Name: a behavioral assay for investigating the role of spatial memory during instinctive defense in mice
Uploaded: 2018-07-21T13:00:00.000+00:00
Duration: 5 min 49 s
Description: Watch this Scientific Journal Video about A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice at JoVE.com

这项工作是由威康信托/皇家学会亨利戴尔奖学金 (098400/Z/12/z), 一个医学研究理事会 (MRC) 赠款 MC-UP-1201/1, 康威信托和盖茨比慈善基金会 (到肺结核), MRC 博士奖学金 (D.E. 和 R. V) 的资助。和勃林格殷格翰全宗博士奖学金 (到 R. V)。我们感谢科斯塔斯卡 Betsios 编程的数据采集软件, LMB 的机械和电气车间, 为建设实验舞台。

所有实验都是根据1986年《英国动物 (科学程序) 法》 (PPL 70/7652) 进行的, 经过当地的伦理认同。
1. 设置行为器具
注意: 行为器具的主要组成部分是巴恩斯迷宫13的变体, 由一个白色的丙烯酸圆形平台 (直径92厘米) 与20等距, 5 厘米直径, 圆形孔, 从平台的边缘径向定位, 5 厘米。.19这些将被关闭与黑塑料插头 (5 毫米深), 而剩余的孔导致一个黑色有机玻璃风雨棚 (维度 15 x 5.8 x 4.7 cm) 老鼠能容易地进入和退出。竞技场的中心区域由一个固定的圆形平台 (直径22厘米) 和外围安装在一个框架上, 通过计算机控制的步进电机可以在360度范围内旋转。平台从地板上升高了45厘米, 以防止老鼠爬下去。参见图 1中的示意图。
<ol><li>将视觉提示附加到平台外围 (2D 或3D 个不同形状、颜色和图案的符号), 距离边缘至少2厘米, 以防止老鼠到达。 注: 可视提示的示例为262厘米2的纯蓝色正方形, 或圆形 (直径15.5 厘米), 用对角紫色条纹填充。</li>
	<li>添加〜2克的床上用品从鼠标的家庭笼到内的庇护所, 作为一个地方嗅觉提示。(可选) 在庇护所入口处放置一个发光二极管 (LED), 以提供当地的视觉地标提示。</li>
	<li>要将鼠标从外部、远端视觉线索中分离出来, 请装配一个八角或圆柱形壁, 在平台表面下延伸40厘米以上和15厘米, 在竞技场周围, 定位10厘米, 远离平台边缘。</li>
	<li>将超声波扬声器放置在平台中心上方50厘米处, 以提供厌恶听觉刺激。</li>
	<li>将一个半透明的屏幕64厘米以上的竞技场, 以支持项目的开销厌恶视觉刺激使用投影仪。</li>
	<li>投影机提供的光线将照亮行为器具;校准它, 使在平台表面的中心有 5-10 lx。将红外线灯和红外线摄像头放在平台上方, 用一个长通滤波器 (> 700 nm) 防止投影仪在获取的视频中闪烁。</li>
	<li>使用商用软件或合适的算法在线跟踪鼠标的位置。为跟踪白色竞技场上的黑鼠, 一个简单的阈值操作, 然后用掩模质心的计算是适当的。</li>
	<li>理想情况下, 将行为器具放在隔音盒内, 防止外界提示影响实验。</li>
</ol>2. 提供天生的厌恶刺激
注意: 下面描述的刺激可以用各种各样不同的软件包生成, 包括 LabVIEW 和 Matlab。
<ol><li>视觉刺激<ol>
<li>投射到屏幕上的刺激, 位于竞技场上方64厘米。刺激包括一个线性地膨胀的黑暗的圈子在灰色背景22 (韦伯对比 =-0.98, 亮度 = 7.95 cd 或 m2)。 注意: 标准圆应该是直接地位于鼠标的位置之上, 对2.6°的视觉角度在开始时, 在 224°/s 200 毫秒到47.4°之间线性扩展, 在偏移前它保持为250毫秒。</li>
		<li>当提供视觉刺激时, 把它放在鼠标上方。这可以通过等待, 直到鼠标通过一个预定义的区域在竞技场, 或通过跟踪鼠标位置在线和使用跟踪坐标来定义圆的中心。 注意: 虽然我们建议将视觉刺激直接投射到鼠标上方, 但如果在鼠标前面投射同样的刺激 (同时仍与地面平行), 也可以引发防御行为。在我们的经验, 这是情况下, 至少在30厘米之前, 鼠标的位置。</li>
