我们衷心感谢加拿大卫生研究院 (NSERC) 和加拿大科学与工程研究所的支持。波斯特得到了亚历山大·格雷厄姆加拿大研究生奖学金 (NSERC) 的支持。

所有的程序都是按照国家研究委员会的《动物保护和使用实验指南》 (第八版; 2011) 和加拿大动物保护委员会关于试验动物的护理和使用指南 (1993) 进行的, 并由西安大略大学动物学委员会动物保育委员会批准。
下面的过程可以应用于实验研究皮层贡献的运动控制在步行猫。
1. 仪器
<ol><li>构造用于评估障碍物记忆的仪器。 注: 该仪器由2.43 米长, 29 厘米宽的走道包围, 18 厘米高清晰丙烯酸墙 (图 1)。一个狭窄的插槽沿设备的一半, 允许25.8 厘米宽 x 3 毫米厚的障碍, 以提高或从人行道上使用杠杆安装在行走表面。</li>
	<li>为了确保动物的注意力保持在进食上, 避免用手抬高或降低障碍物。相反, 障碍可以提高或降低使用实验者的腿移动的杠杆下的走道, 使实验者继续喂养的动物。</li>
	<li>适当地维护杠杆系统, 以确保障碍可以提高或降低无声。</li>
	<li>使用一个小的高架平台 (23 厘米长的 x 23 厘米宽 x 16 厘米高), 在软食物放置, 以指导动物的运动。</li>
	<li>记录所有使用以太网摄像头 (54 帧/秒) 安装在三脚架1.85 米远离走道中线的试验。</li>
</ol>2. 培训程序
注意: 为了获得成功的数据, 在行为测试之前的训练期间, 确保每只动物都能适当地适应测试室和仪器。反复接触到一个新的环境将有助于减少令人吃惊或其他压力的行为。 驯化可能因动物而异, 可能需要1-2 月的训练。根据动物进食的重点和动机, 最初的驯化疗程可达5分钟。随后的会议应旨在增加动物的工作时间 (通常约20-25 分钟)。
<ol><li>获得成熟 (> 6 月的年龄) 国内短毛猫从任何重量或性别的商业实验室饲养员。 注意: 在考虑哪些动物应该包括在研究中时, 食物的工作动机和合作性的性格构成了选择标准。</li>
	<li>使每只动物都能佩戴1米长的皮带。将皮带拴在走道中点上方的架子上。 注意: 这使得动物可以沿着器械的中心部分不带任何张力, 从而鼓励动物留在仪器的这一部分。建立这样的边界有助于使用移动测试对象。</li>
	<li>把动物放在人行道上, 让它从平台上吃软食物。 注意: 这项初步训练的目的之一是确保动物在向前移动时很容易跟随食物平台, 并能舒适地与安全带和皮带行走。使用软性食物作为积极的强化, 鼓励动物在每次训练或测试期间保持专注, 并促进舒适的工作环境。</li>
	<li>确保动物对搬运感到舒适, 包括必须将动物移到人行道起始区域的情况。</li>
</ol>3. 行为训练和测试协议
注意: 障碍记忆被评估在两个范例: 一个视觉依赖障碍记忆任务, 和一个触觉相关的障碍记忆任务。这两种范式都应在最初的培训和随后的测试中使用。
<ol><li>
视觉障碍记忆
	<ol>
<li>要评估视觉障碍存储器, 请将障碍物提升到人行道上 (图 2A)。将平台放置在障碍物的远处。将动物放在人行道的开始区域。</li>
		<li>允许动物接近食物, 只用前腿踩在障碍物上, 以便从站台上进食。</li>
		<li>当动物继续进食时, 降低障碍物, 使其变得与人行道齐平, 以防止任何进一步的视觉或触觉输入。</li>
		<li>在一个可变的延迟期间, 移动食物批转再鼓励动物恢复步行;这种延迟可以小于 1 s 到2分钟以上。</li>
		<li>重要的是, 在没有障碍的情况下进行试验, 以防止习惯性的障碍和学习回避反应的发展。在这种视觉障碍缺席试验中, 在将动物放在人行道的起始区之前, 确保障碍物不会被抬到人行道上。</li>
		<li>观察 hindleg 步在障碍目前和障碍缺席试验, 以验证典型的运动行为和完整的视觉障碍记忆前冷却。确保动物能在无接触的情况下清除障碍, 而在障碍性试验中, 四条腿的跨步明显升高。 注意: 观看训练试验的视频可能有助于验证。</li>
	</ol>
</li>
	<li>
触觉障碍记忆
	<ol>
