<html>简介甲壳类动物的神经肌肉接头有生理和特别是多年来突触生理学提供了重要的贡献。在便于清扫和生存能力可能促进早期的解剖学家和更高生理学家使用甲壳类动物作为实验的准备工作的关键因素。特别是小龙虾很容易从最淡水河流和湖泊以及它们很容易在实验室环境比较冷，咸水环境的甲壳类动物需要维持索取。 一个早在1800年代后期的动物学家了心脏病，尤其是甲壳类（即crayfishes <a href="http://www.biology.ualberta.ca/palmer.hp/thh/crayfish.htm">）和</a> <a href="http://www.biology.ualberta.ca/palmer.hp/thh/huxley.htm">写</a>小<a href="http://www.biology.ualberta.ca/palmer.hp/thh/huxley.htm">龙虾</a> （TH赫胥黎， 1879年）一书。多年担任该文本对这些微生物的指导书，今天仍然是作为一个选择性地对生活的历史，解剖学和生理学方面的crayfishes的综合性著作欢呼。赫胥黎视为模型动物的小龙虾，潜入深处的动物在各方面，因此，他的著作的综合性。时机是有利的，因为生理1880年林格保持蛙心的准备工作（林格，1882a，b）项所需的离子的理解中后期开花。这可能是原因之一，生理实验进展迅速在其他物种以及小龙虾。此外，描述生理盐水维持甲壳类动物的筹备工作已在1936年由Van Harreveld。 令人惊讶的揭幕战中的小龙虾腿肌肉的支配也正在围绕这段历史时期（格拉德别德曼，1887年）特点。但更令人惊讶的是查尔斯Richet在法国小龙虾肌肉生理学研究已经正在进行。事实上，在小龙虾的实验有可能是第一次证明在神经肌肉接头（NMJ）（Richet，1879年;也看到Richet，1881年）的便利。在未来的几十年中，小龙虾NMJs被描述解剖学和生理紧张局势的发展和解剖（范Harreveld和Wiersma，1936年）。 细胞内记录的到来，振兴的领​​域，以解决不同的问题，用锋利的电极（凌和Gerard，1949）。甲壳类动物的肌肉被称为分级收缩（卡茨和Kuffler 1946年，卡茨，1949; Wiersma，1949年），但直到1953年，弼和Katz记录蟹肌纤维短长期便利的跨膜电位。 揭幕战中的小龙虾，四肢肌肉再次强调在1961年时Dudel和Kuffler证明这种肌肉的便利， 并为第一时间发现，突触前抑制的现象（1961a，B; Dudel，1963， 1965a）。他们还报告了本NMJ（1961b）突触传递的量子性质。在过去50年中一直存在相当重视的准备和各种技术，用于监视突触生理学的一个给定的位。对于一个简短超过使用该制剂的调查中，我们开始注意到，整个肌肉是由一个兴奋和抑制轴突可以选择性地刺激支配。阿特伍德（1964年）表明刺激促进兴奋性突触后电位和肌肉紧张的列车。 Irava​​ni（1965）报道取决于地区的肌肉突触反应的区域差异。在此之后不久，Dudel（1965a，b）项记录一起揭幕战上的神经末梢潜力和证明，神经调节 - 羟色胺增强突触传递增加意味着量子内容。 到了这个时候，它的成立，甲壳类动物的肌肉反应谷氨酸和各种氨基酸，以及γ-氨基丁酸（范Harreveld和Mendelson，1959年，罗宾斯，1959年; Kerkut等，1965） 。弗洛里（Bazemore 等人 ，1956年，1957年）和其他（Boistel和弼，1958年）被确定GABA的抑制反应。后来，γ-氨基丁酸是孤立和Kravtiz（克拉维茨和波特，1965年; 克拉维茨等，1963a，B;克拉维茨，1962年）证实，从龙虾的揭幕战制剂轴突。 小龙虾的肌肉提供不仅方便准备，但允许一个研究单一识别运动神经元可以在各种突触后反应的结果在生理和结构层次。开门红肌肉特别是由一个单一的兴奋性运动神经元支配，但不同位置上的兴奋性突触后电位（EPSPS）可以随背表面纤维的50倍（BITTNER，1968a，b）项和高达8折腹肤浅纤维（Irava​​ni，1965年）。 “&gt;开创性的发现，在小龙虾首战NMJ表现在除了短期促进长期便利化（LTF）（Sherman和阿特伍德，1971年），作为一个方面，需要解决这些现象的机理基础。注意，长时程增强（LTP）被发现在脊椎动物的大脑，两年后（布利斯和L MO，1973年）没有引用到原来的小龙虾NMJ现象的发现，从这个时期的属性集中的许多研究者科技型中小企业和LTF使用小龙虾的揭幕战NMJ研究细胞机制（阿特伍德，1973年，1976年，1982年; 阿特伍德等 ，1994;朱克，1973，1974a，B; Bittner和塞维尔，1976年; 帕纳斯等， 1982年a，B，C，D; Dudel 等 ，1983;。