	</ol>
</li>
	<li>听觉刺激<ol>
<li>触发一项由三频率调制的列车组成的刺激, 从17到20赫23 3 s, 总共持续9秒。在平台级别测量的声压级 (SPL) 应在73和78分贝之间的范围内。</li>
		<li>将声卡和放大器连接到舞台上方的超声波扬声器, 以提供听觉刺激。</li>
	</ol>
</li>
</ol>3. 动物:
<ol><li>使用雄性或雌性 C57BL/6J 小鼠, 介于6至24周前。保持在12小时的光循环的动物, 免费进入周和水。</li>
	<li>确保小鼠在行为检测前至少放置72小时, 并在光周期的光阶段对其进行测试。 注意: 我们发现, 单一居住的小鼠的基线探索行为比动物群所居住的小鼠更具可比性。此外, 有外科植入物进行神经活动记录或操作的动物通常需要单独安置, 因此在相同的住房条件下有控制数据集是有益的。</li>
</ol>4. 标准行为测定:
<ol><li>精心清洁的平台表面与70% 乙醇或乙酸去除不必要的嗅觉线索。用清水冲洗遮蔽处, 用70% 乙醇冲洗, 再用清水冲洗, 以减少乙醇气味。</li>
	<li>将平台和遮蔽处彻底干燥, 以免老鼠在潮湿的情况下进入遮蔽处。</li>
	<li>在实验之前, 通过旋转平台, 随机地在平台上为每个试验提供避难所的位置。庇护所的位置将锚定在竞技场上, 但将旋转与行为器具外壳。</li>
	<li>把鼠标带到实验室的笼子里, 把笼子放在测试平台的顶部, 10 分钟的驯化期。</li>
	<li>将鼠标从其主笼中取出, 并避免被尾部检索: 要么是杯状的, 要么让它爬上可以被举起的浓缩物品。通过尾部采摘老鼠已经显示增加焦虑水平, 从而可能影响行为反应的威胁24。</li>
	<li>轻轻地将鼠标放在竞技场的中心, 开始视频采集。</li>
	<li>允许7分钟的适应期, 在此期间没有厌恶刺激。确保鼠标有足够的时间找到和进入避难所至少一次。如果所有四只四肢都在庇护所内, 就把它当作一项入境。7分钟应足以使大多数老鼠找到并进入庇护所, 但如果不发生这种情况, 则将适应期延长5分钟, 直到至少有一个入口被核实。</li>
	<li>触发厌恶刺激 (听觉或视觉) 的交付, 要么通过观察现场视频饲料, 或自动跟踪动物的位置在线和预先定义一个领域的利益触发刺激交付的区域一旦动物到达它。视觉刺激通常是通过在线视频跟踪坐标直接传递到动物的上方。</li>
	<li>要在同一个会话中引出多个转义响应, 请让鼠标主动离开掩蔽处。如果老鼠在最后一次厌恶刺激后没有成功地逃到收容所, 那么至少在六十年代之前, 再提出另一项刺激措施。</li>
	<li>化验结束后, 将动物送回其家笼。如果需要对同一鼠标进行进一步检测, 请在重新测试之前至少等待48小时。</li>
	<li>在测试下一只动物之前, 按照步骤4.1 和4.2 所述清理平台和遮蔽物。</li>
</ol>注意: 步骤5、6和7是标准行为分析的独立变体, 每一个都可以单独执行。
5. 导航策略的行为检测:
注意: 这个化验的目的是确定老鼠用来指导逃逸行为的线索。在这个实验中, 平台的可移动的外部部分是旋转的, 而鼠标在固定的竞技场中心, 它径向地置换了遮蔽处、位于遮蔽处的嗅觉线索以及连接到平台的近距离视觉线索。如果鼠标跟随任何被旋转的线索, 以便导航到避难所, 它将正确地逃离到避难所的新位置, 但是如果它使用任何其他的线索保持到位, 它将导航到以前的庇护所位置。
<ol><li>执行上文所述标准行为分析的步骤4.1 至 4.8, 引出至少一项飞行响应作为基线。</li>
	<li>让鼠标主动离开庇护所, 等待它导航到平台的中心部分。在中心放置一个小碟子与床上用品, 以鼓励探索和保持在中央平台的动物足够长的时间, 允许旋转的平台的外部部分。添加菜和床上用品, 而鼠标是在避难所和成功逃生后, 在平台旋转之前。如果鼠标在逃生响应期间没有进入避难所, 等待它自愿进入避难所, 然后添加菜和寝具。</li>
	<li>使用步进电机旋转平台至少36度 (2 孔), 然后立即使用感官刺激, 以引起逃生反应。这是可能的旋转触发惊吓, 甚至启动一个逃生响应。 注: 从我们的经验来看, 55% 的老鼠将跑出中央 (非旋转) 平台, 而外部部分仍在旋转, 这些试验应排除在分析之外。</li>