<li>要评估触觉障碍记忆, 确保在将动物放在人行道的起始区域之前, 不会将障碍物抬到人行道上 (图 2B)。</li>
		<li>允许动物走向位于障碍物的远侧的食物平台。</li>
		<li>当动物进食时, 将障碍物抬高到食物盘下的人行道上, 防止障碍物的任何视觉输入。</li>
		<li>当食物被向前移动时, 请注意, 动物在踏过前应先用前腿接触障碍物。</li>
		<li>允许动物继续进食, 同时跨越他们的前后腿的障碍。在此期间, 降低障碍, 使其成为冲洗与人行道, 以防止任何进一步的视觉或触觉输入。</li>
		<li>在一个可变的延迟期间, 移动食物再次批转鼓励动物恢复步行。</li>
		<li>重要的是, 在不存在障碍的情况下进行试验, 避免前腿接触, 防止习惯性障碍和学习回避反应的发展。<ol>
<li>在这些触觉障碍缺席试验, 有动物的方法和吃的食物平台, 如步骤3.2.1 所述。然而, 在把食物向前推进的步骤3.2.3 前, 提高和降低障碍 (步骤 3.2.2)。确保允许动物继续进食的类似的延迟期 (步骤 3.2.4) 在运动的最后的继续之前 (步 3.2.5)。</li>
		</ol>
</li>
		<li>观察 hindleg 步在障碍目前和障碍缺席试验, 以验证正常的运动行为和完整的视觉障碍记忆前冷却。</li>
	</ol>
</li>
</ol>4. 视频分析
注: 为了评估障碍记忆, 在初始训练期间进行的分析和随后的测试后, 冷却环植入涉及量化的峰值步高度, 步隙, 以及脚趾和障碍物之间的水平距离在每个步骤的高峰期视觉和触觉范例 (图 2C)。
<ol><li>使用自定义手写脚本分析视频。</li>
	<li>每一次试验, 每步通过标记脚趾最靠近相机的位置来跟踪每个脚印。</li>
	<li>在每个步进轨迹的最高点 (图 2C) 中, 测量峰值台阶高度作为脚趾和人行道表面之间的垂直距离。</li>
	<li>在障碍物的试验中, 将台阶间隙测量为在障碍物的高度上减去的台阶高度。</li>
	<li>此外, 测量障碍-目前试验中每一步的峰值处的脚趾与障碍物之间的水平距离。</li>
	<li>确认在冷却回路植入之前, 障碍记忆能力是完整的, 通过验证在障碍试验中的峰值台阶高度是否提升, 与在无障碍试验中的步进相比。</li>
</ol>5. 冷却回路 (Cryoloop) 植入
<ol><li>根据先前报告的外科手术程序8 (图 3), 在5和7区域 cryoloops 种植体。</li>
	<li>简而言之, 对于每个半球, 执行一个开颅和 durotomy 从斯科特·霍斯利-克拉克坐标20 A15 到 A25, 以揭露 ansate 和侧面沟的接合点。</li>
	<li>从23口径不锈钢皮下注射导管中定位单个冷却回路, 与顶叶区5或7的皮质表面直接接触。</li>
	<li>安全的基础, 每个 cryoloop 的头骨与牙科丙烯酸锚定不锈钢螺丝。</li>
	<li>关闭开颅与额外的牙科丙烯酸;绘制到丙烯酸边缘的皮肤边缘和缝合在一起。</li>
</ol>6. 皮质冷却协议
<ol><li>
实验设置 注意: 在将动物带入测试室之前, 要准备并测试冷却回路。该冷却电路由一甲醇储气罐 (3.2 毫米外径, 1.6 毫米身份证), 往复式活塞泵, 和干冰浴连接通过聚四氟乙烯管 (1.6 毫米外径, 0.5 毫米身份证;图 4)。此外, 还需要一个数字温度计。<ol>
<li>在冰浴中加入500毫升干冰至 200 mL 甲醇。配合油管两端紧贴在入口和出口的一个虚拟 cryoloop, 以完成冷却电路。</li>
		<li>将热电偶插头连接到数字温度计上, 使用由两个热电偶连接器和热电偶线组成的电缆进行连续温度监测。当一端插入温度计时, 确保此电缆的长度足以到达动物的头部。</li>
		<li>使用开关打开活塞泵。 注: 甲醇应从油层中抽出, 通过泵到干冰浴, 在那里流动的甲醇在油管中将冷却到-75 ° c。冷却后的甲醇将退出冰浴, 并通过附着的 cryoloop, 然后返回甲醇库。</li>