Vyshedskiy和林，1997年，B，C）又一个焦点已经了解如何可以产生一个单一的运动神经元支配开门红肌肉中的各种肌肉纤维等多样的突触反应（林德，1974年; G · nzel等人，1993年; Govind等，1994; Iravani，1965年;阿特伍德，1967年; BITTNER，1968a，B;谢尔曼和阿特伍德，1972年，朱克，1974a ;帕纳斯等人 ，1982年a;朱克和海顿，1988年; Dudel，1989，B，C，D）。 突触结构差的突触反应的帐户可以进行调查，通过超微结构分析（Jahromi和阿特伍德，1974年）。由于活动离子差异的措施是能够与钙+和Na +的指标，以及钙+缓冲区（Mulkey和朱克1993年。; 斯罗等人，2002年）轴索注射调查，和这些助焊剂可以是在终端为蓝本（温斯洛等 ，1994; Cooper 等人 ，1996年b）。活动依赖的适应化修改（阿特伍德等人 ，1991年）和神经调节受体亚型（Dropic 等 ，2005年的药理鉴定;。Ruffner等，1999;火花和Cooper，2004; 火花等，2004;泊和库珀，2002;），影响突触小泡池和动力学（劳格斯登等 ，2005;。Southard 等 ，2000;。火花等 ，2003）也审查了这是导致新的问题的方式加以解决。在揭幕战中NMJ的突触传递中的科技型中小企业与膜去极化，钙的作用的概念，导致一些分歧意见（Mulkey和朱克，1991年; Hochner等，1989）。 最近在从单一的运动神经元突触的强度和便利的区域差异，已经解决，似乎是由于当地突触前突触结构的变化和生理（阿特伍德等人，1994年的差异。阿特伍德和库珀，1995年，1996年，B; Cooper 等人 ，1995年，1996年，B）。超微结构分析电子显微研究表明，膨体包含大多数的突触联系（弗洛里和卡希尔，1982年，Cooper 等人 ，1995年）。突触传递的强度下降沿一个终端，这似乎是由于突触结构的复杂性 （Cooper等人，1996年; Govind等，1994）的长度。在突触结构的差 ​​异可能解释的差异的Ca2 +刺激过程中的大量涌入在各种频率 （Cooper等人，1995年，1996年b）的一部分。 由于有地区差异肌肉的表型和生化之间的揭幕战中的肌纤维（G · nzel，等，1993;。Mykles等，2002年），分为 ​​地区，可以解释一个发育调控的肌表型影响和维护的运动神经元（Mykles 等 ，2002）的区域差异。逆行影响的想法已经在青蛙骨骼肌（Nudell和格林内尔，1983年）的调查，在龙虾 （Katz等，1993），在小龙虾（Lnenicka和梅隆，1983年），合理的令人信服的证据。在单个神经元的终端，不影响其他终端的地方性法规是空间中的甲壳类动物的运动神经元很可能因为终端可以测量从1cm到10cm的距离，从对方。与脊椎动物不同，可能包括一个电机单元，在无脊椎动物的肌肉（见1973年阿特伍德，审查）。励磁支配整个开门红肌肉的运动神经元支配担架肌肉更近端的小腿段。开门红肌肉中的肌纤维之间的便利的测量结果表明，这可能与休息中钙水平的差异+离子（Cooper等人，2005年b）和/或可能发行合作（帕纳斯等，1982年a，B） 量子签名之间沿开门红肌肉终端的高和低输出突触结构复杂的差异进行了调查，激活招聘已经在科技型中小企业之间的突触区，但是这已被证明是难以确定（兰开斯特等人，2007年Viele等人，2003年，2006年）。 。之间的高，低产出的突触小泡池，将证明动力学差异来加以规范，因为它是已知的neromodulators低和高输出端子（劳格斯登等 ，2005年差的影响; Sparks和库珀，2004年; Cooper 等人 ，2003年）。 开门红肌肉准备在小龙虾未来的用途是作为丰富的，因为它已被50或100年前。相比其他许多突触的筹备工作的准备还是很耐寒。量子反应，可以直接的突触联系的电生理记录以及囊泡的动态成像，在明确界定的各类终端。的编制并没有失去它的魅力，在单一的兴奋和抑制输入识别神经元。尽管小龙虾没有被遗传操作的实际，研究可能的地址为果蝇的突触蛋白的作用。有许多相似之处，突触功能的果蝇 NMJs（阿特伍德和库珀，1995年，1996年，B），可以由蛋白质注射研究研究（ 他等，1999） 。在运动神经末梢的突触小泡池的监管也是为将来的调查以及机理研究的富人区，了解在科技型中小企业的钙调节（德赛Shah等人，2008年，德赛Shah和库珀，2009）解释许多突触传递的基本原则，其余的奥秘。 方法解剖 克氏螯虾 ，小龙虾，测量体长6-10厘米（阿查法拉亚生物供应公司，Raceland，LA）诱导automize有力地捏在ischiopodite段的第一或第二的步行腿。 <img src="/files/ftp_upload/1595/1595fig1.jpg" alt="图1" /> 转身的腿，直到可以肯定的外（侧方）面临的夹层板。这通常是拱形的一面向上。配售的腿一块纸巾帮助，以便使这些削减的同时，可以打开的准备，很容易。 <img src="/files/ftp_upload/1595/1595fig2.jpg" alt="图2" />用手术刀刀片断路器和持有人的锋利刀片是用来蚀刻的角质层，直到刚刚切割meropodite段在此图所示的模式通过。需要注意的是并非要削减远远远端背侧，腹侧切开的meropodite - carpopodite联合。将代替现在的角质层。 <img src="/files/ftp_upload/1595/1595fig3.jpg" alt="图3" /> <img src="/files/ftp_upload/1595/1595fig4.jpg" alt="图4" />随着手术刀剃须刀刀片的propodite蚀刻角质层，直到切割一方propodite段以上图所示的模式，然后重复对方加入近端的削减。需要注意的是没有切入了开门红肌肉。这是可以做到保持倾斜到接近肌肉，通过角质层切割时刀片。腹切背，周围的腹侧方的连接，要小心不要剪得联合连接近端狭窄，容易折断。将代替现在的角质层。 <img src="/files/ftp_upload/1595/1595fig5.jpg" alt="图5" />应放入盐水的筹备工作。这清扫菜应该有一个底部Sylgard（道康宁）涂层（厚1cm）。Sylgard是用于使昆虫引脚可分为卡仍然持有的准备。在这一点上坚持在尾鳍背角脚，内切，在meropodite窗口。 （＃5）随着细镊子解除略有末端的角质层，剃须刀，切开屈肌纤维远离角质层，切割在远端到近端的方式。提起角质层关闭窗口。 <img src="/files/ftp_upload/1595/1595fig6.jpg" alt="图6" /> <img src="/files/ftp_upload/1595/1595fig7.jpg" alt="图7" />现在削减在meropodite apodeme（肌腱） - carpopodite联合（如下所示）。要非常小心，远离腿部腔拉肌腱，晋级前，只切断肌腱，不是主要的腿部神经，肌腱的内侧是。捏用镊子切断肌腱和解除在尾方向拉屈肌。现在主要的腿神经和伸肌暴露。 <img src="/files/ftp_upload/1595/1595fig8.jpg" alt="图8" />继续propodite段现在削减propodite dactylopodite联合。这里接近肌腱可能削减从角质层附件。拉propodite下来，回尾端腹侧（更紧密的肌肉一侧）段，所以可以看出，尾地区连接的肌肉。这些肌肉切剃刀。要小心不要削减肌肉过于紧密的联合和风险削减到了开门红肌肉运动神经​​分支。现在暴露了开门红肌肉生理盐水。 <img src="/files/ftp_upload/1595/1595fig9.jpg" alt="图9" /> <img src="/files/ftp_upload/1595/1595fig10.jpg" alt="图10" />返回的meropodite区域隔离含有开门红肌肉的兴奋性和抑制性运动神经元的神经束。 meropodite段的最尾端地区腿部的神经束，通常包含一个独立的神经束。这个简短的地区可以看到两束背的包可以用细剪刀切断。砍完，然后拿起＃5镊子轻轻拉远端meropodite段长度的一半左右，直到达到。这种长期的神经分支包含的兴奋首战神经的较大神经束包含了开门红肌肉抑制运动神经元。 <img src="/files/ftp_upload/1595/1595fig11.jpg" alt="图11" /> <img src="/files/ftp_upload/1595/1595fig12.jpg" alt="图12" /> <img src="/files/ftp_upload/1595/1595fig13.jpg" alt="图13" />现在是削减在对角线的方式，例如可以通过放在meropodite背方面，昆虫针在meropodite段的准备工作。这个职位的腹侧开门红肌肉，所以，它面临着观察员（如下图所示）。 <img src="/files/ftp_upload/1595/1595fig14.jpg" alt="图14" /> <img src="/files/ftp_upload/1595/1595fig15.jpg" alt="图15" /> <img src="/files/ftp_upload/1595/1595fig16.jpg" alt="图16" />开门红肌肉块，现在可以通过推动对角质层和propodite腔出的纤维删除剩余接近肌肉纤维。