作者要感谢坎迪斯谢医生的建议和援助，对鸡内耳解剖成像技术和多丽丝吴医生的专业知识，在早期胚胎发育。这项工作是由美国国家科学基金会，美国国立卫生研究院IOS-0642346 T32-DC010775，NIH，NIH R01-DC010796，T32-GM008620和美国能源部GAANN P200A120165的支持。

1。准备好以下的解剖工具和试剂<ul><li>人工脑脊液（人工脑脊液; 130 mM氯化钠，3 mM KCl中，1.2毫摩尔KH 2 PO 4，20毫摩尔的NaHCO 3，3 mM的HEPES，10mM的葡萄糖，2毫摩尔的CaCl 2，1.3 mM的用MgSO 4）用95％O连续输注2/5％CO 2中 ，在室温下。输液，补到2/3的500毫升广口的Nalgene瓶的盖子钻了一个洞。坦克通过管道将被附加的玻璃干起泡，贯通通过的jar盖中的孔的人工脑脊液。调整压力以获得恒定的气泡流，达到约1/3的液体，但力度不够导致溅在轮辋。淹没的Nalgene罐分开个别样品从对方的气体湍流的5毫升玻璃小瓶（最多6个）。学联应含氧为任何组织孵育之前至少20分钟。 </li><li>小硅夹层盘（35毫米×10权衡船毫米的Petri涂层的使用Sylgard的人造橡胶包）用解剖针放入小六角聚苯乙烯（47毫米底部）。 </li><li>两个细尖镊子，一个弯曲尖端钳，一个50毫升烧杯中，和用于组织废物的容器。 </li><li>被拉玻璃微（1.2 OD / 0.9 ID）破碎至约20-50微米的前端开放，并填充与罗丹明葡聚糖胺（RDA，分子量= 3000，Invitrogen公司）中的6.25％的溶液与0.4％的Triton X-100的PBS 。安装细管，以picospritzer和稳定微操作机器人。 </li></ul> 2。微解剖，揭示基底乳头E8 4,5 <ol><li>用弯钳，切割E8胚胎的正下方脑干，直接将头权衡船到有机硅涂层的解剖盘内。 </li><li>用解剖针，头的位置，一个腹面观，所以在耳朵上侧的利益是稍微明显，头部稍微倾斜的背部和指向10-30度，距离CE印表机（ 图2A）。立即用别针固定头。如果双边跟踪需要，在0度，中心的头，或一个完整的腹面观，使双方可以访问。类似地，对于一个左双面喷射，在-10℃至-30度的头部固定从中心使左侧是可访问的。 </li><li>使用50毫升的烧杯中，30毫升的含氧脑脊液转移入菜，完全浸没在组织。 </li><li>轻轻抓住下颌细尖镊子剥离下来，然后取出下眼睑上侧的兴趣。 </li><li>上面的血管组织，包括任何剩余的腭和咽喉。这些软组织剥离露出光滑的表面，在其表面上具有鲜明的椎动脉显影chondrocranium。由这点，特征的白色otoconial质量应该是可见的。 </li><li>仔细解剖周围皮肤的外鼻道，下颌关节软骨及中耳，而离开内耳完整。使用镊子轻轻地切掉软骨结构。 </li><li>删除椎动脉和集中使用斯文扫地运动的血液用细镊子提示的。可以掩盖血和凝血可能会堵塞注射针口。 </li><li>蜗管与相邻的感觉上皮（基底乳头）和声学神经节直接位于下方chondrocranium 6的，并可以被形象化为一个细长的手指状突起从内耳腹侧延伸，与最远侧尖端（顶点）附近的前中线5。有可能是一个小的解剖和/或耳石钙化的（白色），蜗管（ 图2B），可以帮助可视化的顶点池的血（红色）。在的顶点是lagenar的黄斑，有感觉的补丁被认为是前庭功能7,8。 </li></ol> 3。 CN第八听觉纤维的选择性标记<ol><li将注射针蜗管和基底乳头区附近的胚胎表面。 </li><li>慢慢地降低的微量移液器第一穿刺头盖骨。这颅地区是厚度小于周边地区和需要减穿透力。微管现在应该蜗管内。可选:一个小的注射追踪染料，可在这里，以帮助可视化的基底乳头。 </li><li>不重新定位微管的情况下，继续降低，直到它只是违反了深边境的蜗管（ 图1）。在这里执行注射剂，并重复沿基底乳头的长度（ 图2C，2D），在多个区域内不含前庭lagena位于最近端的小费。 10-30磅之间，picospritzer，多次注射。注入染料的流体的体积的变化，并取决于所需的程度上的标签。每次注射应导致的荧光染料 - 拉贝莱德点约为200 - 400微米直径的（ 图2D）。 </li><li>一旦注射完成后，用钳子来横切脑干上面的示例中，使用移液管，放置到一个小罐子的尾部的人工脑脊液的容器内，用95％O 2/5％CO 2的不断灌注。 </li><li>对于每个胚胎，允许20-30分钟的染料转移，这取决于所需的距离上的中心跟踪。 </li><li>删除组织和准备​​组织学切片。在这里所示的例子中，在4％多聚甲醛（在1X PBS）过夜，30％蔗糖（1X PBS）过夜组织浸渍。 </li></ol> 4。 Counterstaining与分析<ol><li> Counterstaining是非常有用的帮助观察者的解剖位置，以及确定特定地区的利益。在此制备一种有效的染液是神经丝免疫标记（ 图1，图3B，3E），这强烈升abels轴突和可以划分在中枢神经系统的轴突大片。 </li><li>分析应该执行整个标记的区域中，，突起可以延长几百微米或更大沿rostrocaudal轴。基本分析的一些例子是:目标错误率，时序和/或发散的突起图形的变化。 RDA-标记的轴突与高分辨率（ 图3A，3D）的外周和中枢神经系统中的可视化。 </li></ol>