		<li>确保泵的设置, 在冰浴内的油管长度, 和从冰浴到假循环的油管长度是最佳的, 使假 cryoloop 温度可以达到一个稳定的状态在-5.0 ° c 左右。 注: 此初始设置期间所达到的温度通常足以达到3.0 ±1.0 ° c 时的测试温度, 而同一系统用于冷却植入的 cryoloop。通过调整泵的转速、增加在冰浴中浸没的油管长度和/或最小化从冰浴到 cryoloop 的油管长度, 可以解决实现足够冷却的困难。</li>
		<li>如有必要, 通过管端管接头和法兰端部的管子端部用翻边工具来延长油管的一端。使用连接器以类似的法兰端连接所需长度的油管。</li>
		<li>验证所有连接是否舒适, 并且不存在泄漏。一旦满意的初始设置, 开关泵关闭, 并删除假 cryoloop;这个电路现在已经为一个试验动物准备好了。</li>
	</ol>
</li>
	<li>
行为测试
	<ol>
<li>将动物放在测试仪器上。滑动线束在头上, 并确保表带舒服周围的动物。系上皮带</li>
		<li>卸下植入的 cryoloop 的保护盖, 露出入口和出口管。适合油管两端紧贴在 cryoloop 的入口和出口管。将热电偶插头连接到数字温度计。</li>
		<li>开始测试阶段与视觉 (步骤 3.1) 或触觉 (步骤 3.2) 障碍记忆试验。跟随对所有四类型的另外的试验 (视觉障碍-当前, 视觉障碍-缺席, 触觉障碍-存在, 触觉障碍-缺席) 以一个随机的方式。 注: 一个典型的测试环节由一个 "温暖" 的试验块组成, 在没有冷却的情况下观察记忆引导的避障以建立基线测量。</li>
		<li>打开活塞泵, 等待 cryoloop 达到3.0 ±1.0 ° c (1-2 分钟) 的温度。然后, 运行一个 ' 凉爽 ' 的试验块后, 活塞泵已接通。在这段试验中, 如果需要, 评估冷却区的贡献, 以记忆为导向。确保 cryoloop 的温度保持在3.0 ±1.0 ° c 整块。 注: 所有四种试验类型应在整个区块中随机穿插。</li>
		<li>在活塞泵关闭后, 运行最后的 "复" 测试块, cryoloop 已恢复到原来的温度。 注意: 在此块中重新建立基线步进行为。同样, 所有四种试验类型都应在整个区块中随机穿插。</li>
	</ol>
</li>
	<li>
清理
	<ol>
<li>当行为测试结束时, 从入口和出口管中取出油管。注意剩余的甲醇可能从油管末端滴下, 可能会刺激动物。</li>
		<li>确保更换防护帽。在把动物送回蚁群之前, 去掉皮带和安全带。修剪油管两端 (3-4 毫米) 使用油管切割器, 以防止在下一个测试日漏连接。</li>
	</ol>
</li>
</ol>7. 验证冷却范围
<ol><li>在行为测试结束时, 请确认使用先前报告的技术8, 将停用的范围限制在每个 cryoloop 下方的皮层区域。 注: 这可以通过跃层映射12或与热成像相机13、14、19进行验证。</li>
</ol>

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作者没有什么可透露的。

所描述的范例采用冷却诱导的离散皮层区 deactivations 的 cryoloop, 以研究记忆制导障碍的运动在猫。视觉和触觉障碍记忆范例是相当简单的动物执行, 因为他们利用自然主义的运动行为, 发生在最小的努力, 当一个动物的动机是跟随一个移动的食物来源。因此, 大部分的培训阶段是专门适应动物的测试室和冷却设备。大多数动物都需要反复接触, 佩戴安全带, 并通过皮带拴住, 然后才能舒适地和自然地在仪器上行走。此外, 在测试过程中, 活塞泵的声音可能会分散或惊吓动物。用假 cryoloop 完成冷却回路, 在初始训练过程中运行泵, 可以使动物适应泵的声音。尽管在测试之前有足够的训练, 在动物变得焦躁不安之前, 测试的时间很可能会很有限。因此, 充分的时间, 以确保适当的设置和故障诊断之前, 把动物进入测试室将优化后续数据收集。