有时，一个结缔组织覆盖可以通过小心使用＃5镊子删除的揭幕战。主要小腿神经沿着开门红肌肉和进入的dactylopodite可以是dactylopodite联合或只是拉着用细镊子开始削减。这主要的腿神经，有时明显相关的血管现在可以拉近端方向轻轻开门红肌肉的长度，然后剪去。 <img src="/files/ftp_upload/1595/1595fig18.jpg" alt="图18" />现在暴露了开门红肌肉没有任何组织在一个细胞内的电极或联络macropatch电极的一种方式。 <img src="/files/ftp_upload/1595/1595fig19.jpg" alt="图19" />为了刺激开门红肌肉的兴奋性神经的准备工作现在转移到录音室，用塑料吸电极设计。在进入会议厅建成的刺激电极可避免使用刺激电极放置一个显微。引脚准备录音碟和地方的神经，它包含在吸电极的兴奋神经的分支。 <img src="/files/ftp_upload/1595/1595fig20.jpg" alt="图20" /> <img src="/files/ftp_upload/1595/1595fig21.jpg" alt="图21" /> （从采取Mykles，DL，Medler，SA，Koenders，A.，和库珀，RL（2002）肌原纤维蛋白异构体的表达是缓慢的小龙虾和龙虾爪和腿开门红肌肉纤维的突触效能与杂志。 205实验生物学（4）：513-522）。 盐水解剖的准备工作是维持在小龙虾生理盐水，修改后的范Harreveld的解决方案（单位：mm：205氯化钠5.3氯化钾13.5 CaCl2.2H2O; 2.45 MgCl2.6H2O;调整pH值7.4的HEPES）。 记录细胞内EPSPS 为了激发诱发反应，选择性地刺激兴奋的轴突草刺激。开门红肌肉的区域是用锋利的细胞内电极（20至30毫欧电阻）与3米氯化钾填补刺穿。可以使用一个标准的头阶段，细胞内记录放大器;然而，我们采用的一种模式Axonclamp 2B（分子器件，美国加利福尼亚州的桑尼维尔市，美国）放大器和1个LU头阶段。不同的刺激条件下，可以通过短期便利（STF）或其他类型的各种所需的响应。科技型中小企业获得通过给予10年或20秒的时间间隔，分别在10年或20脉冲的火车，兴奋神经。列车内的刺激频率可以是多种多样的（40，60和80赫兹）。 INTR脱细胞EPSP的录音进行例行这些标准程序（Crider和库珀，1999年，2000年，Cooper 等人 1995年b; Dudel，1983; Sparks和库珀，2004年，德赛，沙阿和库珀，2009）。 开门红肌肉分为三个一般地区的远端，中央和近端。即使整个开放的肌肉是由一个单一的运动神经元支配，NMJs在结构上是不同的，有特定的区域突触效能的差异，在这三个地区（Cooper等人，1995年a，B ）一般。的肌纤维表型类型也已表明，在这些地区的不同（Mykles等， 2002）。基于这些原因，最远端的纤维使用，因为它们很容易被划定的筹备工作之间的一致性。 <img src="/files/ftp_upload/1595/1595fig22.jpg" alt="图22" />直接录制神经末梢识别地区的中心量子EPSPS 突触膨体可视化的重要染料4 -二- 2 - ASP（Magrassi 等 ，1987），这并不影响突触传递的浓度和聘用倍，（5微米，5分钟的治疗，Cooper 等人，1995年）。管腔宏观补丁记录电极（Cooper 等人 ，1995年c。ST“hmer 等 ，1983）的荧光显微镜，可以直接放置在一个单一的孤立的静脉曲张。为了唤起人们的神经末梢，兴奋的运动神经受到刺激，如上所述。自发以及诱发的量子反应，可以录制沿可视化膨体的字符串，轻轻地降低了管腔，并提高它在每个静脉曲张。 基本上由1981年Dudel，描述了一个宏观的补丁电极;武伊托维奇等人通过突触电位记录。 （1991年）和Mallart（1993年）。 Kimax玻璃（外径：1.5毫米），被拉到和火抛光生产内径从10到20微米的补丁提示。电极内腔充满了沐浴液。该放大器是上述细胞内的录音中使用的相同。通过测试电流脉冲通过电极，电极和密封性能，可确定。密封电阻范围从0.3到1.0 M0hm和电极电阻为0.5〜1.0 M.0。密封电阻可以监视整个录音。 量子事件的直接计数可能与低刺激的频率。对于每一个诱发反应，量子事件的数量才能确定。一系列反应，量子事件总数计算，然后根据这些直接计数估计平均量子内容。计算平均量子含量的一种方法是采取总数的答复总数（德尔卡斯蒂略和Katz，1954），广达和鸿沟。有其他可以使用的方法之一，以及基于对峰值振幅或EPSPS 面积 （Cooper等人，1995年）。

