作者想感谢马蒂Woodlee博士，博士迪安娜阿德金斯，科尔丈夫，莫尼卡答：马尔多纳多，成龙凯恩，博士和蒂姆Schallert援助，在发展中国家的粉丝处理测试，以及为协助开发的Capellini周路处理测试。由美国国立卫生研究院NINDS（NS05689）和美国国立卫生研究院NIA（1F31AG034032 01）的赠款支持。

1。准备测试开始<ol><li>未煮过的意大利面片是用刀片切割长度。对于大鼠，斯金纳品牌粉丝面条直径（1.5毫米）切成7厘米长。对于小鼠，DeCecco品牌capellini面食（直径1.0毫米）削减至2.6厘米的长度（图1C）。因为面食品牌的质地和直径，这可能会影响处理方式各不相同，这是建议，反复测试使用单一来源的面食。 </li><li>为了克服neophobic的反应，并建立在处理技巧，动物暴露在他们的家笼几次在周面食件前测试。 （通常情况下，暴露的5-10天，每次约4件就足够了。） </li><li>如果测试将是管理的一腔家笼以外，如果家里笼移动测试，测试环境的饮食习惯是。 （通常需要3-5次，直到动物很容易吃面食在这种环境下。） </li><li>即使在动物急切地接受面食，他们可能会突然变得沉默寡言当一个大相机是搬到旁边的试验区。是很有帮助的习惯会为一个或更多的摄像头放置在前面的测试环境。 </li><li>因为大多数动物的行为，仿佛面食是非常可口，它是没有必要的食物的限制他们在测试前。然而，去除食物的前几个小时或过夜可方便吃的实验者和照相机面前，并确保它们不会在测试时间吃饱。 </li><li>打破在一块吃面食，审判无效。我们发现，如果动物是从来没有比在测试中使用的，不再在很大程度上避免这种习惯。 </li><li>测试会话，面食件都标有一个特定的时间间隔，使用卡片模板（1.75厘米粉条增量capellini股和0.86厘米增量）的超细提示标志。这有利于在吃面食链运动的可视化。 </li></ol> 2。测试设置<ol><li>动物应家笼或其他仪器unmarred，明确的墙壁的测试，并应在测试过程中会从他们cagemates隔离。 </li><li>虽然老鼠可以实时测试由实行实验者（奥尔雷德等 ，2008），量化，是促进视频录制，可以在慢动作视频播放，使数据收集。我们还没有找到可行的，可靠的量化没有慢动作视频播放，由于他们的爪子和快速运动的小尺寸的鼠标前爪运动。 </li><li>一个具有高帧速率和足够的变焦的摄像头使用。我们用了至少420 S - 1在正常室内照明条件下的快门速度设置为一个16X变焦镜头佳能XL1S相机。轮式三脚架的使用与平移和倾斜的头，有利于相机的定位。 </li><li>老鼠通常在其腰部坐直吃面食和爪子是最容易定位在大约闸室底板水平小幅上升角（图1A）相机看。如果在笼子与刨花测试，去除一些木屑可能是有用的，以优化的观点。 </li><li>小鼠往往在面食吃，向前弯曲角度比老鼠一块。因此，一个开放式的测试室是放置在镜子上方。相机被放置在一个比地面略高的水平，和向下的角度集中在反射镜中的爪子（图1B）。第一面镜子的使用大大提高了清晰度视频播放的爪子。 </li><li>每个面食一块咬声音和，因此，录音是有用核实吃的。 </li></ol> 3。管理面食处理试验<ol><li>测试阶段组成的连续试验，每次审判之一面食吃一块。我们通常每4-5 3-4试验鼠和鼠标每试验记录数据。 </li><li>之前偷拍，被放置到一块面食商会发起的饮食和动物移动到一个适当的相机视图。 </li><li>这是必要的动物，面对镜头和重点，充分放大（〜10 - 12X）查看前爪和所有的数字离散调整。两个爪​​子的关节应可见。至少这台相机，我们发现它很有用，使用自动对焦功能，只审判发病前，在感兴趣的区域缩小。其后，我们切换到手动对焦。 </li><li>只要动物和照相机的位置，第一次审判开始。 A片被丢弃或直接在动物面前垂下，录音开始发病之前吃。 </li><li>此后，试验是在直接继承。这是可取之间的试验，以尽量减少延误，使动物不搬走从最佳观看位置。 </li><li>爪子被视为侧面或失去焦点的试验都没有进球。 </li><li>天，例如，可重复以上测试会话文件前和后处理（例如，中枢神经系统损伤后的恢复。 奥尔雷德等，2008）的时空格局。 </li><li>一个挑战，可以让动物吃在所需的位置，相对于相机。这可能是必要的相机测试前几分钟habituate相机害羞的动物。动物通常可以哄骗到的位置掉落或晃来晃去的理想地区的面食。这可能需要几件来实现这一理想的位置，尽管给定块的数量应尽量减少，以避免饱腹感。 </li><li>几个大鼠的连续测试，我们发现配置轮式三脚架的相机在中间的几个笼子围成一个圈的时候，很方便。 A片下降到每个笼子里的理想地区和相机，然后转移到大鼠，开始先在一个适当的位置吃。这使得更多时间的慢（吃在位置）大鼠成为习惯于相机和实验者存在，他们也可能是刺激吃的更快大鼠刺骨的声音。 </li></ol> 4。前爪调整定量的视频分析和进食时间<ol><li>前爪调整期间吃的分析是一种简单的定量测量，是敏感的单侧损伤后中枢神经系统损伤的保护作用（奥尔雷德等，2008; Whishaw＆科尔斯，1996年，Whishaw等，1997。）和衰老相关的变化在小鼠（坦等 ，2009）。 </li><li>该试验的量化部分面食一块放入口中时开始和结束时最后的面食被释放的爪子和放入口中消失。 </li><li>视频播放的速度（〜50％的实时），或帧帧提前使用，根据需要，确定调整。 </li><li>调整是计算每次爪子发行和重新接触面食片，当有改革的爪子上的一块举行，或当有数字分机前屈或内收外展（图2）。每次调整记录，分别为左，右前爪。 </li><li>没有数字或重新定位的爪子按住运动时爪子整个面食一块幻灯片，调整不计入。运动时，它不是在与面条片（即动作片）接触的爪子也没有计算在内。 </li><li>吃的时候也被记录每次试验中，与饮食的起始和结束时面团放入口中消失开始。我们的记录时间使用视频播放显示功能，但它也可以用秒表记录在正常速度播放。 </li><li>如果动物停留在吃，但保持一个片上举行，时间暂停的总时间中减去。暂停饮食是显而易见的，要么发声咬一个口或停止运动的一块。 </li><li>审判从分析省略，如果在任何饮食的一部分，爪子遮住，或者如果动物打破了一块，因为较短件改变处理模式。 </li><li>一爪子比其他一些大鼠和小鼠始终使更多的调整。对于单方面的中枢神经系统损伤模型，单侧损伤更灵敏的检测，如果受伤影响的爪子，在受伤前的测试调整（奥尔雷德等，2008）。 </li><li>小鼠下降，有时在中间吃面食一块，休息片刻后，把它捡起来，继续吃。在当天的最后审判，可能是由于部分在吃的饱腹感，失去重心，这是最常见的。因为这是常见的，即使在完整的小鼠，我们不要忽略这些试验。然而，一块被丢弃的时间中减去从总的进食时间。 </li><li>在完整的大鼠，下降件非典型和恢复进食往往是非常拖延或缺席。这种试验是从调整和进食时间的分析省略。 </li><li>吃发病前的调整是不计算在内。吃前处理的行为是受中枢神经系统的伤害（例如，Whishaw 等 ，1992;。Whishaw 等 ，1986），但我们还没有延长前爪这一时期的调整，以定量分析方法。 </li><li>这些数据可以概括为：（一）调整每一块爪子，（二）平均每片吃的时间及（c）率的调整（调整/进食时间）的平均数目。 </li></ol> 5。非典型处理模式的视频分析<ol><li>非典型处理模式，可以区分不同的中枢神经系统损伤的保护作用类型（ 奥尔雷德等，2008）和recordi吴这些行为可能是有用的作为一个补充分析。 </li><li>处理模式被认为是非典型的，如果他们经常在完整的动物观察。完整的动物通常会主动与熊掌，“把握爪子”吃，放在面食较低，“指导爪子”口附近（图3A，C），然后放置。同样的爪子通常是在所有试验的把握爪子。往往大鼠位置爪子相距甚远沿第一个长度的面食，一块缩短他们一起移动。老鼠通常口的位置都爪子，接近到另一个举行的把握和指导四肢，甚至在开始进食。在这两个物种，爪子更对称的立场，侧方或数字插话，当一块很短（图3B，D）的移动。在使用期间的饮食指导，持有或推进的面食连续两个爪子。 </li><li>在大鼠和小鼠的爪子位置非典型模式包括：（A）配售的爪子一起，在一个对称的举行，面食一块长（超过原始长度的一半），（B）未将爪子前一块完全消耗（C）对称的位置，开关在饮食中的引导和把握四肢的职务。 </li><li>在大鼠和小鼠的两个附加非典型行为：（A）未为延长时间在一只爪子接触面食一块吃（“不接触”）和（B）使用口渡过面食爪子来重新定位（“口拉”）。 </li><li>两种行为被认为是非典型老鼠，但对小鼠的典型：（A）驼背的姿势和（B）下降面食一块吃后开始。然而，滴在小鼠体内的频率，可能会增加中枢神经系统损伤后。因此，我们在这两个物种记录下降的频率。 </li><li>其他非典型行为已经在大鼠单侧纹状体多巴胺耗竭模型：（A）“铁腕”是在一个静止的，僵硬的立场，把握肢体期间举行吃，（B）反复运动的指导爪子刚性把握爪子，和（C）和面食放入口中的角度（奥尔雷德等人 ，2008头的极端倾斜;各地也看到Whishaw等1997） 。 </li><li>我们观察到，老年小鼠经常作出调整是无效放入口中移动面食的。因此，不会导致在面食一块放入口中可见运动的调整是分开记录。 </li><li>切换引导和把握四肢，联系失败，滴，口拉记录每发生，而所有其他非典型行为记录每试验一次。 </li></ol> 6。代表性的成果从录像分析的结果应包括调整每名审判爪子的数目和每个面食一块吃东西的时候。感觉皮质病变或脑缺血大鼠的结果表明，动物受损（禁忌病变）肢体的效果，至少一个月后受伤等外少调整。单侧多巴胺减少与对侧肢体的调整和增加在患侧肢体的调整等，2008）（奥尔雷德枯竭的结果。从小鼠的研究结果表明岁动物需要更长的时间吃面食件，并进行更多的调整不会导致面食进步放入口中，较年轻的成年小鼠（坦等人，2009年）。这些测试分析，可能还包括非典型处理行为的意见。此外，纹状体损伤和大脑中动脉闭塞导致异常饮食行为等，2008）（奥尔雷德次数增多。 <img src="/files/ftp_upload/2076/2076fig1.jpg" alt="图1" /> 图1：（一）大鼠和（二） 小鼠 。面食处理的姿态。请注意老鼠坐视他们的腰部，而老鼠的预感在面食，需要放置鼠标下方的一面镜子，以查看爪子运动。 （三）面食件的例子，准备进行测试。 <img src="/files/ftp_upload/2076/2076fig2.jpg" alt="图2" /> 图2（AC）鼠和鼠标（DF）的一个例子前爪调整。第一个面板显示调整前的动物，中央面板显示的前爪调整，指示调整爪子的星号（S），和最后一个面板显示面食片的更换。 <img src="/files/ftp_upload/2076/2076fig3.jpg" alt="图3" /> 图3。引导和把握时，面食是（A，C）长和短（B，D）的大鼠和小鼠的肢体位置。

