We thank M. Forney, R. Maged, and K. Hellwinkle for data collection and two anonymous reviewers for comments on a previous version of this manuscript. This research was supported by an NSF award to LDL (IOS-0918268).

所有的程序是由宾夕法尼亚州立大学机构动物护理和使用委员会（IACUC - 协议ID:43242），并遵守所有地方，州和联邦法规。 1.准备<ol><li>购买或构建巴恩斯迷宫，从而确保该目标孔适当大小为感兴趣的物种。对于这个协议，使用八种成人边斑点蜥蜴（ 歌stansburiana）。确定使用其他种类的研究足够的样本大小。 <ol><li>如果构建巴恩斯迷宫，购买任何标准，无孔圆桌会议（106公分），高架76.2厘米掉了一地。 </li><li>使用卷尺，标记隔开26厘米桌子周围的周边10等距点，并从该表的边缘6.35厘米。使用10洞，而啮齿动物迷宫通常有多个孔。这样做允许在逃逸选项的数量的减少。 </li><li>使用孔锯位（2.54厘米，直径），钻出来所有10个目标孔和沙子锋利的棱角。 </li><li>挂载4枚架子支撑钉用螺丝每孔下，使用家庭外壳作为维度指南。 </li></ol></li><li>将迷宫在安静的房间明亮的顶灯和可用的导航（门，柜等 ）空间线索。 </li><li>装入自动对焦，广角镜头的摄像头（11.3厘米×3.99厘米）或其他适当的相机直接在迷宫的上方。可以肯定，从视频馈送视场包含整个迷宫。 </li><li>将计算机和观察员椅至少有1米的迷宫。 </li><li>标记在地板上的迷宫的腿部用永久性标记或磁带。这允许迷宫在试验的过程中的任何微小移动的校正。 </li><li>随机分配孔为"1"，而其他的孔数之一连续通过"10"。永久性标记，写号沿外侧面迷宫为便于参考。确保该数字不是迷宫内的动物中可见。 </li><li>随机指定目标孔使用一个随机数发生器的主题。 </li></ol> 2.培训试验注:爬行动物具有可以从文献中获得的优选的体温。如果动物成为在多个训练试验低迷，迷宫可能是太爽了，这可能会影响行为。一个小空间加热器或热磁带上的迷宫的下侧能够充分提高迷宫表面温度，体温的最佳的热指示，以保持最佳的体温和行为表现。 <ol><li>如果受试者被安置考场外，把他们家的外壳动物进入室内测试前至少30分钟。可以肯定，以便测试之前保持体温提供家外壳（ 例如 ，加热灯，高温胶带）的热量。 <ol><li>打开跟踪软件和视频输入摄像头应用程序。自定义跟踪软件为Matlab和图像处理工具箱写的;其他跟踪软件商用。 </li><li>从视频源评估的视场。如果迷宫不在视野，检查迷宫是在地板上线之内。 </li><li>打开包含信息的日期工作表，开始训练试验，主题ID，分配目标孔，培训运行试验次数，注释和观察者的缩写/结束时间。 </li><li>随机化，其中受试者将被测试的顺序。考试科目有系统地。 </li></ol></li><li>从它的家笼中删除第一主体并轻轻放在迷宫的中间的主题。将在动物塑料桶;由研究人员这样做可以防止无意识的方向偏差。允许30秒来适应。 </li><li>安装迷宫下动物的笼子，正下方为签订目标孔，通过滑动机箱顶部到销坐骑。可以肯定，在家里笼姥岩石直接进洞，这将作为一个鲈鱼当动物下降到洞下。