historical view and physiology demonstration at the nmj of the crayfish opener muscle

在这里，我们目前的一些关键重要的发现开门红神经肌肉接头（NMJ）甲壳类动物的准备和说明，仍然有很大的借鉴这种模式的准备。在了解历史中，人们可以明白为什么即使在今天这个NMJ仍然提供了丰富的的操场上解决问题，关于前和突触后的功能和可塑性。为细胞内，以及作为外的电生理和影像的可行性和易于访问终端是显着的优势。在高和低输出端子的落后水泡动力学和融合的调制机制是乞求作进一步调查。准备还提供了一个计算的评估和操作的可测试的模型系统，研究在理论模型的突触功能的关键变量，例如在短期促进钙的动态。活动区和统计性质的量子释放的突触的复杂性也是一个开放区域为将来的调查，实验和计算。

Watch this Scientific Journal Video about Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle at JoVE.com

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

首战小龙虾腿肌肉是其历史的重要性，并在实验的多功能性肌表型，突触生理学和可塑性。

小龙虾开瓶器肌肉NMJ的历史观和生理学示范

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

historical-view-physiology-demonstration-at-nmj-crayfish-opener

Research

JoVE Journal

Biology

21.2K 次观看.  University of Kentucky. 首战小龙虾腿肌肉是其历史的重要性，并在实验的多功能性肌表型，突触生理学和可塑性。

视频: Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

果蝇幼虫NMJ夹层

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

科研

生物学

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

果蝇幼虫NMJ免疫

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

电生理方法记录从果蝇幼虫NMJ突触潜力

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

鼠标提睾肌肌肉制备的微循环活体成像

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

组织分流和冻结的骨骼肌疾病模型

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

生理学实验室演示：肾小球滤过率在一个老鼠

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...