通过调节泵的转速可以解决实现足够冷却的困难。然而, 应注意增加的压力, 可能导致油管被迫关闭的进口或出口管的 cryoloop。另外, 在冰浴中浸入的油管的长度可能会增加, 以便有更多的时间来冷却试管内的甲醇流动。此外, 确保从冰浴到 cryoloop 的出口点的长度尽可能短, 将减少冷却损失。然而, 这个距离也必须足够长, 以允许在给定的行为范例中有足够的运动范围。油管可与灵活的泡沫包装绝缘, 以优化冷却效率。这样的包装也可以防止在油管上形成的冷凝物掉落在动物身上, 这可能会刺激或惊吓动物。在测试期间, 确保在 cryoloop 的入口和出口管上的油管的舒适适合, 可以使连接 cryoloop 困难。戴上腈或乳胶手套可以更好地控制油管。确保动物是舒适和耐心的, 而实验者附加油管是必不可少的。食物可以用来保持动物的静止和含量。
当某一特定区域被停用时, Cryoloops 可以经常冷却, 产生高度可重现的行为变化。通过评估同样的任务在存在和没有皮质失活在同一动物, 总数量的动物使用可以减少。此外 , 冷却的程度可能纵进一步指定皮层对特定行为的贡献。例如, 单侧和双侧 deactivations 可以在同一动物中进行, 以检查行为的可能侧效应。此外, 冷却的程度可以变化, 以检查层流贡献。通过冷却 cryoloops 在皮质表面到3.0 ±1.0 ° c, 所有六层皮质直接地在每个循环之下被冷却到 < 20 ° c, 抑制神经元扣球活动22。另外, cryoloops 可以被冷却到8.0 ±1.0 ° c, 选择性地冷却仅 supragranular 皮层层数在这临界温度之下20° c。评估行为, 这种肤浅的皮层失活以及完全皮质失活可能允许椎 dissociations 皮质功能21。
尽管具有这样的通用性, 但在实验设计时应考虑以下限制因素。虽然冷却是一个很好的方法来停用所有的细胞类型在一个给定的皮质区域, 它不能提供一种手段的细胞特异性, 可以实现与 optogenetic 的失活技术。此外, 冷却需要至少四十五年代之前, cryoloop 温度稳定在3.0 ±1.0 ° c 的临界温度的功能钝化。因此, 应将实现功能停用所需的时间跨度的考虑纳入到实验性的选择协议中。
总的来说, 冷却系统需要最少的维护。应定期检查冷却回路的油管和连接器是否有泄漏。应每周更换储罐内的甲醇, 以确保甲醇不受微粒物质的污染。植入 cryoloops 也需要最少的维护。边缘是定期清洗的3% 过氧化氢溶液后, 手术磨砂解决方案。随着适当的使用和护理, 植入 cryoloops 可以冷却多年例行。这些皮质冷却程序可以适应其他行为模式10,11,12或电生理记录准备13,14替代动物模型15,17,18,22。

在自然的、不平坦的地形上, 关于障碍物的感官信息可以通过视觉或触觉获得, 可以迅速地改变运动以躲避。步进运动的细致协调涉及多个皮层区域1,2。例如, 电机皮层3,4和顶叶皮层5,6,7在复杂的运动任务中被牵连, 如避障。在足动物中, 避免障碍所需的阶跃调制必须伸展至前腿和后腿。如果向前运动被延迟在前腿和 hindleg 障碍间隙之间 (可能出现作为动物踏入小心地通过一个复杂, 自然主义的环境跟踪猎物), 关于在记忆中维护的障碍的信息被用来指导一旦走了, hindleg 就会越过障碍物。
实验技术旨在解除离散皮层区域可以用来研究皮层贡献的记忆引导障碍运动。冷却诱发的皮质失活提供了一种可逆的, 可靠的, 可重现的方法来评估皮质对显性行为的贡献8。Cryoloops 由不锈钢管制成, 是特定于皮层的兴趣, 确保高度选择性和离散的基因座的失活。一旦植入, 冷冻甲醇泵通过管腔的 cryoloop 冷却的皮层区域直接下方的循环到 < 20 ° c。在这个临界温度下, 突触的传递在大脑皮层的区域被抑制。这种钝化可以通过停止甲醇的流动来逆转。该方法已用于研究皮层对感官处理和行为的贡献9,10,11,12,13,14,15,16,17, 以及跳眼移动的马达控制18和内存引导障碍运动19。
该协议的目的是使用可逆冷却诱导 deactivations 评估的参与, 顶叶皮层区的运动协调在猫。特别地, 记忆引导障碍运动被检查与或没有活跃顶叶皮层。这些方法已经被用来成功地证明了顶骨面积5在记忆制导避障中的作用, 在行走 cat19。

