我们要感谢Urmas Arumae共享的胶原蛋白协议和尤塔Fey和厄休拉·欣茨优秀的技术援助。此外，我们想感谢基督教F.阿克曼，乌尔里克·恩格尔和尼康在德国海德堡大学的医学影像中心和约阿希姆·基尔希请帮助视频shoting的。部分资金通过德意志研究联合会（SFB 592）的工作。

1。胶原蛋白凝胶的制备<ol><li>准备一个股票介质，含455微升10倍MEM，112微升7.5％ 碳酸氢钠 ，50μL谷氨酰胺和NaOH。的浓度和的NaOH的量取决于对大鼠尾胶原制备（见1.2），最终体积为1,000微升股票介质。 </li><li>制备鼠尾胶原根据到Ebendal（1）。胶原溶液在4℃下存放不能观察到甲降解的胶原溶液。新一批准备后，估计需要的股票介质（见1.1）NaOH的浓度。使用的pH指示剂的培养基的量和浓度的NaOH滴定。 800μL酸性鼠尾胶原蛋白210μL的股票介质混合时，把一个红色阴影。如果的NaOH的浓度太低，则胶原溶液会变黄，由于介质的pH指示剂。介质和混合添加的胶原蛋白无产品ucing泡沫。在冰上执行这些步骤。该聚合反应发生后，在2小时之内的胶原蛋白溶液和库存介质已被混合和新的解决方案是在37℃下温育测试前开始实验。 1.3）混合800μl的胶原蛋白溶液与210μL，该股中期（见1.2）。将100μl的该溶液中的孔中的8孔室滑动。孵育幻灯片，在37℃，5％CO 2和潮湿的条件下的细胞培养箱中。准备下的胶原蛋白凝胶的细胞培养板凳和无菌条件下。 2小时内的胶原蛋白凝胶化。 </li></ol> 2。解剖胚胎SCGS的<ol><li>收获胚胎的时间子宫的孕鼠（E16-18），直到进一步的需要，让他们在PBS。 </li><li>解剖一个胚胎的羊膜。杀头的胚胎尾部的锁骨水平。修正了在硅基底菜头与“昆虫针”。头的腹侧有面对到你的D你看颌下区。的固定能够更轻松地准备。 </li><li>卸下颌下区域左和右的皮肤，并与它的子皮下的结缔组织。 </li><li>删除颌下唾液腺从自己的位置，以清除到舌骨，infrahyoidal的肌肉与胸锁乳突肌的观点。 </li><li>删除这些肌肉，仔细清除到喉头和气管的观点。取出气管和喉部。颈动脉椎前肌肉双方可见。 SCG是位于颈动脉分叉处，有一个椭圆形。小心地取出核，并将其放置在PBS。轻轻地取出神经节，如果他们坚持他们的清扫工具。这是有益的使用钳子的针保持器，以及在SCG去除安装有一个“昆虫针”。 </li><li>一旦在PBS中，存储的神经节，在室温下在PBS中，直到您解剖enouGH节。然后，清洁的神经血管和结缔组织。执行SCG下的层流工作台放置在解剖显微镜下的清扫和清洗。 </li><li>最后，将取出的神经节到凝胶化的胶原蛋白凝胶的帮助下更容易处理的注射器安装在注射器针头。 </li><li>在37℃下，用5％的CO 2和潮湿的条件下孵育SCG的外植体对凝胶化的胶原蛋白在细胞培养箱。 </li></ol> 3。 SCG外植体的处理<ol><li>经过1-2小时的潜伏期，包括SCG用100微升的Neurobasal细胞培养液中含B27的补充，谷氨酰胺和抗生素外植体含有胶原蛋白的。立即室玻片的孔中包含的体积为200微升（加入100μl凝胶和100μl培养基）。添加另一200μl的培养基中，但现在含有NGF（60纳克/毫升），以促进轴突生长。因此，最终的NGF浓度为30纳克/毫升。 </li><li><ol class="lalpha"><li>探讨SC迁移的发生，增加额外的因素，直接在体外日0（DIV0）。双人所需浓度（2）含NGF的介质中溶解的因素。 </li><li>为了分析种姓的影响，已经开始沿着轴突迁移，添加额外的因素DIV3直到DIV4（潜在的停止的实验）。为此，小心地取出180微升的解决方案，在DIV3，并加入200μL新培养基中NGF和其他因素的的双浓度（2）。 </li></ol></li></ol> 4。延时成像使用倒置显微镜进行分析。可以使用的各种目标，定义的字段的视图和放大倍率。然而，一个重要的方面，是所使用的目标的工作距离，SCG外植体的成像进行通过载玻片和胶原凝胶。 