	<li>清洁平台和遮蔽处, 并将鼠标返回到其家庭笼子, 如步骤4.10 和4.11 所述。</li>
</ol>6. ' 动态环境 ' 行为检测:
注: 本试验旨在评估小鼠如何适应其防御行为对环境突变的影响。在这些实验中, 在引出至少一个转义响应后, 会更改掩蔽位置, 并允许鼠标在触发随后的转义响应之前访问它。如果鼠标完美地更新了庇护所的新位置, 并在其以前的位置计算出庇护所的缺勤情况, 它应该逃到新的位置。否则, 要么它没有记住庇护所的新位置, 要么记住了这两个地点, 而更喜欢逃到旧的地方。
<ol><li>在上文所述步骤4.1 至4.8 之后执行此项化验, 至少以一个飞行响应作为控制。</li>
	<li>一旦鼠标自愿离开避难所, 拆除庇护所, 并重新定位它在一个新的洞在平台的另一端。</li>
	<li>允许鼠标探索环境, 直到它进入它的新位置的避难所。</li>
	<li>当老鼠自愿走出庇护所时, 用厌恶刺激来呈现它, 观察它的反应。</li>
	<li>使用听觉刺激的实验, 因为它的持续时间较长, 在长期的威胁下, 动物可能会对其他地点执行额外的逃生反应, 如果他们没有找到庇护所在第一。</li>
	<li>清洁平台和遮蔽处, 并将鼠标返回到其家庭笼子, 如步骤4.10 和4.11 所述。</li>
</ol>7. ' 无庇护所 ' 行为分析:
注意: 这项试验的目的是了解环境中的庇护所的缺失是如何影响防御行为的表现的。
<ol><li>堵住所有20个洞。在这个实验中, 竞技场上没有可用的庇护所。</li>
	<li>清洗和烘干平台后 (步骤4.1 和 4.2), 执行步骤 4.4-4.6。</li>
	<li>允许7分钟的适应期。</li>
	<li>展示视觉刺激并观察防御反应。缺乏庇护所促进了冰冻行为, 通常持续持续的视觉刺激。使用一个缓慢扩大的斑点 (e.对应2.6°的视觉角度的圆圈, 在 11.2°/s 上线性扩展到 47.4°, 在偏移前保持1秒) 以促进对冻结反应的观察。</li>
	<li>如果每个鼠标需要多个响应, 请使用至少六十年代的相互刺激间隔。</li>
	<li>在没有庇护所的情况下获得所需的响应数后, 在其中一个孔下引入遮蔽处, 并让老鼠探索环境至少5分钟. 5 分钟的时间, 大多数老鼠都能找到并进入新引入的庇护所。, 但如果不发生这种情况, 则将此期间延长5分钟, 直到至少验证一个条目为止。</li>
	<li>重复步骤4.8 和 4.9, 并观察在环境中存在庇护所时执行的转义响应。</li>
	<li>清洁平台和遮蔽处, 并将鼠标返回到其家庭笼子, 如步骤4.10 和4.11 所述。</li>
</ol>8. 数据分析:
注意: 以下每个数据分析步骤都可以独立执行。
<ol><li>在厌恶刺激的表现之后, 逃逸反应很容易被识别为突然加速。量化的滞后时间, 以避免刺激演示和头部旋转运动的开始, 针对目标的逃生或加速度运动的开始, 谁先来。</li>
	<li>冰冻反应的特点是动物的完全静止, 除了呼吸运动25, 持续至少 0.5 s7。测量厌恶刺激的表现与静止期开始之间的时间延迟。</li>
	<li>对于每个转义响应, 通过计算在飞行过程中小鼠与遮蔽处的距离和实际位移的比值来量化飞行路径的线性度。</li>
	<li>对于每个转义响应, 通过测量动物指示其飞行的孔与通向遮蔽处的孔之间的距离来计算精度。由于有20个孔, 每个孔关闭目标代表减少10% 的准确性 (e., 2 孔关闭目标可以表示为80% 精度)</li>
	<li>对于 "导航策略" 的行为分析, 在平台旋转之前量化相对于掩蔽位置的响应的准确性。平均精度与前旋转精度没有显著差异, 表明该动物没有使用任何旋转的感官线索来导航到逃生目标。</li>
	<li>对于 "动态环境" 行为分析, 将所引出的每个转义响应分类为指向新的或旧的遮蔽位置, 或两个都没有。评估模式如何沿引发的防御行为的迭代进行演化。</li>
	<li>对于 "无遮蔽" 行为的分析, 比较鼠标的速度后, 显示厌恶视觉刺激的存在和缺席的庇护所, 并计算的延迟启动的防御反应。另外, 为了响应每个情况的刺激, 计算触发冻结响应的概率。</li>
</ol>

