<p class="jove_content"&gt;支持这项工作是由神经系统疾病的国家机构和国立卫生研究院中风壁间方案。我们还想感谢他的早期贡献的方法，并在图3的数据乔治Mentis博士。</p

荧光葡聚糖已被用作解剖工具和成像神经元活动1-4。领域等 。（2009）发表了一份协议，为应用重点是作为一个模型系统的脊髓轴突束离子和电压敏感染料。在这里，我们描述了荧光和/或离子敏感的染料， 在体外 optophysiological和形态学研究申请截止前根，背根或脊髓神经束更详细的过程。 1。 I型和II型移液器开始拉了一个酒精灯的火焰7两个短节的聚乙烯管材（PE90，粘土亚当斯品牌）生产圆锥提示移液器。一个吸管（I型），应该是一个小技巧，可以紧紧握住目标根或道（外径：0.2-0.4毫米，内直径0.1-0.3毫米）短（3-7毫米）。然后拉第二移液器（类型-Ⅱ）与I型吸取尽可能较长（8-12厘米）的薄，轴和一个非常精尖（外径：0.2-0.3毫米，内径：0.1-0.2毫米），可以插入其尖端。第二更薄移液器将用于人工脑脊液（ACSF）和染料溶液从I型吸管分别吸和介绍。 2。定位型我吸取解剖和分离脊髓8,9和它放置在整个加载过程与，冰鲜学联（〜16°C）的灌流洗澡，。放置I型电极支架（H1/12电极支架，Narishige）吸取其背面的开口，这样可以很容易地通过软管连接到一个注射器（1毫升的U-100胰岛素注射器，流式细胞迪肯森或类似）（PharMed BPT的，科尔-帕默，AY242002; 10厘米）（ 图1A）。 3。染料中的应用<ol><li>使用注射器，先画进入I型吸管（图1B，2A）学联其次填补（ 图2B）轴突束或根。软管，然后应该从背面的开口。 II型移液器连接到另一个注射器，并插入到I型吸取轴突束或根。因此，剩余的脑脊液只涵盖轴突道（ 图1c; 2C - D）II型移液器，吸学联的注射器的使用。然后退出I型吸管II型移液器。监视了几分钟，确认密封良好，轴突束或神经脑脊液水平（见第3节，在“良好的密封故障排除”）。 </li><li>溶解在学联的染料或双蒸水0.2％的Triton X-100的II型吸管吸引到它，同时注意不包括气泡。插入II型移液器含有染料，分为​​I型吸管到剩余学联解决方案（ 图2E）。慢慢释放染料进入脑脊液，使用温和的正压力（ 图2F）。要小心不要引入任何气泡或导致靶组织内尖端的位移。撤出后，足量的染料已被释放（3-5μL染料溶液）II型移液器。 </li></ol> 4。孵化该组织在6至20小时，根据实验类型之间的暗孵育。一旦有足够的时间已经允许灌装，轻轻拉离离开组织成像或组织学准备组织电极。 故障排除<ol><li>如果组织的目标并不容易进入到I型吸管尖或松动一旦进入，然后头直径大小是错误的。如果发生这种情况，拉，并使用不同的吸管。 </li><li> I型吸管脑脊液前加入染料，确保足够的脑脊液仍然如此，它可以达到II型吸头（ 图2D）。否则，气隙存在，一旦染料中添加。如果发生这种情况，不添加染料和删除从移液器的轴突束，然后绘制它与II型移液器达到足够的脑脊液。 </li><li>如果在I型吸管增加学联水平后，这是与II型吸管吸气，这意味着该组织的密封不好。使用不同的电极，染料，否则将在标注过程中稀释。 </li><li>如果气泡被引入到I型吸管，同时注入染料，推进II型枪头接近泡沫，轻轻地运用弱的负压力，使用所附的注射器，排出气泡。 </li></ol> 5。代表结果为了说明该方法的应用，我们同时加载的运动神经元，感觉传入脊髓的interneurons有三个不同的DEXT跑了共轭荧光染料（ 图3A）。正如图所示。被标记为3B，3类神经元;感觉传入（红色），投射到腹侧索（绿色）和运动神经元（蓝色）的interneurons。 <img alt="图1" src="/files/ftp_upload/4008/4008fig1.jpg" /> 图1。示意图显示移液器I型和II型装配 。 （一）一个类型，我移液器（淡蓝色）是1电极支架放置，以便它的背面的柔性橡胶管（二）开幕式可以很容易地连接和融入其中1 II型吸管可以被插入（三）。 <img alt="图2" src="/files/ftp_upload/4008/4008fig2.jpg" /> 图2。示意图显示的荧光染料的运动神经元的加载过程 。 （A）型我吸管放在附近的腹侧根被填补。 （二）移液器学联绘制根吸管，吸力。 （C四）灵活的橡胶管断开I型吸管后端和应用II型吸管吸去学联金额减少。 （五）II型分为I型吸管其余脑脊液中含有溶解染料的吸管插入尖。 （英法）与染料慢慢地填写I型吸管，然后取出-Ⅱ型吸管。 <img alt="图3" src="/files/ftp_upload/4008/4008fig3.jpg" /> 图3。小鼠离体脊髓灌装过程中的应用 。 （一）示意图显示了三个I型吸液管，用于填补在不同的脊髓神经元类。运动神经元（蓝色;梯级蓝葡聚糖）和骶感觉传入（红色;德州红葡聚糖）分别通过腹和背根，回填。荧光葡聚糖用于装载脊髓的interneurons（绿色），其轴突通过腹侧索（VF）的提升。 1毫克的每个葡聚糖染料（10,000兆瓦;分子探针）被溶解在6μL蒸馏水含有0.2％的Triton X-100（二）。从脊髓中的神经元都回标记A.注意标记的感觉传入，主要与同侧几对侧预测，骶脊髓节共聚焦图像。标记的运动神经元是同侧的填腹根，而标记的interneurons对侧侧填充（箭头）。校准栏200微米。

