这项研究得到了资助，在促进科学（日本学术振兴会），批准援助赠款由教育部，文化，体育，科学和技术在日本，为日本社会科学的研究的援助从鸟类和日本科学技术厅，来自日本的神经信息学中心（NIJC），日本理化学研究所和科研优先领域，结合脑研究（师恩）赠款援助赠款援助的波峰的援助 - 由文部科学省在日本。我们感谢他们的协助，在这部电影奥哈拉公司和圆子林。

协议<ol><li>高架十字迷宫试验中使用的仪器是在A +的配置，包括对面的相互垂直的两个张开双臂（25 × 5 × 0.5厘米），两个中心（25 × 5 × 16厘米的封闭武器）平台（5 × 5 × 0.5厘米）。张开双臂有一个非常小（0.5厘米）的墙，以减少跌倒的人数，而封闭的武器有高（16厘米）的墙包围的手臂。整个装置是高出地面50厘米（大原公司，东京），是放置在一个空的圆形罐（100厘米直径，高35厘米，通常用于Morris水迷宫任务）保护小鼠下降或在实验过程中企图逃跑。该仪器是由塑​​料材料。该平台是白色的墙壁都是透明的。是在高架十字迷宫设备的材料和颜色的变化。 </li><li>小鼠与12 h光/暗周期（上午7:00灯）内，如前面所述（隆夫和宫川，2006A）。行为测试之间进行的上午9:00和下午6:00。所有实验小鼠转移到行为测试室30分钟前开始一审habituate行为测试室条件。测试顺序是平衡整个基因型。有两个测试试验的目的而使用的做法动物。其一是，以确保一切罚款录音系统。另一种是尽可能保持统一的测试条件。也就是说，在整个会议的第一个鼠标将与他人（即，测试业务，并从以前的试验中没有气味的线索事先没有噪音）没有这样的审判实践经验有所不同条件。根据京都大学的动物研究委员会的指导方针保持动物。 </li><li>行为测试室（170 × 210 × 200厘米，小原公司，东京）是隔音和光照强度保持在100勒克斯。鼠标被放置在中心区与走向一个封闭的手臂其头部的迷宫。高架十字迷宫实验是使用一台摄像机连接到电脑，这是一个远程设备控制记录。条目的数量（一个条目定义为鼠标的大规模进入手臂中心）到每个手臂和所花费的时间在张开双臂的记录和这些测量焦虑样行为的索引服务。 </li><li>小鼠被允许自由移动约10分钟的迷宫。每个鼠标接收一个在我们的测试电池的试验。用于采集和分析的行为数据（图片EP）的应用程序是基于对公共领域的图像J计划（韦恩Rasband在美国国立精神卫生研究所和在<a href="http://rsb.info.nih.gov/ij/">可用</a> http://rsb.info.nih.gov/ <a href="http://rsb.info.nih.gov/ij/">/ IJ</a> ），这是刚宫川（通过奥哈拉公司，日本东京）修改。 </li><li>移动的距离，到每个手臂的条目数量，时间花费在每个胳膊，和图片EP方案计算到张开双臂的条目％。 </li><li>每次试验后，所有的武器和中心区的清洁与超级次氯酸水，是一种高效去除异味剂和相对较弱的气味本身相比，其他的清洁解决方案，以防止对嗅觉线索为基础的偏见。因此，我们可以控制嗅觉线索的条件下进行测试。 </li></ol>

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C., Wahlsten, D., Dudek, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+mouse+behavior:+interactions+with+laboratory+environment.">Genetics of mouse behavior: interactions with laboratory environment.</a> Scienc. 284, 1670-1672 (1999).</li></ol>