此时，在导航中使用的家用机柜用于使用热或化学线索是无关紧要的。 <ol><li>啮齿动物迷宫用干净的外壳作为主题的逃脱。然而，鼓励迷宫下安装动物的家外壳训练试验期间进入孔。 </li></ol></li><li>轻轻地从迷宫的顶部取下塑料桶。 </li><li>先从摄像头应用程序的训练试验的记录。 <ol><li>允许受试者对探索迷宫10分钟。如果有什么事情发生不典型，注意，这在工作表中。 </li><li>如果受试者落入或运行从桌子上，轻轻返回受试者对迷宫的中间。继续定时和记录，如果动物是出迷宫30秒或更小的。如果主体是走出迷宫超过30秒的，中止审理。记录在工作簿的说明部分这些信息。 </li><li>如果被摄体下降打入球门洞无助，因为在考虑试用在那个时候。 </li><li>如果主题不降入目标孔，考虑到审判结束后10分钟已经过去了。 </li></ol></li><li>如果10分钟后，这个问题并没有下降到球门洞，用手轻轻地引导动物，头先，到合适的目标孔。 </li><li>停止视频和跟踪软件。保存录像。 </li><li>允许符合其家用机箱休息至少2分钟，在机箱仍然安装在迷宫之下。 </li><li>使用喷雾瓶和纸巾，擦拭迷宫的顶部1:10肥皂与水的稀释肥皂混合物。做导航迷宫的时候所以排除了使用化学线索。有些品种，特别是那些大量使用化学感受信息，necessitate用热水迷宫的额外漂洗，以消除所有的气味线索。 </li><li>从迷宫下卸下外壳，要么重复训练与审判同一个人或一个新的个体。每个主题可以每天最多5训练试验运行中，与个人的试验之间30分钟间隔。 </li><li>重复这个过程，直到主体下降到它的目标孔无助，在3个不同的训练试验，表明已发生的目标位置的学习。三个训练试验不需要是连续的。如果受试者达到这一标准，受试者移动到探测试验，以评估空间记忆。 </li></ol> 3.探头试验注意:一旦一个主题达到标准，这个问题已经学会了如何定位的目标。然而，在这点上，目前还不清楚，如果主题是使用了空间的策略或一些其他导航策略导航。探测试验受试者在训练试验达到标准后为这个和s测试应执行的一天。 <ol><li>旋转迷宫180°，保证了迷宫的腿在地板上的标记内。迷宫的转动允许本地线索直接识别的目标是在直接冲突的更稳定的空间线索。 </li><li>重复步骤2.1 - 2.5.2，省略迷宫下安装主体的外壳家里步骤2.3。否则，不得使用迷宫探险时从家里外壳散发嗅觉或其他线索。 </li><li>在探测试验中，受试者可以自由地探索迷宫的完整的10分钟来评估的探索行为。 10分钟后，停止视频和跟踪软件。 </li><li>拆下迷宫主题，并返回到其家笼。 </li><li>清洁用稀释肥皂混合物迷宫的顶部。 </li></ol> 4.行为的措施注:对于培训TRIALS，包括行为的措施，例如延迟至在目标孔到达，制成的错误数（一nongoal孔的调查;动物的鼻子必须是1厘米孔的内），和时间比例的正确象限花费迷宫。 <ol><li>对于训练试验，确定搜索策略。一个直接的战略被定义为降入了球门孔少于3个错误。串行策略是沿迷宫的周边3个或更多个连续迷宫孔检查。随机的策略是用3个或更多的错误迷宫孔非串行检查。 注:有关探测试验，行为措施包括延迟，在空间上正确的孔到达，调查空间校正洞前所犯的错误数量和时间在迷宫的空间正确的象限花的比例。 </li><li>对于非随机的选择空间校正目标孔，使用无需更换抽样的更严格的评估时计算机会率。 </li></ol>