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没有利益冲突的声明。

第八次神经的早期发展研究已部分限制，因为很难确定胚胎从多个不同的神经轴突产生。一些研究探讨了指导听觉和前庭感觉细胞和神经节细胞的命运在早期发育过程中的分子信号，5,11,12，但还没有得到确定的调节中央神经支配的进程。声神经节细胞预测报告通常描述外围过程到感觉上皮13日至15日 ，而被称为中央工艺主要核突出。中央突出第八细分在胚胎发育过程中的研究也常...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>聚苯乙烯称盘</td><td> Fisher Scientific则</td><td> 02-202-101 </td><td></td></tr><tr><td>培养皿中，35 X 10毫米</td><td> Fisher Scientific则</td><td> 50820644 </td><td>使用硅解剖菜</td></tr><tr><td> Sylgard的有机硅弹性体套件</td><td>世界精密仪器</td><td> SYLG184 </td><td>外套Petri网进行解剖盘</td></tr><tr><td>解剖销</td><td>各个</td><td></td><td>举行胚胎发生在解剖</td></tr><tr><td>氯化钠</td><td>各个</td><td></td><td>脑脊液配方的一部分</td></tr><tr><td>氯化钾</td><td>各个</td><td></td><td>脑脊液配方的一部分</TD&gt; </tr><tr><td> KH 2 PO 4 </td><td>各个</td><td></td><td>脑脊液配方的一部分</td></tr><tr><td> 碳酸氢钠 </td><td>各个</td><td></td><td>脑脊液配方的一部分</td></tr><tr><td>葡萄糖</td><td>各个</td><td></td><td>脑脊液配方的一部分</td></tr><tr><td> 氯化钙 </td><td>各个</td><td></td><td>脑脊液配方的一部分</td></tr><tr><td> 硫酸镁 </td><td>各个</td><td></td><td>脑脊液配方的一部分</td></tr><tr><td>为脑脊液的容器。建议半透明的广口罐，500毫升（16盎司）的Nalgene盖。 </td><td> CPLabSafety </td><td> QP-PLC-03717 </td><td>钻孔开口顶部的盖子为玻璃bubling干到渗透液</td></tr><tr><td>空5毫升玻璃小瓶或类似的透明瓶</td><td>美国制药合作伙伴，公司</td><td> 6332300105 </td><tD&gt;使用在脑脊液孵化到样本保持独立，相​​互借鉴，从鼓泡流</td></tr><tr><td>坦克Carbogen增（95％O 2/5％CO 2）的通过管道连接到鼓泡</td><td>各个</td><td></td><td>连接通过管道输液进入脑脊液玻璃干起泡</td></tr><tr><td>玻璃干起泡</td><td>各个</td><td></td><td>进入脑脊液注入Carbogen增</td></tr><tr><td>弯尖镊子</td><td>世界精密仪器</td><td> 501008 </td><td>从鸡蛋中取出胚胎头</td></tr><tr><td>细尖镊子</td><td>世界精密仪器</td><td> 501985 </td><td>对于显微解剖</td></tr><tr><td> 50 mL烧杯</td><td>各个</td><td></td><td></td></tr><tr><td>罗丹明葡聚糖胺（RDA） </td><td> Invitrogen公司</td><td>各个</td><td>荧光轴突示踪</td></tr><tr><td>的Triton X-100的</td><td> ICN Biomedicals </td><td></td><td></td></tr><tr><td>磷酸盐缓冲盐水（1X PBS） </td><td>各个</td><td></td><td>标准的实验室试剂</td></tr><tr><td>薄壁玻璃毛细管，0.9，1.2 OD ID 4英寸（100毫米）长</td><td>世界精密仪器</td><td> TW120F-4 </td><td>加载RDA。每个毛细管两个玻璃微</td></tr><tr><td>针/移液管拉马</td><td>大卫KOPF工具</td><td>型号720 </td><td>设置:热16.4，电磁阀2.2 </td></tr><tr><td> Picospritzer </td><td>帕克仪表</td><td>各个</td><td>附加细管玻璃微</td></tr><tr><td>微操作机器人</td><td>成重</td><td>各个</td><td></td></tr><tr><td>解剖显微镜与荧光</td><td>各个</td><td></td><td></td&gt; </tr><tr><td> 4％多聚甲醛</td><td>各个</td><td></td><td>标准的实验室试剂</td></tr><tr><td>抗神经丝抗体，可选</td><td>密理博</td><td> AB1991 </td><td>按照组织的协议，制造商推荐的</td></tr><tr><td>低温恒温器和相关材料的切片</td><td>徕卡</td><td>各个</td><td></td></tr><tr><td>外荧光显微镜成像</td><td>蔡司，各种</td><td></td><td></td></tr></tbody></table>