所有的程序获得批准使用动物保健委员会和日本京都大学的。

尽管小鼠基因组已经测序，大多数基因的功能尚不清楚。 （奥斯汀等人，2004年;相叶等，2007）在小鼠的特定基因的缺失或其他操纵基因改造技术让。的突变小鼠（隆夫和宫川，2006年b;隆夫等，2007）的行为进行分析，然后确定特定基因上的一个特定的行为的影响。 高架十字迷宫测试的所有目前可用的动物模型焦虑的最流行的测试（1997年罗杰斯和Dalvi，克劳利，2007年）。这种焦虑行为测试已被用于筛选和转基因和基因敲除小鼠（克劳利，1999年）分型和药物发现（霍格等1996年;。克劳利，2007年）。高架十字迷宫试验有一个筛选（Rodgers和Dalvi，1997; Mechiel Korte和德波尔，2003;克劳利，2007年）;抗焦虑药物较强的预测有效性抗焦虑药物的增加，具体anxiogenic药物减少，条目的数量成张开双臂和所花费的时间。总项得分和总距离，被认为是一般的活动有用的索引。总条目评分也是焦虑指数，和条目，并在每个手臂所花费的时间的百分比构成初级焦虑指数（1997年，罗杰斯和Dalvi，Mechiel Korte和德波尔，2003年）。开启和关闭的武器被认为是引起同样的探索驱动，因此避免张开双臂被认为是一个更高层次的恐惧“（Rodgers和Dalvi，1997）归纳的结果。它被认为是造成开放和高架空间的恐惧，对小鼠的厌恶探索迷宫的张开双臂。 1984年，汉德利和Mithani高架X（加号）以上所述的迷宫测试的初步工作报告。原来的测试仪器提出了高出地面70厘米，由两个封闭，两个张开双臂，每个测量长45厘米，宽10厘米。在最初的研究中，他们打开/总ARM条目比“（汉德利和Mithani，1984年）。随后，其他指数包括的条目进入封闭式和开放式的武器和大鼠（Pellow等人，1985年; Pellow和文件，1986年）的封闭与开放的武器所花费的时间和小鼠（1987年，制表） 。 高架十字迷宫测试的变化包括延长都张开双臂（50 × 10厘米）和高大的围墙和一个封闭的武器打开屋顶的封闭武器（50 × 10 × 40厘米），整个迷宫升高到了50厘米（Pellow等，1985; Pellow和文件，1986年）的高度。目前，在我们的实验室，高架十字迷宫测试仪器配置在A +的形状，与对面的相互垂直的两个张开双臂（25 × 5厘米，与一个很轻微的，0.5厘米，墙上）两个封闭的武器（25 × 5 × 16厘米），并提出了高出地面50厘米（宫川等人，1996年，真锅等，2000;宫川等，2001;西格等，2004;森岛等人，2005年;宫本等，2005;箭头等，2006年;服部等，2007;尼曼等，2007;佐野等人，2008年，堀井等人，2008年，福田等，2008;池田等人，2008年）。被放置在鼠标+（5厘米× 5厘米）的中心，并允许自由探索迷宫。虽然5分钟的录音是共同的，行为记录在我们的协议为10分钟，以增加检测表型的机会。开放和封闭的高架武器诱发探索冲突（Mechiel Korte和德波尔，2003;克劳利，2007年）。 高架十字迷宫试验的措施，在实验过程中记录观察员出席了会议。在我们的实验室中，测试是一个视频摄像机连接到一台计算机和行为数据（图片EP）收购，并使用图像EP方案分析与记录。到与总手臂条目数量的张开双臂，张开双臂与封闭的武器花费的时间条目的数量，提供焦虑样行为的措施。 我们评估了超过90株突变小鼠基因工程使用在电影中的协议，并有超过5000个小鼠（包括野生型和突变型小鼠）的原始数据的大集。在我们的测试电池中，野生型窝通常用来作为对照。作为背景应变，C57BL/6J小鼠被广泛使用。我们收集C57BL/6J小鼠在行为测试数据。 C57BL/6J小鼠在高架十字迷宫测试获得的值如下（N = 914，平均SEM）的总距离：1547.55 14.27厘米;所花费的时间时间：56.49 2.42秒（张开双臂），384.02 3.13小号（封闭的武器），花了161.90 2.17秒（迷宫的中心）;的时间比在张开双臂：9.19 0.36％，在封闭的武器花费时间的比例：63.82 0.52％;条目数：7.64 0.21（张开双臂）， 24.32 0.28（封闭式武器）;％开放的胳膊条目：21.9 0.05％封闭武器条目：78.1 0.05。 C57BL/6J小鼠在张开双臂花更少的时间比在封闭的武器（P &lt;0.0001，N = 914，配对T T美国东部时间）。这表明，C57BL/6J小鼠倾向于避免张开双臂，和时间花费在张开双臂的焦虑样行为的有效指标。此外，测试顺序是平衡整个基因型，因为审判的数量会影响在张开双臂和中心平台所花费的时间。也就是说，指数均在第3和第4小鼠的增加相比，第一组（P = 0.0089，N = 476）（未发表数据）。随着我们的协议，超过1661小鼠的分析表明，在笼子里的测试小鼠的顺序，并没有显着影响的开放臂停留的时间（未发表数据）。也就是说，从笼中采取的第一个小鼠的表现没有显着差异从第二，第三或最后的老鼠。 虽然都用于评估类似焦虑的行为，高架十字迷宫试验和光/暗转换测试的结果并不总是一致（Holmes等，2000; Tujimura等，2008;岛等，按）例如，脑特定钙调磷酸酶基因敲除小鼠花费的时间减少的金额，光在光/暗转换测试室，但时间的增加量在高架十字迷宫测试（宫川等人张开双臂。 ，2003）。我们行为的测试电池的因子分析表明，高架十字迷宫试验和光/暗转换测试评估焦虑样行为的不同方面，如明亮的空间焦虑在光/暗转换测试和开放空间的焦虑，在开放的现场测试（隆夫和宫川，2006年b;山崎等人，2006年）的行为。因此，光/暗转换测试和高架十字迷宫试验，包括在我们的行为测试电池。 Crabbe和他的同事们报告说，控制变量和特征的突变体的实验可能产生的结果，具体到一个特定的实验室（克拉布等，1999）。程序实验室之间存在的差异，使其难以复制或它们之间的比较结果。建立协议的视觉文档，将促进更好地理解实验程序，允许在实验室和不同菌株的突变小鼠的行为表型的比较中使用的协议的标准化评估使用这些测试。我们以前发布的光/暗转换测试（隆夫和宫川，2006A）的电影协议。同样，其他的协议，如开放的现场测试，看电影，porsolt强迫游泳试验，目前正在作为未来视频杂志文章的出版和恐惧空调测试，我们在我们的行为的测试电池使用。