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The authors declare that they have no competing financial interests.

当对空间学习和记忆试验进行测试时，但是也有一些在一些在协议的主要步骤解决几个重要概念问题。首先，受试者必须证明他们正在学习的目标孔的位置在训练试验的过程。达到预设标准表明已发生的目标孔位置的学习。如果患者不学习目标孔的位置，有没有可行的办法来再确定一个导航的策略。如果动物们达到标准，探测试验是确定哪些线索在迷宫导航过程中优先考虑的重要。在探索试验，地方和空间线索相互冲突，迫使一个主题导航使用的唯一线索的一个优先考虑。因此，探针试验为其中球杆的类型被召回和迷宫导航期间优先评估重要。因为专注于空间与当地的线索，它消除可能被用于导航的其他潜在线索是重要的;这些线索可能成为研究的潜在的变乱。特别是在鳞类爬行动物，嗅觉线索起到择偶食品19起主要作用，避免掠夺和位置。因此，消除了试验间嗅觉暗示重要的是要排除嗅觉暗示而在迷宫以下。 虽然这个协议与成年男子边斑点蜥蜴13效果很好，修改迷宫和协议有可能需要在年轻的蜥蜴或在不同物种（LaDage，未发表的数据）测试空间学习和记忆的时候发生。例如，少年蜥蜴（ 即年龄超过9个月的侧面斑点蜥蜴），即跳，或树栖物种可能倾向于逃离了迷宫，很少考虑到陷入球门洞品种。在一些情况下，各地的迷宫壁或封闭的迷宫可以CIR有助于cumventing此问题，只要空间提示仍然从迷宫的表面是可见的。此外，修改的任何类型的迷宫当任务的生态相关性必须加以考虑。侧面斑点蜥蜴，它逃入裂缝在岩石和入地下小孔，在巴恩斯迷宫表现良好。然而，这往往会保持不动的物种可能无法在巴恩斯迷宫任务，除非暴露在激励（ 例如 ，模拟捕食未遂）诱导迷宫探索表现良好。同样地，可以作出其它的修改，以改善包括垂直地定向迷宫树栖物种的任务的性能，提高了孔的直径较大的物种，并且使用孔的皮而不是对不陷入孔物种。以前的研究已经表明，改变迷宫的属性可以创建在性能20-24变异;因此，进行修改巴恩斯迷宫的时候，自然他的差异保守党和感兴趣的物种的行为，必须加以考虑。 虽然这个协议的工作足够好于边斑点蜥蜴探测空间学习和记忆，有一定的局限性和困难，执行的协议。首先，边斑点蜥蜴必要大量训练试验要达到标准，可能是因为没有激励出台，鼓励探索，并从迷宫中逃脱。正因为如此，特别是当与啮齿动物的过程中训练试验的性能相比，书面协议是相当耗时的。引入厌恶刺激来增加动力逃脱迷宫可以规避这一问题，并降低要达到的标准训练试验的数量。此外，由于研究的缺乏的实验测试对于空间记忆和爬行动物的学习，该协议和迷宫​​修改可能不适合或适用乐所有物种。修饰如上面所建议的那些可能有助于在设计用于感兴趣的物种的适当迷宫和协议。 这里所描述的协议允许在一个小蜥蜴空间学习和记忆的实验测试。虽然爬行动物定向和导航已经在该领域7,8,25被证实，鲜为人知的是作为用于哪些线索和导航过程中优先考虑。少数研究提示为优先航行过程中的爬行动物实验检验已经用不同的范式和物种趋于排除有关跨这个分类群的空间学习和记忆的普遍性。然而，在至少一个物种和整个研究规范的测试设备和协议，将在探测的空间学习和记忆的概括帮助。一旦这个基础已经建立，其他的协议的更详细的应用将是适当的。对于instanc即，引入更多明显的局部或暗示训练期间减少空间线索的可靠性可能诱发上利用当地的线索为在迷宫中导航的更大依赖。另外，男女可以优先考虑不同的线索，从而评估对这项任务的女性也可照亮线索优先有趣的中间性的差异。这样的修改，因为这些可能揭示空间和本地线索的显着性和稳定性如何在导航过程中调节内存和随后的调用和使用这些线索的线索优先级。