1/10-30分钟记录帧速率，显示效果不错（2）。然而，该方面具有被调整到的科学问题。一个正常的CCD摄像头可以被用于图像采集。对于时间推移成像的细胞培养孵育室具有被连接到显微镜的阶段。孵育在成像过程中的组织在37℃下，用5％的CO 2和潮湿的条件下。孵化系统成像开始前一小时开始。这允许显微镜份（ 例如物镜）包括腔室滑动调整的温度和防止温度引起的漂移。定义的特定区域的分析（内的外植体和不同外植体之间），与显微镜的软件和软件控制的机动阶段（多位置设置）用于同时分析不同的应用条件下的SCG外植体的帮助。对于延时成像野生型组织，以及从转基因动物的组织，可以使用标志着在SC（S100B：GFP）（3）。对于成像荧光基团的荧光光源，以实现在micropscope设置。使用标准的过滤器。 5。 SC迁移距离的量化<ol><li>在终点（DIV4例如），修复胶原蛋白凝胶的4％PFA室幻灯片3-4小时。连续洗的胶原凝胶和10分钟，用PBS中的5倍。应用免疫组化（2）的标准协议。如果你是新的编制SCGS，验证身份的神经节作为交感神经节对酪氨酸羟化酶（TH）的一个共同的标记儿茶酚胺细胞的免疫组化。期间免疫组化（初级抗体后）中，外植体的含胶原凝胶转移到24孔板中，其中有一个更大的量，这是有益的洗涤凝胶。 </li><li>免疫组织化学染色后，小心地将胶原凝胶在载玻片上。小心取出剩余的解决方案，让胶原蛋白干/缩小到两个方面。这使得很容易测量的距离（2）。在此之后，装入样品。 </li><li>最后，测量斐济（NIH）软件的帮助下，迁移距离。为此需要任何特殊的插件。为了让正确的测量单位为μm，检查正确的成像元数据和设置在斐济，图像和规模。使用斐济还定量细胞。 </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+expressed+in+mouse+transgenic+lines+mark+subsets+of+glia,+neurons,+macrophages,+and+dendritic+cells+for+vital+examination.">Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.</a> The Journal of Neuroscience. 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S., Massamiri, T., Karwa, A., Warner, B., MacDonald, J., Hemenway, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+imaging+of+cell+death+using+an+Hsp90+ligand.">Noninvasive imaging of cell death using an Hsp90 ligand.</a> Journal of the American Chemical Society. 133 (9), 2832-2835 (2011).</li><li>Shcherbo, D., Souslova, E. A., Goedhart, J., Chepurnykh, T. V., Gaintzeva, A., Shemiakina, I. I., Gadella, T. W. J., Lukyanov, S., Chudakov, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+and+reliable+FRET/FLIM+pair+of+fluorescent+proteins.">Practical and reliable FRET/FLIM pair of fluorescent proteins.</a> BMC Biotechnology. 9, 24 (2009).</li></ol>

