道根结条，以检查传导失败机制

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

多尾根结鱼巨噬细胞的快速隔离

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...