这项工作是由美国糖尿病协会（ADA），格兰特JF1-10-14（MDiF）。我们要感谢在华盛顿州立大学的实验室动物资源的人员。作者感谢援助尼尔·佩顿，博士从数据统计分析

小鼠和大鼠的TNTs之决心通过增量热板镇痛米iHPAM，公司生命科学印地安人条约理事会（伍德兰希尔斯，CA）]。该设备由几个部分组成：加热系统下方和有机玻璃观察室以上的铝板（10×20厘米）;热控制单元，数据采集软件（印地安人条约理事会第＃软系列8），PC（个人计算机）和脚踏板，遥控启动，停止或重置控制的单位。供热系统还允许开始/停止/手动重新加热过程中，通过在设备前面的键盘。可以设置不同的加热速率，待机/截止温度的iHPAM。 1。设备设置<ol><li>试验前的动物，在视觉上的单位进行检查，以证实所选择的加热速率（ 例如 ，6°C /分钟），初始待机状态，并最终切断的温度（ 例如 28°C和55°C时，分别）。 </li><li>检查所有连接​​。保持结果的电子副本，单位必须连接到计算机上运行的软件系列8。另外，可以打印出硬拷贝结果通过串口单位。 </li><li>测试设备。按下脚踏开关一次，以确保软件系列8条记录的数据：初始待命，最后截止温度和在需要达到的温度切断秒的时间。 </li><li>为进一步控制测试，在实验中所涉及的调查可能需要体验到自己的设备。撤回的手，从金属板，在感知疼痛的时刻（46-48℃），既不能造成伤害皮肤，也不产生任何后不适。 </li><li>一旦单位已经成立，并从步骤1.3和1.4的记录结果，他们可以进行比较。这是有助于发现潜在的问题，可以使用单位无效。例如，初始待机/晋级决赛小康坦佩之间的差异ratures和本单位的实际阅读，和/或由单位和实际的板手撤离所需的温度显示温度之间的差异。 </li></ol> 2。正常动物测试无论是年轻或成年Wistar大鼠[产后（P）年龄21-25天，或任何遗传背景或性别的老鼠（P21-25）可以使用。在我们的实验中使用的小鼠的应变C57BL/6J小鼠。 <ol><li>前一天晚上进行的实验，放在笼子上的iHPAM放在替补驯化的动物。两个小时之前进行测试，开启了iHPAM熟悉本单位的背景噪音的动物。在这一点上，可以设立iHPAM和测试，以验证正确的功能（ 参见步骤1）。整个测试过程中提供的食物和水自由采食 ，除了当动物在观察室（ 见步骤2.1）。 注意：这是小鬼ortant不认为在行为实验的实验室条件下，默认情况下，控制。特别应注意遗传和环境因素可能影响行为研究的结果（ 审查7）。 </li><li>轻轻地放在正常小鼠/大鼠在有机玻璃观察室，对本单位的加热板的顶部。让动物驯化到温暖的表面（在28°C），直到动物显示了一个舒适的行为。 注：在此条件下，正常成年大鼠显示了初步的探索行为，对偶尔站在观察室的盖子，而嗅探。几分钟后，老鼠可能显示休闲较量梳理和宽松的行为。有时，老鼠奠定了一个宽松的身体姿势（容易扩展），而随便嗅闻环境。正常小鼠通常比老鼠更活跃。然而，小鼠显示几分钟后，宽松的探索行为，我n的观察室。观察要么逃逸行为（ 例如 ，跳跃，试图逃跑，从观察室）或防守的态度（ 例如 ，尖叫），这是罕见的。同样重要的是要注意的小鼠可以沟通的痛苦从一个鼠标到另一个8。因此，它可能是重要的试验鼠放置在单独的笼子里。 </li><li>地方反映，在观察室的后面，允许在各个角度的意见。拿着数码摄像机三脚架可能的帮助，以确定确切的时刻，在nocifensive（避免痛苦）的行为发生。 </li><li>一旦该动物是在观察了板腔热舒适按脚踏开关。当加热板温度6°C /分钟的速度，正常的动物表现出nocifensive的行为，涉及任后，爪子*通常温度从46到48℃3,5,9,10肆虐。因此，动物是典型的去除形式observ约3分钟后ATION室。 注：观察大鼠或小鼠，即典型的nocifensive响应，后肢舔爪子，而晃动和/或解除，终止的热刺激是不够的。