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<td>Elevated plus maze</td>
<td>Tool</td>
<td>O&#x2019;hara & Co.</td>
<td>(none)</td>
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elevated plus maze for mice

尽管小鼠基因组完全测序，大部分在大脑中表达的基因的功能尚不清楚。突变小鼠的行为分析，可确定特定基因上的一个特定的行为的影响。如果目标基因表达在大脑中，表型的突变小鼠的行为可以澄清的遗传机制的正常行为。高架十字迷宫试验是最广泛使用的用于测量焦虑行为的测试之一。测试是基于开放和高架地区的小鼠的天然反感，以及其自然的自发的探索行为以新颖的环境。该仪器由越过中间互相垂直张开双臂和封闭的武器，和一个中心区。小鼠获得所有武器，并允许它们之间自由移动。张开双臂张开双臂所花费的时间到的条目数是用来作为开放空间诱导小鼠焦虑指数。不幸的是，实验室之间存在的程序上的差异使得它难以复制和实验室之间的结果进行比较。在这里，我们提出了一个详细的电影，高架十字迷宫试验证明我们的协议。在我们的实验室中，我们已经评估了超过90株的使用在电影中的协议的突变小鼠。作为一个公共数据库，我们现在建设的一部分，这些数据将被披露。可视化的协议将促进更好地了解整个实验过程的细节，允许在不同的实验室使用的协议和不同菌株的突变小鼠的行为表型的比较标准化的评估使用这个测试。

Watch this Scientific Journal Video about Elevated Plus Maze for Mice at JoVE.com