许多神经变性疾病如阿尔茨海默氏症的本与认知能力逐渐下降，通常并发与大脑1-4的降解。为了测试脑损伤和退化对认知过程的影响，临床研究已经利用模型啮齿类动物和测试设备和协议的标准化的优点。特别是空间学习和记忆过程已经通过几个标准范式，如Morris水迷宫，迷宫巴恩斯和旋臂迷宫评价（这些和其他范式进行了全面审查，见5,6）。这些空间学习和记忆范式的丰富历史已经证明相当成功，使研究人员能够了解许多方面和中人类的记忆，大脑功能和疾病关系的细微差别。 虽然认知过程的评估在临床研究中被检查退出Ë一段时间以来，针对nonmodel物种的认知能力的研究是比较新的。研究人员在nonmodel物种学习认知通常是在认知过程的生态和进化相关兴趣，尤其是在生存和繁殖的环境。在爬行动物一些研究表明，先进的认知能力，特别是空间记忆，可能依据一些行为，特别是关于导航和定向。然而，尽管许多研究已经证实，爬虫可位移7,8-之后重新调整，认知机制基础方向调整行为尚未梳理开。正因为如此，一些研究试图通过实验评估航行在9-17空间学习和记忆的重要性。在这些研究中的方法啮齿类范例和协议，有时修改后用于在爬行动物使用后都主要设计的，但是这些研究曾在评估空间内存变量成功。有研究显示空间学习和记忆中的一些物种11-17，而其他研究没有发现这样的9,10的证据。因此，在爬行动物中的作用或空间学习和记忆的存在，在导航过程中，目前尚不得而知。 一个问题可能有问题时，实验评估爬行动物的空间学习和记忆是任务的生态意义。爬行动物是一个特殊的分类群啮齿动物截然不同，表明在生态学，行为和生理变化很大。在整个行为爬行动物的种属差异可能会影响空间认知能力的评估，特别是当所用的模式不打入一个自然的行为。例如，在通常寻求庇护的小缝里种，空间能力可以很容易地使用巴恩斯迷宫，而这个迷宫可能不是理想的选择模式评估在通常静止不动一个物种。同样，大多数鳞类爬行动物都没有水产，因此Morris水迷宫不得用于测试空间学习和记忆的合适选择（但见15）;然而，这个迷宫可能是龟16测试空间能力的理想选择。最后，该组的生理学必须考虑，因为爬行动物ectothermic和适当的温度的维护，特别是在基片的，必须在检测过程中被考虑。 该协议这里介绍的范式被用于探测空间学习和记忆成人边斑点蜥蜴（ 歌 stansburiana）13日 ，一只小蜥蜴，通常从大鳄出逃到岩石18小裂缝。知道该物种的自然历史与行为的这一方面，我们所使用的传统的巴恩斯迷宫的变形测试对于空间学习和记忆。巴恩斯迷宫我SA旱地迷宫，通常用于在啮齿动物模型测试空间认知。我们修改迷宫在从啮齿动物迷宫几种方式，在设计和协议（下面描述）。我们的迷宫由一个圆形平台具有10个孔相互沿平台的周边等距离（ 图1）。这里描述的方案涉及参加训练试验学习目标孔的位置的受试者，那么，一旦受试者获知目标孔的位置，探针试验用于导航到目标中，以确定空间内存使用。

<table border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Barnes maze</td>
			<td style="width:239px;">TSE Systems</td>
			<td style="width:176px;">302050-BM/M</td>
			<td style="width:183px;">Available from other vendors. Alternatively, a Barnes maze can be constructed from a standard, non-porous round table.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat tape</td>
			<td>Big Apple Pet Supply</td>
			<td></td>
			<td>May also use a small space heater situated on the floor under the maze.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pet keeper for small animals</td>
			<td>Petco</td>
			<td>1230204</td>
			<td>Housing enclosure that can be mounted under the maze.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nickel plated shelf support pegs</td>
			<td>Newegg</td>
			<td>241941</td>
			<td>Pegs attached to underside of maze. Secures enclosure to maze during trials.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LifeCam Studio webcam</td>
			<td>Microsoft</td>
			<td>Q2F-00013</td>
			<td>Available from other vendors. Other brands of webcams may also be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tracking software</td>
			<td style="width:239px;">Code custom written for Matlab 
			and the Image Toolbox</td>
			<td></td>
			<td>Video tracking software. Other tracking software such as VideoMot 2 from TSE Systems can be used.</td>
		</tr>
	</tbody>
</table>