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在这段视频中，我们将演示如何粉丝和Capellini处理测试，分别在大鼠和小鼠进行。这些测试是一种简单，定量的方法来研究在正常动物的爪子和两位数的功能，损害大脑的电机系统后，或在衰老过程中。慢动作的爪子和两位数的动作用于处理未煮熟的面食小块，使研究者能够量化每个爪子作出的调整，以及观察实验引起的中枢神经系统损伤后，可能会出现的异常行为的视频分析。可以反复做的测试会话在不同的日子，大多数动物在几分钟之内完成会话的所有试验。因此，测试是适合纳入到一个更大的电池测试或实验设计。也可以用来测试在运动技能学习（徐等 ，2009），的研究，但在这里我们提出了一个协议，在随着时间的推移建立技能检测变化。 目前，熟练深远的测试是在大鼠灵巧的前肢功能最常用的测试（例如，Whishaw 等 ，1986; Ballermann 等 ，2001;。蒙托亚等人 ，1991年）和小鼠（法尔＆Whishaw，2002年;坦南特和琼斯，2009年贝尔德等人 ，2001年）。到达测试允许每个肢体分离的测试，而意大利面处理是一个双手的任务。然而，测试两种测试在熟练的前爪功能和技能收购障碍的敏感性相似，涉及在前肢的运动皮层的代表 （许志琴等，2009）的可塑性。在我们的经验，达到测试执行更为复杂和艰苦的，一些实验性的问题，目前的测试可能会提供一个更有效的替代。然而，不像这里描述的测试，技能达到测试已使用了几十年（1934年，彼得森），他们现在包括一个多功能高度敏感的措施和测试参数（Ballermann 等 ，2001;法尔＆Whishaw，2002年;蒙托亚等人 ，1991年Whishaw 等，1992）。相比之下，粉丝和Capellini处理测试延长机敏食品处理大鼠的行为（Whishaw和Coles，1996）比较近的刻画。在管理和分析，这些试验无疑可以进一步完善和扩展，例如，可以更精确地量化数字和双手的精细运动模式和量化的舌头，嘴和下巴的协调运动。