<ol>
	<li>Nance, D. M., Burns, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dextrans+as+sensitive+anterograde+neuroanatomical+tracers:+applications+and+pitfalls.">Fluorescent dextrans as sensitive anterograde neuroanatomical tracers: applications and pitfalls.</a> Brain Res. Bull. 25, 139-145 (1990).</li><li>Glover, J. C., Petursdottir, G., Jansen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dextran-amines+used+as+axonal+tracers+in+the+nervous+system+of+the+chicken+embryo.">Fluorescent dextran-amines used as axonal tracers in the nervous system of the chicken embryo.</a> J. Neurosci. Methods. 18, 243-254 (1986).</li><li>McPherson, D. R., McClellan, A. D., O'Donovan, M. 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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+functional+impairment+of+sensory-motor+connectivity+in+a+mouse+model+of+spinal+muscular+atrophy.">Early functional impairment of sensory-motor connectivity in a mouse model of spinal muscular atrophy.</a> Neuron. 69, 453-467 (2011).</li><li>Blivis, D., Mentis, Z., O'Donovan, J. M. G., Lev-Tov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+sacral+neurons+involved+in+activation+of+the+lumbar+central+pattern+generator+for+locomotion+in+the+neonatal+rodent+spinal+cord.">Studies of sacral neurons involved in activation of the lumbar central pattern generator for locomotion in the neonatal rodent spinal cord.</a> Soc. Neurosci. Abstr. 564.8, (2009).</li><li>O'Donovan, M. 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<p class="jove_content"我们没有透露。</p