这是通过按下脚踏板。此时，该软件记录板的温度和板立即冷却下来，直到达到待机温度（28℃）（0.5分）。此温度即一个唤起任何nocifensive的反应被视为有害的热量阈值（TNT），正常（对照组）的动物。 *有时，特别是在小鼠，nocifensive行为有害的温度可能先被发现在一个或两个前爪（ 例如 ，舔而坐），几秒钟前后腿的爪子。然而，前爪子舔是一个正常的修饰行为的组成部分。因此，只有后腿爪子的反应进行评估。小鼠中的场合可能新郎以及他们的后腿的爪子。然而，正常的疏导做N加时赛最后几秒钟，不涉及密集舔，清洗后它的爪子通常结束。 </li><li>轻轻地从板的动物，并放置在其各自的笼子里。其他控制动物（正常），然后重复的热量阈值测量。 注：TNTs之可以在单一的动物，每隔1-2分钟确定没有从动物观察室。在大鼠或小鼠TNT决定，可反复几次，在数天。这是特别有用的动物个体的平均TNT炸药以及其随着年龄的变化来确定。 </li><li>控制动物TNT表示为三个或更多的阈值±标准差的平均值。 </li><li>获得的数据进行统计分析，比较不同的治疗组小鼠或大鼠。正态分布数据，可以适当地使用参数的程序，包括方差分析和治疗组HOMOskedastic;当这些条件都得到满足下列方法可应用于。纽曼两两比较测试，以检测即多次测量的差异，重复/个人未经处理或在不同时间点（ 例如，在图3中的数据）TNTs之方差分析。配对的t检验可用于比较药物治疗前与后两个组数据， 即 TNTs之; 配对 t-测试可用于确定在TNT变化的差异，药物治疗与车辆处理的动物（ 如在图4中的数据）。 </li></ol> 3。糖尿病动物测试菌素（STZ）诱导的糖尿病大鼠是最常用的动物模型研究的PDN的机制，并评估镇痛药物和疗法11,12。大鼠由单一的腹腔剂量（60毫克/公斤），可以对STZ b糖尿病é热痛觉测试在糖尿病发病11后的第三周。重要的是要注意，并非所有的糖尿病大鼠发展的PDN。然而，动物，发展痛觉过敏（50％），可以很容易地确定。事实上，他们的TNTs之都低于正常*。 *要确定TNTs之STZ糖尿病大鼠，起板温度15°C而不是28°C时使用。这些变化的原因是，大多数糖尿病动物与PDN的预期是痛觉过敏2,11。因此，他们将不得不比正常热痛阈值低（ 例如，&lt;45°）。 <ol><li>一名患糖尿病的老鼠被放置在测试仪器上的观察室。动物被驯化的板面，直到动物显示了一个舒适的行为。 注：通常情况下，糖尿病大鼠不显示大部分正常行为。糖尿病大鼠通常机能减退，无精打采，常常表现出限制运动和疏导缓慢。这是罕见的，以观察对观察室的盖子榜上。几分钟后，糖尿病大鼠可能冷静留在室的一个角落里，与周围的休闲嗅探。 </li><li>一旦糖尿病的动物是在观察室的舒适，板被加热步骤2.3可能会出现糖尿病大鼠nocifensive的行为，涉及任后/前爪子。这通常发生在非有害板温度（ 例如 ，&lt;45℃），或室与6°C / min的温度升高不超过5分钟后。 注：一个年轻的成年糖尿病大鼠nocifensive反应不正常大鼠有所不同。然而，唤起这种行为的最低温度显着降低。 </li><li>见步骤2.4 </li><li>以上三个实验当天的阈值的平均值表示是°C±SEM被视为有害的diabeti的热量阈值Ç动物。 注：一个类似的范例解释为正常或糖尿病大鼠可以遵循确定的热反nocifensive热阈值的止痛效果。 </li></ol> 4。测试镇痛药物的抗伤害性的属性（ 如止痛药），以及最低有效剂量的药物，如参数的测定，可以很容易地获得通过上述步骤解释。据预测，适当管理，以大鼠/小鼠的镇痛剂量会显着增加其TNTs之。这是在动物与3热痛觉特别敏感。然而，为了避免在解释结果的偏见，研究者在观察动物的行为所涉及的必须不知道或知道如果动物已处理或不。此外，被观察者的注射药物，可准备另一个研究者和标记A，B，等剂量提供打印输出，列出每个人剂量确定的动物。 