the vermicelli and capellini handling tests: simple quantitative measures of dexterous forepaw function in rats and mice

啮齿类动物的饮食行为上刻画的发现，他们使用的协调前爪的动作来操纵粮食件。这项工作时，我们已经扩展开发一个简单的前爪灵巧单侧损伤和年龄相关的变化是敏感的定量测量。鼠害学习娴熟的前爪和数字运动管理薄面食件，他们急切地消耗。我们前面描述的量化大鼠粉条处理方法，并表明，这些措施都非常敏感，从单方面的缺血性病变，大脑中动脉闭塞和单侧纹状体多巴胺耗竭[奥尔雷德，RP，阿德金斯，DL，Woodlee，吨前肢障碍，丈夫，立法会，马尔多纳多马，凯恩，JR，Schallert，T.＆琼斯，TA粉处理测试：一个简单的定量测量大鼠灵巧的前爪功能。<em&gt;研究神经科学。方法</em&gt; 170，229-244（2008）]。这里我们提出一个更详细的协议，在本次测试的大鼠与小鼠，Capellini处理测试新开发的版本进行比较。大鼠和小鼠同时处理未煮熟的粉条或capellini面条，分别短，与定位，以优化爪子运动一个摄像头录像。慢动作视频播放，可以识别定义为任何明显的拆卸和更换的爪子，或任何数量的数字，面食一块吃开始后，前爪调整。前爪调整，每件平均每每个测试环节的试验。反复测试允许在前爪的灵巧，随着时间的推移变化敏感的定量分析。预测试习惯和处理实践的协议，以及非典型处理模式特征的程序，进行了阐述。由于大鼠和小鼠进行的面食处理测试略有不同，这些测试得分在管理和特定物种的差异突出。所有动物的使用是按照协议由得克萨斯大学奥斯汀分校的动物护理和使用委员会批准。

Watch this Scientific Journal Video about The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice at JoVE.com

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

粉丝和前爪灵巧Capellini处理测试的啮齿类动物的自然倾向的优势，利用娴熟的前爪和数字运动操纵的食物。动物的录像，在处理生干面食的短链。慢动作视频播放，可量化的前爪调整。

粉条和Capellini处理测试：在大鼠和小鼠的灵巧的前爪功能简单的定量措施

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

the-vermicelli-capellini-handling-tests-simple-quantitative-measures

Research

JoVE Journal

Neuroscience

20.9K 次观看.  University of Texas at Austin. 粉丝和前爪灵巧Capellini处理测试的啮齿类动物的自然倾向的优势，利用娴熟的前爪和数字运动操纵的食物。动物的录像，在处理生干面食的短链。慢动作视频播放，可量化的前爪调整。

视频: The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

科研

神经科学

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 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 ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

小鼠用树脂叶毒素和疼痛测试进行脑血管内治疗

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

视网膜原酸和皮质化突触功能的电生理学研究

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

小鼠体内合成蛋白的定量测量

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

大鼠运动皮层中的激光诱发脑损伤

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...