<p class="jove_content"我们在这里描述为简单和成本效益的协议，引入神经元，神经和脊髓束的染料。此方法涉及确定的解剖途径暴露的装载过程持续时间为一个高度集中的染料溶液。逆行标记这一点背景与目标站点的结果相比，微量注射，洗澡的应用和电技术。然而，该方法仅限于中枢神经系统的轴突束可以分离的站点。此外，由于染料扩散到沿轴突到达细胞体，加载时间将取决于从染料应用到神经元胞体部位的距?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td colspan="2"<strong&gt;材料名称</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品目录号</strong</td&gt;<td<strong&gt;评论</strong</td&gt;</tr&gt;<tr&gt;<td rowspan="2"&gt;管</td&gt;<tdPE90&gt;（IDxOD：0.034“x0.050”华尔街Thikness：0.008“）</td&gt;<td&gt;粘土亚当斯品牌Intermedic</td&gt;<td&gt; 427421</td&gt;<td&gt; I型和II型移液器</td&gt;</tr&gt;<tr&gt;<tdNSF-51（IDxOD：1/16x1/8;华尔街Thikness：1/32）</td&gt;<td&gt; PharMed BPT的，科尔 - 帕默</td&gt;<td&gt; AY242002</td&gt;<td&gt;挠性管</td&gt;</tr&gt;<tr&gt;<td&gt;注射器</td&gt;<td&gt;1毫升胰岛素注射器的U-100</td&gt;<td&gt;流式细胞迪肯森</td&gt;<td&gt; 329650</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;针</td&gt;<td&gt; 19G X1-1/2“金属轮毂针</td&gt;<td&gt; MONOJECT</td&gt;<td&gt; 200136</td&gt;<td&gt;连接​​II型移液器注射器</td&gt;</tr&gt;<tr&gt;<td&gt;</td&gt;<td&gt;酒精灯</td&gt;<td&gt;</td&gt;<td&gt;</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;旋塞阀</td&gt;<td男露儿滑适配器&gt;三通阀</td&gt;<td&gt;百特医疗用品公司</td&gt;<td&gt; 2C6241</td&gt;<td&gt;使用用注射器</td&gt;</tr&gt;<tr&gt;<td rowspan="3"&gt;持有人</td&gt;<td&gt;的H-1电极支架</td&gt;<td&gt; Narishige</td&gt;<td&gt; H-1/12</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;永磁立场</td&gt;<td&gt; Narishige</td&gt;<td&gt; GJ-8</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;微操作机器人</td&gt;<td&gt; Narishige</td&gt;<td&gt; H-3333</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt;镊子</td&gt;<td&gt;科技杜蒙瑞士00632-11</td&gt;<td&gt;杜蒙</td&gt;<td&gt;的JF-5训练班</td&gt;<td&gt;</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"*这些建议的材料。可以换成任何兼容的产品</p

retrograde loading of nerves, tracts, and spinal roots with fluorescent dyes

逆行标记神经元，是一个标准的解剖方法1,2，也被用来加载到3-6神经元钙和电压敏感染料。一般来说，染料应用于固态晶体或局部压力注射用玻璃吸管。但是，这可能导致稀释染料和减少标签的强度，尤其是当几个小时的染料扩散。在这里，我们展示了一个简单和低成本的技术引入使用聚乙烯吸吸管，充满了染料溶液的神经元的荧光灯和离子敏感的染料。这种方法提供了可靠的方式维持在整个装载过程中的轴突接触高浓度的染料。

Watch this Scientific Journal Video about Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes at JoVE.com

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

我们描述了一个简单和低成本的技术引入神经元或神经束，使用聚乙烯吸吸管荧光灯和钙敏感染料的高浓度。

逆行神经，大港，并用荧光染料的神经根装载

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

retrograde-loading-nerves-tracts-spinal-roots-with-fluorescent

Research

JoVE Journal

Neuroscience

12.2K 次观看.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. 我们描述了一个简单和低成本的技术引入神经元或神经束，使用聚乙烯吸吸管荧光灯和钙敏感染料的高浓度。

视频: Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

突触小泡池，利用苯乙烯染料的光转化的研究

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

科研

神经科学

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

蟹电压敏感染料Stomatogastric神经节神经元的光学成像

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

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 ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

促进存活神经干细胞与纤维蛋白和生长因子鸡尾酒和分化的严重脊髓损伤后

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

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 ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

成像膜电位与基因编码荧光电压传感器的两种类型

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

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 ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

脊髓损伤 wistar 大鼠跑步机的作业训练

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.