根据被测试的药物，不同剂量的一个潜在的镇痛可管理的正常或糖尿病动物5-15分钟前行为测试。必须包含适当的控制（ 即注射用车辆的年龄相匹配的动物和/或无PDN的糖尿病大鼠）和测试使用相同的条件下（ 例如，同一起跑线板的温度）。慢性影响的药物，以及可测试。 5。代表结果板温度最低的唤起任青年非正常治疗的大鼠或小鼠后肢爪子nocifensive反应，确定以下步骤2-1到2-6。正如图1所示，正常的年轻成年大鼠和小鼠TNT分别为47.2±0.2°C和47.5±0.5°C，分别。 我们有OBSErved既不显着的日变化，个人TNTs之（ 图2A），也不在同年龄的人（ 图2B）TNTs之显着变化。 TNT炸药，由于性别的差异并没有观察到（ 未显示 ）。 年轻的成年STZ糖尿病大鼠的TNTs之以类似的方式确定为正常大鼠（步骤3.1至3.3）。然而，起板的温度设定为15°C取代28°C。 STZ糖尿病大鼠nocifensive行为进行了评估，个人和11天注射STZ后开始每天时尚至少三次。正如图3所示，TNT STZ糖尿病大鼠（P &lt;0.01）显着下降，成为明显的两个星期后注射STZ。 STZ糖尿病大鼠的平均TNT炸药为45.6±0.1°C组（n = 16 14-23天的集中值）。 糖尿病大鼠参展显着低于诺玛TNT炸药L者认为有PDN的。这些老鼠被用来测试急性镇痛抗伤害性物业（化合物A）。确定一个化合物或单独测试前5分钟车单剂量注射的正常和糖尿病大鼠TNTs之。 如图4所示，单个化合物的剂量腹腔内显着增加糖尿病大鼠TNTs之时相比，与车辆单独治疗组（45.6°C 与 47.6°C，分别）。的化合物A的反nocifensive效果中也观察到正常大鼠;虽然显着（P &lt;0.01），这是不太明显（47.4°C 与 48.0°C，分别）。 <img alt="图1" src="/files/ftp_upload/3785/3785fig1.jpg" /> 图1。 TNTs之正常年轻的成年大鼠或小鼠的测定：年轻的成年大鼠（P21-24）或小鼠（P20-21）TNTs之在iHPAM测试步骤ABOVE。共有12只和25只小鼠进行了测试。在数天，并进行测量结果进行了汇总。结果以平均值±标准差表示。记录在这些老鼠的TNT炸药的最低和最高值分别为46.2°C和48.6°C分别。在对小鼠的情况下，最大和最小TNT值分别为48.8°C和44.9°C，分别。 <img alt="图2" src="/files/ftp_upload/3785/3785fig2.jpg" /> 图2。年龄依赖大鼠和小鼠的TNTs之：一）绘制四个正常大鼠（N = 4），21天的年龄确定在5天，每天至少三次TNTs之二）TNTs之同窝小鼠的决定表示一次。年龄。每个测定点上的数字代表使用的窝数。 <img alt="图3" src="/files/ftp_upload/3785/3785fig3.jpg" /> 图3。糖尿病大鼠成为痛觉过敏3糖尿病发病后的EKS：6组（n = 6）年龄匹配的年轻成人STZ糖尿病大鼠TNTs之测定糖尿病诱导后11天开始每一天。显示每天的TNT炸药的平均值（平均值±标准差）。在正常范围内（红线）之间的11-13天注射STZ后确定的糖尿病TNTs之。两周后，STZ注射TNTs之则显着下跌至45.1±0.4℃（P &lt;0.05）。 <img alt="图4" src="/files/ftp_upload/3785/3785fig4.jpg" /> 图4。急性反nocifensive效果的化合物B STZ糖尿病大鼠。年轻的成年正常大鼠（蓝组 ，n = 12）和年龄匹配在iHPAM测试车辆后腹腔注射STZ糖尿病痛过敏大鼠（绿组 ，n = 6） （暗杠）或化合物（光棒）。平均每个治疗（车辆控制或化合物A处理）TNT±标准差显示。星号表示统计差异（P &lt;0.05）。