assessing spatial learning and memory in small squamate reptiles

临床研究已经利用了各种范式来评估认知能力下降，通常瞄准的空间学习和记忆能力。然而，在nonmodel物种的认知过程的利益，通常是一个生态环境下，也成为研究的一个新兴领域。特别是，在在爬行动物的认知过程的兴趣不断增加，虽然对爬行动物认知实验研究是稀疏的。少数爬行动物的研究已经进行实验的空间学习和记忆测试使用了修改后在使用爬行动物啮齿类动物的范例。然而，生态的生理学和该分类群组的行为的重要方面，必须对于基于空间的认知测试时考虑。在这里，我们描述了旱地巴恩斯迷宫的修改和相关的测试协议探测空间学习和小鳞类爬行动物记忆能力时可以提高性能。所描述的范例和亲cedures成功与男性侧面斑点蜥蜴（ 歌stansburiana），这表明空间学习和记忆可以在这个分类群与生态相关设备和协议进行评估使用。

该协议允许基于空间的导航的小蜥蜴实验评估。先前的研究成功地利用这个协议探测男性侧面斑点蜥蜴13空间导航。在该特定的研究中，雄性被训练以导航到一个目标孔，并且一旦准则为止，进展到探针试验，以评估导航到一个目标孔时优先的线索。 从研究的代表性成果，这里提出，表明虽然蜥蜴做学习的目标位置，每一个人达到标准所需的训练试验的数量是相当可变的，范围从17 - 81训练试验（平均:48.29 ）。因为变量数目训练试验中达到不同个体准则，试验分为位数和平均。在多次训练试验INDIVI偶花更少的时间来寻找目标孔，特别是训练试验的第一和第四四分位数之间，表明受试者学习目标孔（p值= 0.012， 图2）的位置。在探测试验中，迷宫旋转从而相互矛盾的地方和空间线索。个人赴空间正确的目标孔的速度也优于偶然性，（P &lt;0.001， 图3）和迷宫（P &lt;0.001）的空间正确的象限度过了大部分时间，这表明空间线索被划分优先级导航过程中。 <img alt="图1" src="/files/ftp_upload/55103/55103fig1.jpg" /> 图1.巴恩斯迷宫设计小蜥蜴。巴恩斯迷宫（106厘米口径），高架76.2厘米掉了一地。十等距离目标孔沿周边设置并且间隔开26厘米。目标孔为2.54cm厘米，直径允许SMA<html> LL蜥蜴下降到安装在桌子底下的家笼。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55103/55103fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55103/55103fig2.jpg" /> 图2. 训练试验。潜伏期（S）±SEM发现在训练试验（n =每四分7个人）的目标孔。因为变量数目训练试验中达到不同个体准则，试验分为四分的学习块和平均。个体表现出在延迟的减少，以找到目标孔之间最明显的训练试验（p值= 0.012）的第一和第四四分位数，这表明个体的学习目标孔的位置。从LaDage 等数据。 2012。ig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/55103/55103fig3.jpg" /> 图3. 探测试验。错误的选择空间校正目标洞前的数 + SEM（N = 7）。在探测试验（P &lt;0.001）个人非随机选择了正确的空间目标孔，表明航行过程中使用的空间战略。虚线表示机会性能。从LaDage 等数据。 2012年<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55103/55103fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Assessing Spatial Learning and Memory in Small Squamate Reptiles at JoVE.com

Assessing Spatial Learning and Memory in Small Squamate Reptiles

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

评估空间学习记忆小鳞类爬行动物

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

assessing-spatial-learning-and-memory-in-small-squamate-reptiles

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.1K 次观看.  Penn State Altoona. This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

视频: Assessing Spatial Learning and Memory in Small Squamate Reptiles

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

线虫的积极丁酮学习，短期，长期联想记忆检测

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

科研

神经科学

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

含量的运动能力，学习和记忆的赤字果蝇</em神经退行性疾病>模型

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

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'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 ...

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

使用近红外光谱MazeSuite和功能研究学习空间导航

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

海马胰岛素显微注射和<em在体内</em&gt;微透析过程中空间记忆测试

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

新生大鼠于A气味偏侧学习模型解剖神经回路托换记忆的形成

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

海马神经元活动的可视化免疫组化空间后学习神经发育障碍的小鼠模型

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

果蝇调理作为学习和记忆的措施

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...