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结果部分中包含的大部分数据是淡水河谷et. al 2017 中提供的数据的子集。

我们在这里描述的测试在技术上是简单的执行, 除了平台旋转实验, 可以很容易地实现在标准巴恩斯迷宫。然而, 可能会出现一些并发症: 一方面, 老鼠似乎害怕进入庇护所, 这可能是由于没有充分清洁或干燥的庇护所。另一方面, 老鼠可能会在庇护所里呆很长一段时间。这是关键的这些实验, 动物从来没有从避难所手动删除, 因为这可能扰乱路径整合以及困扰动物和改变他们对威胁的反应。我们建议在90分钟后终止实验, 必要时再进行48小时的重新测试。另一项相关的实际考虑是, 当鼠标研究平台的边缘时, 如果它的头部倾斜在边缘上, 它可能无法检测到视觉刺激, 因此我们建议在这种情况下对刺激进行预扣。此外, 小鼠可能会在罕见的场合下跳下平台。我们建议结束实验, 并在48小时后重新测试, 积极离开平台的老鼠有可能在将来再做一次, 可能不得不被排除在研究之外。最后, 根据我们的经验, 大多数老鼠会对视觉刺激 (34/36 只老鼠) 做出反应。然而, 如果老鼠对视觉刺激没有表现出任何类型的反应 (惊吓, 飞行或冰冻反应), 他们应该被排除在研究之外。
需要考虑的一个重要问题是, 感官线索的控制通常是空间记忆检测中的一个问题, 因为通常很难消除所有可能的污染线索来源。我们的实验设置减少了外部视觉提示, 周围的迷宫与墙, 它被放置在一个声音阻尼柜内的声音隔离。为了最大限度地减少嗅觉暗示, 我们建议在实验之间彻底清洗70% 乙醇或醋酸的设置。
我们的行为分析增加了以前研究空间导航26的方法, 使用本能的防御行为来探测空间记忆并聚焦于动物行为学场景。这里描述的程序和标准的 BM 检测之间的一个关键区别是缺乏培训课程。在我们的化验中, 适应期确保老鼠至少一次和多次访问避难所, 我们以前已经证明, 这足以记住2的庇护所位置。我们认为, 尽管缺乏训练, 但在寻找避难所的成功率和准确性方面的一个重要原因是, 在实验中绝不会被动地取代动物, 从而使路径整合成为可行的导航策略。然而, 我们注意到我们的检测处理在一个单一的会话中形成和评估的记忆, 我们没有测试的长期记忆的庇护所位置, 这通常是标准 BM 实验的目标。最后, 通过使用离散固有的厌恶刺激, 而不是通常使用的风扇或嗡嗡声在会议期间, 我们的化验提供了很好的实验控制两种行为可能使用不同的导航策略: 觅食和避难所定向逃生。我们认为, 使用这些化验与现代神经科学技术的记录和操作神经活动可以提供重要的洞察力如何计算神经回路的行为。