没有利益冲突的声明。

外周神经系统的发展，是一个令人兴奋的过程。当开发完成，轴突ensheathed SC的沿其整个长度，可以在人类中，往往是超过百厘米的。为此所需​​的SC的正确数量的必须建立在开发过程中，也有在SC沿着向周边延伸的轴突，以确保完整的轴突覆盖。这成立有髓神经也为unmeylinated的轴突。在这两种情况下，所有的轴突接触公务员事务局局长，取决于他们的支持。为了研究SC发展，需要测定轴突室考虑...

<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> 10倍MEM </td><td> Gibco公司</td><td> 21430 </td><td></td></tr><tr><td>碳酸氢钠（7.5％） </td><td> Gibco公司</td><td> 25080 </td><td></td></tr><tr><td>谷氨酰胺</td><td> Gibco公司</td><td> 25030 </td><td></td></tr><tr><td>氢氧化钠</td><td>默克</td><td> 109137 </td><td></td></tr><tr><td> NGF </td><td>罗氏公司</td><td> 1058231 </td><td> R＆D＃556-NG-100 </td></tr><tr><td> Neurobasal培养基</td><td> Gibco公司</td><td> 21103 </td><td></td></tr><tr><td> B27补充</td><td> Gibco公司</td><td> 17504 </td><td></td></tr><tr><td>抗生素</td><td> Gibco公司</td><td> 15640 </td><td></td></tr><tr><td>的d PBS </td><td> Gibco公司</td><td> 14040 </td><td></td></tr><tr><td>昆虫针</td><td> FST </td><td> 26002-20 </td><td></td></tr><tr><td>针头</td><td>布劳恩</td><td></td><td> BD＃300013 </td></tr><tr><td> 8井室幻灯片</td><td>实验室TEK </td><td> 177402 </td><td></td></tr></tbody></table>

analyzing murine schwann cell development along growing axons

周围神经的发展是一个有趣的过程。神经元发出的轴突支配特定的目标，这在人类的神经元胞体距离超过100厘米。在开发过程中的神经元的存活取决于目标源性生长因子，但也支持雪旺氏细胞（SC）的。为此的SC ensheath轴突从突触或神经肌肉接头的神经元的细胞体（或外周神经系统从中央到过渡）的区域。雪旺氏细胞的神经嵴衍生工具，作为前体以及新兴轴突迁移，直到整个轴突覆盖公务员事务局局长。这表明外周神经系统的发展的SC迁移的重要性，并强调研究该过程的必要性。为了分析SC发展，需要设置下到南海，也包括他们的生理底物的迁移，轴突。由于宫内发育<eM&gt;在体内作用时间的推移成像，但是，是不可行的在胎盘脊椎动物，如鼠标（小家鼠 ）。为了规避这一点，我们调整了颈上神经节（SCG）植技术。神经生长因子（NGF）治疗后，SCG外植体，轴突，其次是SC的前体从神经节沿轴突迁移到外围扩展。该系统是来自内源性SC池和他们一起迁移自己的生理轴突增长的同时，SC的美丽。该系统是特别有趣，因为SC的发展沿着轴突可以通过时间的推移成像分析，开放进一步深入了解SC迁移的可能性。

轴突生长促进从SCG外植体处理后，神经生长因子（4）（电影计划，S1 图1计划）。这个过程是很容易可见的任何倒置显微镜可以后跟延时成像（电影S2）。如果科学家是从小鼠胚胎解剖SCG我们强烈建议通过一个简单的抗酪氨酸羟化酶（TH）免疫组化验证的技术。 TH是儿茶酚胺能神经元（在这种情况下，交感神经元）和一个共同的标记也标签轴突（ 图2B）。轴突?...

Watch this Scientific Journal Video about Analyzing Murine Schwann Cell Development Along Growing Axons at JoVE.com

Analyzing Murine Schwann Cell Development Along Growing Axons

在这里，我们描述了雪旺氏细胞（SC）迁移试验中，SC的方向发展，延长轴突。

分析小鼠沿着轴突生长的雪旺氏细胞发展

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

analyzing-murine-schwann-cell-development-along-growing-axons

Research

JoVE Journal

Neuroscience

11.5K 次观看.  University of Freiburg. 在这里，我们描述了雪旺氏细胞（SC）迁移试验中，SC的方向发展，延长轴突。

视频: Analyzing Murine Schwann Cell Development Along Growing Axons

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

科研

神经科学

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

视网膜神经细胞发育和成熟的单细胞的剖析

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

定量细胞迁移检测方法的鼠肠神经前体细胞

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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 ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

分析树突状形态的列和图层

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.