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+alterations+in+thermal+nociceptive+threshold+and+the+distribution+of+immunoreactive+calcitonin+gene-related+peptide+and+substance+p+after+neonatal+administration+of+capsaicin+in+the+rat.">Developmental alterations in thermal nociceptive threshold and the distribution of immunoreactive calcitonin gene-related peptide and substance p after neonatal administration of capsaicin in the rat.</a> Neurosci. Lett. 97, 57-62 (1989).</li></ol>

我们已决定不披露身份在这些实验中使用的镇痛。这里显示的结果的一部分已经在71 届科学会议的美国糖尿病协会（圣迭戈加利福尼亚州）和糖尿病研究（葡萄牙里斯本） 第 47 届欧洲协会的年度会议。

同样经典热板测试，以量化的热痛觉过敏4,13，这里描述痛觉检测允许快速和可靠的方法来量化nocifensive在大鼠和小鼠的行为。然而，相反的经典试验，热板的增量方法非侵入性的，几乎无压力。虽然一些限制是有必要进行测试（ 即动物的，必须是在观察室），大鼠或小鼠习惯于类似的地区（ 例如 ，房屋笼）。 所描述的条件下，正常的年轻成年大鼠和小...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>增量热板镇痛仪</td><td>印地安人条约理事会公司生命科学</td><td> ＃PE34部分</td></tr><tr><td>软系列8 </td><td>印地安人条约理事会公司生命科学</td><td> ＃Series8部分</td></tr><tr><td>菌素</td><td> Calbiochem公司</td><td> 572201 </td></tr></tbody></table>

rapid determination of the thermal nociceptive threshold in diabetic rats

痛性糖尿病神经病变（PDN）的特点是由伤害性刺激的敏感性增加， 即痛觉过敏 ，正常无害的刺激，过敏和异常性疼痛即 。痛觉过敏和异常性疼痛研究已经在许多不同的啮齿动物模型的糖尿病2。然而，由于Bölcskei 等 ，在动物模型中的“ 痛苦 ”的决心表示挑战，由于其主观性质3。此外，使用传统的方法来确定有害的热刺激的行为反应通常缺乏重复性和药物敏感性3。例如，采用热板法Ankier 4，退缩，撤回和/或任何后肢和/或前爪子舔量化为恒高（52-55℃）热刺激反射潜伏期。然而，热刺激痛觉过敏的动物不再现重新显示的差异柔性延迟使用这些超阈值温度3,5。由于最近Bölcskei 等6所述的方法，这里所描述的程序允许的决心，在小鼠和大鼠的热痛觉阈值的快速，灵敏，重现性好（TNTS）。该方法使用缓慢增加热刺激，主要应用于小鼠/大鼠足底表面的皮肤。方法是抗-痛觉特别敏感，学习期间如痛觉过敏状态的PDN。娄的程序的基础上详细公布的，由Almási 等 5和Bölcskei 等 3。这里所描述的程序，已批准的实验动物护理和使用委员会（LACUC），莱特州立大学。

Watch this Scientific Journal Video about Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats at JoVE.com

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

在这里，我们描述了一个快速可靠和简单的程序来确定的最低温度时大鼠或小鼠显示nocifensive的的行为，<em&gt;即</em&gt;“<em&gt;热痛觉阈值</em&gt;（梯恩梯）。这种方法适用于任何一个缓慢增长的热刺激，使精确，重现性与最低估计TNTs之应力的动物。

糖尿病大鼠的热痛觉阈值的快速测定

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

rapid-determination-thermal-nociceptive-threshold-diabetic

Research

JoVE Journal

Neuroscience

27.4K 次观看.  Wright State University. 在这里，我们描述了一个快速可靠和简单的程序来确定的最低温度时大鼠或小鼠显示nocifensive的的行为，即“热痛觉阈值（梯恩梯）。这种方法适用于任何一个缓慢增长的热刺激，使精确，重现性与最低估计TNTs之应力的动物。

视频: Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

评估大鼠蔗糖渴求育成的一个通用方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

科研

神经科学

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

局部和全局的方法评估热痛觉<em&gt;果蝇</em&gt;幼虫

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. 
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of na&#239;ve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

吗啡诱发的痛觉过敏和镇痛耐受小鼠中利用热和机械伤害性的评估方式

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

在自发活动的测定<em&gt;果蝇</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

快速<em&gt;原位</em使用寡核苷酸探针对大鼠多聚甲醛为前缀的大脑5-羟色胺综合征&gt;杂交

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Measurement &amp; Analysis of the Temporal Discrimination Threshold Applied to Cervical Dystonia

The temporal discrimination threshold (TDT) is the shortest time interval at which an observer can discriminate two sequential stimuli as being asynchronous (typically 30-50 ms). It has been shown to be abnormal (prolonged) in neurological disorders, including cervical dystonia, a phenotype of adult onset idiopathic isolated focal dystonia. The TDT is a quantitative measure of the ability to perceive rapid changes in the environment and is considered indicative of the behavior of the visual neurons in the superior colliculus, a key node in covert attentional orienting. This article sets out methods for measuring the TDT (including two hardware options and two modes of stimuli presentation). We also explore two approaches of data analysis and TDT calculation. The application of the assessment of temporal discrimination to the understanding of the pathogenesis of cervical dystonia and adult onset idiopathic isolated focal dystonia is also discussed.

Methods for the measurement and analysis of the temporal discrimination threshold are presented, and its application to the study of the pathogenesis of cervical dystonia are discussed.

宫颈肌张力障碍的时间分辨阈值测量 &amp; 分析

The temporal discrimination threshold (TDT) is the shortest time interval at which an observer can discriminate two sequential stimuli as being ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

小鼠初级少突胶质的快速特异磁分离

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

电刺激在大鼠中建立急性庞廷梗死

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

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) ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

基于旋转加速大鼠漫射性无羟基脑损伤的诱导

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...