本能的防御行为主要被认为是硬线刺激-反应, 如在鱼和两栖动物的 C 开始运动, 这使动物远离威胁源1。然而, 如果他们灵活地考虑到有关当前环境的信息, 防御行为可能会更适应。这种灵活性的一个例子是, 在面临威胁时, 由啮齿类动物所显示的从逃逸到冰冻的转变, 这取决于环境2、3中是否存在庇护所的事先知识。其他在先天行为上的灵活性的例子包括根据猎物和它的庇护所之间的距离调整飞行起始阈值或逃逸速度4、距离威胁5、6和以前的经验11,12、根据厌恶刺激7的感官特性选择不同的防御策略, 甚至在面对诸如饥饿8 等竞争动机时抑制防守行为,9,10。行动选择对环境空间特征获取知识的依赖性使得小鼠本能的防御行为成为研究目标选择、空间记忆和导航的有力模型。在这里, 我们描述了一个常用的行为任务的适应, 巴恩斯迷宫 (BM) 空间记忆分析13, 以确定空间环境对小鼠防御行动选择的影响, 以及他们的导航策略时,逃向避难所。
用于研究小鼠学习和空间记忆的标准巴恩斯迷宫由一个圆形的 ~ 90 厘米直径平台和20个同样间隔的孔组成, 其中19个是闭合的, 一条通向地下的避难所, 老鼠寻找, 以避免开放领域平台的环境。通常, 微弱的厌恶刺激 (蜂鸣器, 明亮的光, 风扇) 在整个试验期间连续使用, 使环境厌恶和因而促进进入风雨棚14。在最常用的化验15,16, 动物有一个适应试验, 它被引导到避难所, 由实验者在被放置在平台上后立即手动。接下来是一个4天的购置期, 每天允许鼠标在迷宫中自由地航行3分钟, 之后, 如果在勘探期间没有到达, 它又被手动引导到避难所。试验的最后阶段是一个探针试验在5天 (虽然一个长期七天记忆试验也通常执行), 当动物探索迷宫与所有孔闭合了。学习和长期记忆是量化的时间, 以寻找避难所和戳在错误的洞在获取期间, 并在接近封闭的遮蔽孔在探针试验。典型的结果表明, 在采集过程中到达遮蔽处的误差和滞后时间的减少, 以及在探针试验15中包含闭合目标孔的象限中所花费的超概率比例。
虽然 BM 分析的几个变种以前被描述了17,18,19, 我们这里描述的范例有三根本性变动从标准试验。首先, 动物被留下来探索迷宫和自己找到避难所, 和测试是在同一届会议上, 在发现庇护所后不久, 在动物从事探索行为的时期。虽然这个设置不测试遮蔽位置的长期记忆, 它被设计成一个自然主义的场景, 模仿新的领土的探索, 在捕食的威胁下。此外, 它允许测试动物如何适应环境中的剧烈变化, 如景观的突变。第二, 我们的试验的一个关键方面是, 实验者从来没有强迫动物进出庇护所, 这可以迷惑动物和排除路径集成20作为一个可行的导航策略21。路径集成是一种导航策略, 它使用自运动提示 (如本体和前庭信号), 这些信号是由电机流出的积分引起的, 用于更新动物的当前位置并导航到目标, 这是不可能的, 如果动物被实验者被动地移动。第三, 我们使用先天厌恶视觉22和听觉23刺激, 以获取逃生的庇护所, 这是很容易区分与持续觅食行为, 并允许评估和量化的具体导航策略使用在防御迫在眉睫的威胁。我们建议, 这种分析将有助于解剖空间记忆在选择和实施防御行为的作用, 更广泛地研究目标导向导航和短期空间记忆。这里描述的协议是由淡水河谷和al介绍的。在 2017年, 我们参考读者, 更深入地讨论实验和结果的基本原理。