Elevated Plus Maze for Mice

高架十字迷宫试验是使用最广泛的测试般在小鼠焦虑行为的测量。在这里，我们目前的电影进行测试的详细程序。

对小鼠高架十字迷宫

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Research

JoVE Journal

Biology

90.2K 次观看.  Graduate School of Medicine, Kyoto University. 高架十字迷宫试验是使用最广泛的测试般在小鼠焦虑行为的测量。在这里，我们目前的电影进行测试的详细程序。

视频: Elevated Plus Maze for Mice

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

光/暗转换测试小鼠

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

科研

生物学

Morris Water Maze Experiment

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered non-fat milk or non-toxic tempera paint, where they must swim to a hidden escape platform. Because they are in opaque water, the animals cannot see the platform, and cannot rely on scent to find the escape route. Instead, they must rely on external/extra-maze cues. As the animals become more familiar with the task, they are able to find the platform more quickly. Developed by Richard G. Morris in 1984, this paradigm has become one of the "gold standards" of behavioral neuroscience.

The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.

Morris水迷宫实验

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered ...

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek&#39;s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: 
 
&#9679; Set up reactions and thermal cycling conditions for a conventional PCR experiment 
&#9679; Understand the function of various reaction components and their overall effect on a PCR experiment 
&#9679; Design and optimize a PCR experiment for any DNA template 
&#9679; Troubleshoot failed PCR experiments

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

聚合酶链反应:基本协议加上故障排除和优化策略

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

牛奶收集方法小鼠和里夫斯&#39;麂鹿

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

在新生小鼠静脉注射

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

亚克隆加插入（SPI） - 一种新型重组工程方法基因打靶载体的快速构建

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

的策略，以验证胼胝质介导的Plasmodesmal浇注在热带反应中的作用

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful in cases of attenuated, oligo- or mono-symptomatic forms of CF usually diagnosed later in life, and of CF-related disorders such as congenital bilateral absence of vas deferens, idiopathic chronic pancreatitis, allergic bronchopulmonary aspergillosis, and bronchiectasis. In both clinical and preclinical settings, the test has been used as a biomarker to quantify responses to targeted therapeutic strategies for CF. Adapting the test to a mouse is challenging and can entail an associated mortality. This paper describes the adequate depth of anesthesia required to maintain a nasal catheter in situ for continuous perfusion. It lists measures to avoid broncho-aspiration of solutions perfused in the nose. It also describes the animal care at the end of the test, including administration of a combination of antidotes of the anesthetic drugs, leading to rapidly reversing the anesthesia with full recovery of the animals. Representative data obtained from a CF and a wild-type mouse show that the test discriminates between CF and non-CF. Altogether, the protocol described here allows reliable measurements of the functional status of trans-epithelial chloride and sodium transporters in spontaneously breathing mice, as well as multiple tests in the same animal while reducing test-related mortality.

Here, we present a protocol to measure nasal potential difference in mice. The test quantifies the function of transmembrane ion transporters such as the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel. It is valuable to evaluate the efficacy of novel therapies for cystic fibrosis.

鼻电位差量化小鼠跨上皮离子转运

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

从小鼠中分离心肌细胞的安特格拉德灌注方法

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating ...

Terminal H-reflex Measurements in Mice

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits after injuries such as spinal cord damage or stroke. An increase of the H-reflex response, together with symptoms like non-voluntary muscle contractions, pathologically augmented stretch reflex, and hypertonia in the corresponding muscle, is an indicator of post-stroke spasticity (PSS).
In contrast to rather nerve-unspecific transcutaneous measurements, here, we present a protocol to quantify the H-reflex directly at the ulnar and median nerves of the forepaw, which is applicable, with minor modifications, to the tibial and sciatic nerve of the hindpaw. Based on the direct stimulation and the adaptation to different nerves, the method represents a reliable and versatile tool to validate electrophysiological changes in spasticity-related disease models.

The clinical evaluation of spasticity based on the Hoffmann reflex (H-reflex) and using electrical stimulation of peripheral nerves is an established method. Here, we provide a protocol for a terminal and direct nerve stimulation for H-reflex quantification in the mouse forepaw.

小鼠终末H反射测量

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits ...