这个项目的资金提供了ARRA资金从NINDS授予NS045647。

1。培训和测试所需的材料<ol><li>测试室。任何容器大到足以容纳一个动物和两个香味杯。我们的实验室利用修改操作性室（医学Associates公司）的与地板条删除，以腾出空间香味杯（ 图1）。 </li><li>香水杯和杯托。杯足够广泛和深入，以便挖掘和一些机制/架（ 图2），以稳定他们。 </li><li>香味砂。我们的测试协议使用先前建立的气味，反应偏差不显示在大鼠（可可，咖啡，罗勒，茴香）4。每一个有香味的沙无气味，110克干净的操场“沙盒”沙1克气味的比例混合，并可以储存在密闭的容器内长达6个月。 </li><li>补强。我们利用水果作为增强循环谷物片。我们建议让动物强化（通过放置在他们的家笼）3至5天之前TRA矿业克服恐新的反应。 </li><li>停止手表或计时器。 </li></ol> 2。动物处理前的培训<ol><li>处理。由于动物将被放置在从培训/测试室反复，我们建议处理动物训练前的三至五天，以了解它们与人类接触。 </li><li>激励。为了激励上执行这项任务的动物食物匮乏，一定程度上是必要的。食物限制开始前至少3天的训练和测试，并可能会维持在90％的免费进料重量的动物。通常周，每天15-20克的量是足够的，但体重应检查每一天，而食品的限制。 </li></ol> 3。挖掘培训训练的动物挖沙可以完成八个交易日的平均时间超过4天的时间。在挖TRA矿业，室应设立由两个香味杯的机制/持有人发出的气味杯安装到腔室。下面详述的目标，塑造动物消耗增强剂完全挖掘沙检索的一般程序。 <ol><li> 1 - 腔习惯和杂志的培训。完全填充的气味杯的加强件（砂）。动物放置到该装置中，30分钟，让动物自由摄取增强剂。 </li><li> 2 - 沙习惯。完全填充的气味杯具强化到每一个杯子，倒入少量无味的沙子填充不超过一半的杯子。动物放置到该装置中，30分钟，让动物自由摄取增强剂。 </li><li> 3 - 表面挖。填写的气味的混合均匀的增强剂和U沙杯具，留下多个强化剂暴露。动物放置到该装置的30分钟让动物自由摄取增强剂。在这个过程中，消耗的暴露的增强剂，动物应发现额外的强化下面的表面的砂。动物应该从使用它的嘴，用它的前爪，从沙中删除每个增强剂。 </li><li>第4次会议 - 挖掘。填充的气味杯与加强件1份3份无气味砂的混合物，留下暴露1-2增强剂。动物放置到腔室中，30分钟，让动物自由摄取增强剂。动物应该开始用它的前爪挖，发现更多的强化，他们从沙中取出。开始强化后，有10分钟的时间内发现的动物不应当予以协助（研究员发现的动物学习的时间，直到1增强剂）。 </li><li> 5 - 充分挖掘。填写杯1增强剂3个部分无味沙的混合物的气味，不留增强剂暴露。解放军策的动物进入装置30分钟，让动物自由摄取增强剂。动物应当予以协助的需要。有些动物可能需要本次会议的多个实例，以证明挖行为。 </li><li> 6 - 试验结构成瘾。将三个强化深，中，浅部在沙中每个气味杯。 ，直到所有三个是取自各一杯，协助根据需要，将动物室。一旦所有被检索到，将动物保持装置为15-30秒，并在此步骤中所描述的气味杯复位。然后，将动物放回室，重复上述过程，直到已完成的动物共有四个试验。动物应该变得习惯于被放置在保持装置的开始挖掘时立即放置到腔室中。动物也应表现出充分的挖掘行为和检索的气味杯底部的增强剂。 </li><li>7 - 单一的强化。将一个单一的加强件在每个杯子中​​的砂的深度的一半。动物放置到腔室中，并允许它来检索这两种增强剂，然后将其放置到所述保持装置中，持续30秒。重复此过程共四个试验。动物应该开始挖掘速度更快，需要研究人员从最基本的援助。 </li><li>会议8 - 模拟歧视的测试。填写杯作为会议7。给动物30秒，同时检索强化。将动物的夹持装置时，都被检索或30秒已经过去了。重复此试验的次数，将用于在测试阶段。动物应学会挖迅速放回室。 </li><li>其他会议。根据初步歧视测试中，实验者可能要执行额外的会议，以了解动物的气味。老鼠往往会在最初表现出恐新症，宁愿熟悉的气味与加固10。 </li></ol> 4。歧视测试<ol><li>研究者应选择的香味配对，香味会诱饵与强化（正确区分），不会。的歧视和逆转，天，试验的最大数量，以及是否受伤前预先测试，如其他决定，将在很大程度上基于对实验的问题（见下面的附加注释）。 </li><li>在大鼠可以测试和手的研究人员记录的数据。研究人员应记录每次试验：正确或不正确的选择，延迟开始挖掘，和老鼠超时试验。 <ol class="lalpha"><li>管理试验的数量应该是在研究人员的自由裁量权（见下面的附加注释）。 </li><li>审讯时间应该是在研究人员的自由裁量权（见下面的附加注释）。 </li><li>在resear雪儿随机试验结构（左 - 右位置的气味杯），有条件的，就是在一排不超过3个试验是在一个给定的侧。这将有助于减少副作用的偏见。 </li></ol></li><li>为了发起每次试验中，将动物进入腔室面向后方，从每个杯子等距。配售的动物之一杯的机会可能会增加发展的偏见。 </li><li>如果正确的动物歧视，允许检索和消耗增强剂，然后将其删除夹持装置，持续30秒。如果超时或选择不正确，请删除它立即之前，“纠正”本身，并将其放置到设备，持续30秒。 </li><li>重复此过程，所选择的试验次数。 </li><li>一旦达到预先设定的标准（ 即 80％的准确率，每天连续三天），动物的动物可能会移动到下一个歧视/反转和重复上面的过程。 </li></ol&gt; 5。其他注意事项<ol><li>在我们的实验室测试发现，培训和测试工作，最好使用二分之一的水果循环谷物片，以避免饱食。 </li><li>在培训过程中，一些动物可能需要比别人更多的帮助。我们建议这样做是必要的，以达到挖掘行为。然而，一旦测试开始，我们从来没有试图进行干预，改善大鼠的挖掘行为。 </li><li>不同的实验室，不同的总的试验次数可能就足够了。在我们的实验室测试6，8，和12次试验，每节1节，每一天，并发现了8个试验设置的偏好。 </li><li>不同长度的审判可能需要在一些实验装置（ 例如 ，动物的下的应力范式可能需要增加试验次数或更少的总试验）。 </li><li>应使用一个预先设定的标准，以确定当动物已经掌握了歧视。在我们的实验室测试要求的accurac超过80％（5/6的试验，试验7/8，10/12试验）y的每一天，连续3天。然而，更高的标准（85％）也可以使用。 </li><li>预设的阈值/最大值应被用于在一个给定的气味略去天的最大数目。这将在很大程度上取决于研究的问题。例如，我们的实验室研究TBI的结果TBI，老鼠都打不标准的先进性，移动/反转后12天的测试一种新的歧视。 </li><li>决定是否前测或后测是一个问题，在很大程度上取决于实验的方向。预测试允许一个，以执行存储器动物伤后的评估，这可能是取决于损伤的类型有关。然而，后测试让一个负责审查收购率差异的群体之间的歧视。我们的实验室已经并将继续同时使用。 </li></ol>

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没有利益冲突的声明。

在这段视频中，我们已经展示了如何挖掘任务可以使用成本相对较低的材料，进行了一定的经验，测试可以进行相当迅速（约10分钟/动物）。这使得任务很容易地纳入到现有的测试电池为脑外伤，中风和其他脑损伤。挖掘任务也很强大，因为它可以评估几种不同的认知方面的，包括以前的学习，反复，和新的收购（ 例如 ，设置转移）的评估。最重要的是，挖任务已被证明是有效的在评估额叶功能大脑受损后。此外，类似的挖范式已经用于研究在大鼠海马损伤4值转移，从而可以很容易地进行研究TBI。 像这样的任务的主要优势是其他形式的认知能力评估通常是测试中最脑损伤螺栓IES。目前存在着缺乏简单，快速收购任务脑损伤评估正面介导的认知。以实验TBI的字段，有强烈的依赖迷宫 ​​作为唯一的评估的认知9。歧视和选择的行为是从正面介导的认知脑损伤后需要进行评估的重要方面。这些都已经在许多不同的模式（延迟匹配样本，差加固率低的响应，努力为基础的决策等）行为的实验分析领域的研究，但缺乏脑损伤的研究14-16。新的疗法用于治疗脑损伤的发展需要感觉，运动和认知功能在多个模式的验证。像这样的任务可能会成为非常重要的全面评估认知功能治疗服务。 虽然挖掘任务重评估脑损伤后的功能提出了正确的方向迈出的一步，它还是有一些限制。它的目的是简单的建设，管理和分析，但是，这意味着它可以测量有上限。目前，两种选择都可以评估在给定的范例。此外，挖任务处理等方面的正面功能障碍，如冲动，可能会干扰动物的决策能力是不够敏感。 ，虽然挖任务已被证明是成功的，我们在年轻的动物脑损伤评价，仍然有几件事情是可以做的，其任务是进一步完善和改进。这些措施包括：评估特殊人群（年龄，性别，应变），直接比较，它是改编自操作性的范例，这个任务与其他任务，评估额叶功能障碍，损伤后进行直接的比较，探索的其他onal的任务产生的数据进行分析和比较。这项任务旨在解决的问题在我们的领域，以多种方式脑损伤的额叶功能测试。然而，一个任务可以测量所有的脑损伤认知过程的影响。因此，我们主张继续发展和改进的任务，如在本协议中的描述，更全面地评估脑损伤后的功能障碍。在这样做时，我们可以评估跨多种措施，增加效益为脑损伤的药物和治疗的发展。

创伤性脑损伤（TBI）和中风引起的脑损伤是在美国的死亡和残疾的主要原因之一。总之，这些疾病导致的共有近250万的发生率，与近50万死亡或减值6-7。尽管这个社会沉重的代价，只有极少数的治疗方法已被开发治疗脑外伤或中风。在一定程度上可以归因于缺乏可用的治疗方案行为评估不足的新的药物和治疗8。特别是，有一个任务，准确，灵敏地评估治疗效果的各种认知障碍，脑损伤后的临床前研究文献的缺乏。目前，大多数的认知进行评估，评估空间记忆的迷宫任务，如Morris水迷宫9。这种缺乏测试有多种原因，包括所花费的时间全面表征和评价认知功能障碍，严重依赖昂贵的设备和专业技术，利用操作性的任务，和这些评估可以执行的速度。为此，我们已经适应了一个基本的香味歧视的任务，挖任务，以评估认知脑损伤。挖掘任务的设计是一种廉价，快速，有效的工具，以评估脑损伤后认知功能障碍从一TBI或中风。 的挖掘任务依赖于学习和决策的基本操作性原则。因为它只有两个选择决定模式，这个任务最简单的歧视，可以代表一个。在下面的协议，以及设置任务的详细信息中的训练方法进行说明。我们发现，这个任务是评估正面介导的认知功能障碍相比单方面顶叶赤字的最强的，但它尚未下进行测试的模型海马损伤5。在理想的情况下，这个任务将是适合于额赤字恢复治疗或治疗受伤的有效性进行评估。然而，这是可能的，修改这个任务也可以被用来探索更复杂的决策行为，如冲动和广义匹配。

<table border="1"><tr><td> 材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>操作性商会</td><td>医学联营公司</td><td> ENV-008CT </td><td>数量：1的标准操作性室，没有外部设备。 </td></tr><tr><td>有机玻璃</td><td>当地的五金商店</td><td> N / A </td><td>数量：1张切成适当的大小，以适应室。 </td></tr><tr><td> PVC管端盖</td><td>当地的五金商店</td><td> N / A </td><td>数量：2 /气味杯设置。 </td></tr><tr><td></td><td></td><td></td><td> 试剂</td></tr><tr><td>沙</td><td>当地的五金商店</td><td></td><td></td></tr><tr><td>各种香料</td><td>当地的杂货店</td><td></td><td></td></tr><tr><td>补强</td><td>当地的杂货店</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> *设备被列入每室建设。 </td></tr></table>

the dig task: a simple scent discrimination reveals deficits following frontal brain damage

在人类大脑受损后的认知功能障碍是最常见的致残原因，还缺乏脑损伤的啮齿类动物模型用来评估认知行为的任务。借贷从操作性文学，我们的实验室利用一个基本的香味歧视范例1-4 以评估在正面损伤大鼠的赤字。此前我们已经简要地描述了挖掘任务，并表明，额叶脑损伤的大鼠表现出严重的赤字跨多个测试任务5。在这里，我们提出了一个更详细的协议完成这个任务。大鼠被放置到一个腔室和允许区分两个香味砂，其中一个包含一个加强件。试用期结束后的大鼠正确的歧视定义为在正确的香味沙挖，不正确的歧视，或30秒的经过。正确识别的影响，恢复和消耗强化。大鼠二从室内被立即删除scriminate不正确的。这可以继续通过各种逆转和新颖的气味。初步分析是每个气味配对（每个气味正确的累积比例）的准确性。从挖掘任务的一般结果表明，它是一个简单的实验准备，可以单方面顶叶损伤的大鼠相比，大鼠双侧额叶皮质损伤评估的赤字。挖掘任务也可以很容易地纳入到现有的认知测试的电池。更多的任务，像这样的使用可能会导致更精确的测试额叶功能损伤后，可能导致治疗选择治疗。根据机构动物管理和使用委员会批准的协议，所有的动物进行使用。

主要有两个变量，可以被记录在挖掘任务：正确/错误和延迟。然而，非应答也可以被记录和分析，因为它们可能是重要的，当考虑动机。这些可以被用来生成分析精度（＃正确/总的试验），响应偏置11，和延迟等的措施。这些措施可以用来评估几种不同类型的学习，这取决于设计的实验（见上述附加注释）。预测试的动物，允许研究员评估内存是个有学问的歧视。拨回的学到的歧视，让一名研究人员看到的动物能够适应不断变化的应变。学习新颖的歧视，使研究人员能够在不同的气味配对，以及评估新型学习能力比较学习。 根据设计的研究（见上面附加注释），这些措施可以分析在重复测量设计，可以折叠成一个单一的变量。以前，我们进入累积比例正确的措施，以便分别分析每个香味歧视5倒塌精度。发生这种情况，总结正确的歧视和总的试验次数除以每个歧视。一系列的单向主体间的方差分析（方差分析），然后用来检查组的差异，为每个受伤后的气味歧视。然而，更多的权力，利用先进的统计技术，如混合效应模型进行分析。一个另一种方法是利用信号检测理论，对数据进行分析。在人的决策文学，这是一个常用的方法，并允许研究人员检查的大多数数据从一个给定的对象在一个单一的图形和快速评估的准确度，偏差和赤字12。分析方法的选择将是Largely驱动实验的问题，是否要跨类型的学习比较，评价一个单一类型的学习，或适用于对数据的决策方法。 双侧额叶脑损伤，实验室研究发现，老鼠在几乎所有类型的学习在这个任务中遭受巨额赤字。正面受伤的动物出现赤字在内存中的一个以前学过的歧视和困难学习，香味配对的拨回（ 图3）。前腰动物也有困难，学习新颖的歧视（ 图3）。根据以前的研究“第13条 ，也有一个大的增加，变化中的正面受伤的动物（ 图3）。 相反，单方面顶叶受伤，在我们的实验室已在此任务中的所有歧视，没有赤字。在我单方面大鼠的研究显示赤字莫里对先前学习的歧视，也获得了逆转歧视非常迅速（ 图4）。他们学会了新的香味配对的非常快（ 图4）。此外，单方面受伤的组内的变异是非常低，与大多数大鼠进行最佳的任务。 <img alt="图1" src="/files/ftp_upload/50033/50033fig1.jpg" /> 图1。在我们的实验室进行测试在一组的标准操作性室（医学Associates公司）进行了调整，在这个任务中使用删除连续四个楼层，酒吧开辟空间两个香味杯。 <img alt="图2" src="/files/ftp_upload/50033/50033fig2.jpg" /> 图2。测试在我们的实验室中使用PVC管端盖（6厘米高，直径为5.5厘米）的气味ç跌宕一块有机玻璃（10×22厘米）的两个钻的圆形孔（直径为5.5厘米）的香味杯架。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/50033/50033fig3highres.jpg" src="/files/ftp_upload/50033/50033fig3.jpg" /> 如图3所示。该图显示了典型的表现为假（蓝色）与正面受伤（红色）大鼠在几个不同的阶段的测试两种不同格式的。在面板A中，数据被表示为与白色的圆点表示的数据点为个别各组大鼠的条形图。在板B中，相同的数据被绘制在​​一个线图。在受伤前的歧视收购上无显着差异。不过，受伤后，在同一歧视，假手术组大鼠在93％累积％的正确执行，而受伤的大鼠在65％累积％执行正确的。当逆转的歧视给药，注射ured影响降到机会表现。同样，在一个新的气味歧视正面损伤大鼠新颖的配对学习有困难，住在下面的机会表现。 <a href="https://www.jove.com/files/ftp_upload/50033/50033fig3large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/50033/50033fig4highres.jpg" src="/files/ftp_upload/50033/50033fig4.jpg" /> 图4。在几个不同的阶段，在两种不同的格式进行测试，该图显示了典型的表现为假（蓝色）与单方面parietally受伤的大鼠（绿色）。在面板A中，数据被表示为与白色的圆点表示的数据点为个别各组大鼠的条形图。在板B中，相同的数据被绘制在​​一个线图。没有任何的歧视群体之间的差异。单方面的jured大鼠进行，或在某些情况下，略高于假水平，损伤后的。 <a href="https://www.jove.com/files/ftp_upload/50033/50033fig4large.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about The Dig Task: A Simple Scent Discrimination Reveals Deficits Following Frontal Brain Damage at JoVE.com

The Dig Task: A Simple Scent Discrimination Reveals Deficits Following Frontal Brain Damage

在这个协议中，一个新的应用程序的香味歧视。挖任务是一个合理的廉价的任务，可以被用来评估大脑受损后的正面-介导的认知。

挖掘任务：一个简单的气味歧视揭示额叶脑损伤后的赤字

Cognitive impairment is the most frequent cause of disability in humans following brain damage, yet the behavioral tasks used to assess cognition in ...

the-dig-task-simple-scent-discrimination-reveals-deficits-following

Research

JoVE Journal

Neuroscience

13.5K 次观看.  Southern Illinois University at Carbondale. 在这个协议中，一个新的应用程序的香味歧视。挖任务是一个合理的廉价的任务，可以被用来评估大脑受损后的正面-介导的认知。

视频: The Dig Task: A Simple Scent Discrimination Reveals Deficits Following Frontal Brain Damage

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

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

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 ...

科研

神经科学

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

染色质免疫沉淀从以下轴索损伤的背根神经节组织

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

在阿尔茨海默氏病模型小鼠学习和记忆障碍的Morris水迷宫测试

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

调查轻度创伤性脑损伤海马电路功能的改变

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

一个简单的替代，以立体定向注射miRNA的脑特异性敲除

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

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 ...

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

Here, a method for isolation of rat brain microvessels and for the preparation of membrane samples is described. This protocol has the clear advantage of producing enriched microvessel samples with acceptable protein yield from individual animals. Samples can then be used for robust protein analyses at the brain microvascular endothelium.

一种简便、可再生的新分离大鼠脑微血管膜样品制备方法

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...