selective tracing of auditory fibers in the avian embryonic vestibulocochlear nerve

鸡胚是一种广泛使用的外周和中枢的神经节细胞预测模型的研究。在听觉系统，听觉选择性标记的第八次颅神经轴突内将提高研究听觉中枢电路的发展。这种方法是具有挑战性的，因为多种感官的内耳器官的第八次神经。此外，可靠区分的标记，听觉与前庭禽流第八次神经轴突内还没有得到确定。听觉和前庭通路不能区分功能在早期胚胎中，感觉诱发反应不存在之前形成的电路。中心突出的第八次神经轴突已经在一些研究中追踪，伴随着但听觉神经轴突标签的标签从其他的第八次神经组件2,3。在这里，我们描述的方法，顺行追踪声节有选择地拉贝升听觉的第八次神经轴突内。首先，在为期8天的鸡胚沉浸在氧合人工脑脊液的前头部的局部解剖，解剖标志确定的蜗管。接着，罚款拉玻璃微被定位成注入少量罗丹明葡聚糖胺进入管和相邻的位于较深的区域中的声学的神经节细胞。在注射30分钟后，听觉神经轴突集中到后脑被跟踪，以后可以可视化的组织学准备。这种方法提供了一个有用的工具，周边听觉中枢电路形成发育研究。

第八次神经和神经本身的解剖结构的组成部分是复杂的，错综复杂的（ 图1，图3）。通过选择性地跟踪从声学的神经节细胞所产生的纤维，可以干净地跟踪段的第八次神经，以及初级听觉传入脑干内区别于他们的前庭同行（ 图2，图3）。同样地，这种技术可用于研究声学的神经节细胞的周缘凸部（ 图3G），或修改，以研究从个别前庭器官所产生的凸起。 ，因...

Watch this Scientific Journal Video about Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve at JoVE.com

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

在这里，我们描述了一个显微切割技术，用荧光染料注射到声早期鸡胚神经节选择性地跟踪听觉神经轴突的神经纤维和后脑。

禽流感胚胎听神经听觉神经纤维的选择性跟踪

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

selective-tracing-auditory-fibers-avian-embryonic-vestibulocochlear

Research

JoVE Journal

Neuroscience

9.1K 次观看.  University of California, Irvine. 在这里，我们描述了一个显微切割技术，用荧光染料注射到声早期鸡胚神经节选择性地跟踪听觉神经轴突的神经纤维和后脑。

视频: Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

科研

神经科学

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

病毒遗传背景明确的神经回路跟踪

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

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 ...

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 ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

人 Intraepidermal 神经纤维内线粒体三维成像及分析

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

影像学检查HT能纤维在小鼠脊髓使用CLARITY / CUBIC技术

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

体外大鼠结肠传入纤维的电生理特性

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

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 ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

通过自由浮动免疫荧光测定,在皮外神经纤维中定位阿尔法核糖核酸聚合体

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...