<table><tbody><tr><td>Infrared iluminators TV6700</td><td>Abus</td><td>-</td><td></td></tr><tr><td>DLP projector Infocus IN3126</td><td>Infocus</td><td>0001740992-00000001</td><td></td></tr><tr><td>Ultrasound speaker Pettersson L60</td><td>Pettersson Elektronik</td><td>-</td><td></td></tr><tr><td>Amplifier QTX PRO240</td><td>QTX</td><td>-</td><td></td></tr><tr><td>Soundcard Xonar D2</td><td>Asus</td><td>90-YAA021-1UAN00Z</td><td></td></tr><tr><td>HP Z840 desktop</td><td>HP</td><td>F5G73AV</td><td></td></tr><tr><td>100 micron drafting film</td><td>Xerox</td><td>3R98145</td><td></td></tr><tr><td>Near infrared camera: Basler acA1300-60gmNIR</td><td>Basler</td><td>106202</td><td></td></tr><tr><td>National Instruments BNC-2110</td><td>National instruments</td><td>777643-01</td><td></td></tr><tr><td>LabVIEW 2015 64-bit</td><td>National instruments</td><td>-</td><td></td></tr><tr><td>Custom made Barnes maze</td><td>MRC Laboratory of Molecular Biology mechanical workshop</td><td>-</td><td></td></tr></tbody></table>

a behavioral assay for investigating the role of spatial memory during instinctive defense in mice

进化选择了一系列的防御行为, 这对所有动物物种的生存都是必不可少的。这些行为通常是由于对固有的厌恶感官刺激的反应而引起的刻板印象, 但是它们的成功需要足够的灵活性来适应不同的空间环境, 这种变化可以迅速改变。在这里, 我们描述了一种行为分析, 以评估学习空间知识对小鼠防御行为的影响。我们已经适应了广泛使用的巴恩斯迷宫空间记忆分析, 以调查老鼠如何导航到一个庇护所, 在逃避反应的先天厌恶感官刺激在一个新的环境, 以及如何适应环境的急性变化。这一新的检测是一个动物行为学范式, 不需要训练和利用的自然探索模式和导航策略的小鼠。我们建议, 这里所描述的一系列协议是研究目标导向行为和刺激触发导航的有力手段, 它应该对本能行为和空间记忆领域都感兴趣。

暴露于听觉或视觉刺激下的老鼠在刺激和开始飞行之间的短暂潜伏期引发了快速的逃逸反应。从视觉刺激中逃生的平均潜伏期是 202 16 ms (n = 51 反应从26个动物) 和明显地更长为听觉刺激: 510 @ 61 ms (n = 36 个动物的答复, p< 15 t 测试在视觉和听觉之间刺激图 2A)(步骤 8.1)。逃逸反应被准确地定向到掩蔽处 (视觉刺激的平均准确度: 97.2 1.4% 和95.0 的听觉刺激, 没有明显的不同的两种类型的刺激, p = 0.1655 t 测试;图 2B)(步骤 8.4), 飞行轨迹接近一条直线 (平均位移比, 以距离为遮蔽的视觉刺激 107.3, 1.3% 和 113.9 @ 1.5% 的听觉刺激;图 2C)(步骤 8.3)。从刺激中观察到短暂的潜伏期对于区分刺激诱发的行为和自导的运行是很重要的, 而飞行轨迹的高线性度表明逃生响应有到达避难所的目标, 而不是简单地离开刺激。
刺激演示后, 旋转的外部部分的迷宫, 而鼠标是静止的中心平台 (范围 = 36° 90°, 平均 56°) 成功地引出逃生响应 (步骤 5)。所有的老鼠都逃到了前面的庇护所, 与旋转前的试验相比, 飞行弹道的精度和线性度没有降低。平均精确度, 测量在参考在原始的风雨棚位置, 在自转之前是 96.3 "1.3%" (n = 25 反应从8个小鼠), 并且在自转以后稍微更高 (100 @ 0%, p = 0.009 t 测试在前和之后自转之间)。平均线性相对于原始的风雨棚位置是 109.4, 5.0% 前自转 (25 反应从8小鼠) 和 109.1 @ 2.1%, 后自转 (n = 8 反应从8小鼠), p = 0.957 t 测试在前和后自转之间,图3A和 B)。这些结果表明, 近端视觉线索不用于引导飞行到避难所 (步骤 8.3-8.5)。
改变避难所的位置后, 获得一个成功的逃生响应 (步骤 6) 导致小鼠访问新的庇护所地点后不久的变化 (平均时间访问新的庇护所 = 33.1 s, 范围 = 4-82 s)。随后的声音刺激顺序试验产生了逃逸到新位置的概率逐渐增加 (第一次试验的 44.4%), 第五试验 (n = 9 只动物,图 3C) 在所有动物中达到100%。这些实验探讨了更新掩蔽位置内存以适应环境变化的动态 (步骤 8.6)。
在7分钟的适应期间到迷宫没有风雨棚 (步 7), 5 s 长的视觉刺激的介绍导致了结冰行为而不是逃命 (结冰可能性 = 95.2%, 平均结冰时间 = 7.9 @ 2.7 s, n = 7 动物)。随后在同一届会议上介绍了一个庇护所, 并展示了长期的视觉刺激, 使逃生反应可靠, 表明小鼠可以根据避难所的存在灵活地切换防御策略 (图 3D) (步骤8.1 和 8.7)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56988/56988fig1.jpg"> 图 1: 行为竞技场的示意图.从顶部 (右) 和侧面 (左) 观看行为竞技场的主要组成部分的图画。一个很大一部分的平台可以旋转, 包括遮蔽和附加的视觉线索。黑暗的圈子在顶部看法是坑可以附有的孔, 是所有相同的 (例子风雨棚位置被表明)。整个竞技场都被一个隔音的柜子包围着。<a href="/files/ftp_upload/56988/56988fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56988/56988fig2.jpg"> 图 2: 转义响应是快速准确的目标定向行为.(A) 从视觉或听觉刺激开始时逃逸的潜伏期。(B) 逃生时航行到避难所的准确性, 测量值为 100%, 如果鼠标直接跑到避难所, 而离避难所的每个洞的距离减少 10% (e., 80%, 如果首先到达的洞是两个洞从正确一个)。(C) 在逃逸过程中, 鼠标的位移是根据动物初始位置和目标位置之间的线性距离绘制的, 表明飞行接近线性。框和胡须显示数据从10-90 个百分点和其余的数据点显示为圆圈。红色的颜色图是来自听觉刺激的数据 (来自15个动物的36反应), 黑色来自视觉刺激 (n = 51 反应从26个动物)。<a href="/files/ftp_upload/56988/56988fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56988/56988fig3.jpg"> 图 3: 老鼠不使用近端视觉线索或与遮蔽有关的线索来指导逃逸反应.(A)图 2C所示的位移图, 用于对平台进行前后的响应。(B) 在飞行前和轮换后条件下逃生时的航行准确性。旋转后的准确度是参照避难所的初始位置来衡量的, 并表明小鼠回到原来的庇护所位置而不是新的位置。(C) 针对新的避难地点的逃逸反应的一部分是针对刺激数字绘制的。在5的刺激之后, 所有的动物都直接逃向新的避难所 (n = 9 只动物)。(D) 对动物的平均速度, 以响应视觉刺激与庇护所 (红色) 或缺席 (蓝色), 显示逃生和冰冻反应, 分别。速度已规范化为基线, 并对反应时间 (定义为飞行的开始或冻结响应, 虚线)。阴影区域显示 SEM. (n = 7 个动物)。对于2A 和 2B, 该框和胡须显示的数据从10-90 百分点和其余的数据点显示为圆圈。红色图是从听觉刺激的数据在平台自转之前 (n = 25 反应从8个动物) 和蓝色从反应在旋转平台以后 (n = 8 反应从8个动物)。<a href="/files/ftp_upload/56988/56988fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice at JoVE.com

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

一种研究空间记忆在小鼠本能防御中作用的行为分析法

本协议描述了一种基于巴恩斯迷宫测试的行为分析方法, 研究了空间环境知识对本能防御行为的修正。

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

a-behavioral-assay-for-investigating-role-spatial-memory-during

Research

JoVE Journal

Behavior

9.4K 次观看.  UCL Sainsbury Wellcome Centre for Neural Circuits and Behaviour. 本协议描述了一种基于巴恩斯迷宫测试的行为分析方法, 研究了空间环境知识对本能防御行为的修正。

视频: A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

双-H迷宫:一个强大的行为测试学习和记忆的鼠害

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

科研

行为学

Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and validated for rat use. The Radial Water Tread (RWT) maze test of spatial memory (designed specifically for mice and requiring no swimming) has been shown previously to successfully distinguish between controlled cortical impact-induced TBI mice and sham controls. Here, a detailed protocol for this task is presented. The RWT maze capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the sides of an apparatus (thigmotaxis). The walls of the maze are lined with nine escape holes placed above the floor of the apparatus, and mice are trained to use visual cues to locate the escape hole that leads out of the maze. The maze is filled with an inch of cold water, sufficient to motivate escape but not deep enough to require that the mouse swim. The acquisition period takes only four training days, with a test of memory retention on day five and a long-term memory test on day 12. The results reported here suggest that the RWT maze is a feasible alternative to rat-validated, swimming-based cognitive tests in the assessment of spatial memory deficits in mouse models of TBI.

Here we present a protocol for a mouse-specific test of cognition that does not require swimming. This test can be used to successfully distinguish controlled cortical impact-induced traumatic brain injury mice from sham controls.

使用径向水痕迷宫评估外伤性脑损伤小鼠模型中的空间记忆障碍

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and ...

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

利用巴恩斯迷宫和困境中的破坏范式分析小鼠的空间学习和社会行为

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

基于 IntelliCage 的阿尔茨海默病多基因模型认知功能的自动、长期行为检测

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

一种新的小鼠目标识别与目标定位行为测试

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various ...

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

一种用于小鼠运动技能、情绪和认知重复评估的行为测试电池

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

Nest Building Behavior as an Early Indicator of Behavioral Deficits in Mice

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.&#160;

Here, we present a protocol to quantify nest building behavior in mice, which is known to be impaired in several neurological disorders and diseases. This protocol examines the utility of four materials and offers the opportunity to quantify the rater agreement in scoring, improving the validity and reliability of the assay.

巢穴建筑行为作为小鼠行为缺陷的早期指标

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents ...

研究具有改良恐惧条件反射的小鼠的主动防御行为

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

作者聚焦：探索恐惧学习和灭绝中防御行为的神经相关性

To study flight behaviors in a fearful context, we introduce a modified fear conditioning protocol. This protocol ensures that mice consistently exhibit flight behaviors during cue presentation in the fear conditioning.

改良的恐惧条件反射诱导小鼠的飞行行为

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active ...

一种研究空间记忆在小鼠本能防御中作用的行为分析法

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此视频中的章节

双-H迷宫:一个强大的行为测试学习和记忆的鼠害

使用径向水痕迷宫评估外伤性脑损伤小鼠模型中的空间记忆障碍

利用巴恩斯迷宫和困境中的破坏范式分析小鼠的空间学习和社会行为

基于 IntelliCage 的阿尔茨海默病多基因模型认知功能的自动、长期行为检测

一种新的小鼠目标识别与目标定位行为测试

一种用于小鼠运动技能、情绪和认知重复评估的行为测试电池

巢穴建筑行为作为小鼠行为缺陷的早期指标

改良的恐惧条件反射诱导小鼠的飞行行为

改良的恐惧条件反射诱导小鼠的飞行行为

改良的恐惧条件反射诱导小鼠的飞行行为