<table><tbody><tr><td>Camera</td><td>IDS Imaging Development Systems GmbH</td><td>Model: UI-5240CP-C-HQ</td><td></td></tr><tr><td>Intake tubing</td><td>Restek</td><td>25306</td><td>Unflanged end is submerged in the methanol reservoir while the flanged end is connected to the pump</td></tr><tr><td>Pump</td><td>Fluid Metering, Inc.</td><td>Model: QG 150</td><td></td></tr><tr><td>Nalgene Dewar vacuum flask</td><td>Sigma-Aldrich</td><td>F9401</td><td></td></tr><tr><td>Teflon tubing</td><td>Ezkem</td><td>A051754</td><td></td></tr><tr><td>Microprobe thermometer</td><td>Physitemp</td><td>Model: BAT-12</td><td></td></tr><tr><td>Flanged tube end fittings</td><td>Valco Instruments Co. Inc.</td><td>CF-1BK</td><td>Assorted colours available for colour coding. Packages include the same number of washers as fittings</td></tr><tr><td>Washers</td><td>Valco Instruments Co. Inc.</td><td>CF-W1</td><td>Extra washers</td></tr><tr><td>Flanging kit</td><td>Pro Liquid GmbH</td><td>201553</td><td></td></tr><tr><td>Tubing connector</td><td>Restek</td><td>25323</td><td></td></tr><tr><td>Tubing cutter</td><td>Restek</td><td>25069</td><td></td></tr><tr><td>Male thermocouple connector</td><td>Omega</td><td>SMPW-T-M</td><td>Used to make cable connection to thermometer</td></tr><tr><td>Thermocouple wire</td><td>Omega</td><td>PP-T-24S</td><td>Used to make cable connection to thermometer</td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>n/a</td><td></td></tr></tbody></table>

reversible cooling-induced deactivations to study cortical contributions to obstacle memory in the walking cat

在复杂的、自然主义的地形上, 关于环境障碍的感官信息可以用来快速调整运动动作以躲避。例如, 在猫, 关于一个即将到来的障碍的视觉信息可以调节步避。运动适应也可以发生独立的视觉, 因为突然的触觉输入到腿部由一个预期的障碍可以修改的步的所有四条腿, 以避免。这种复杂的运动协调涉及上结构, 如顶叶皮质。该协议描述了使用可逆的, 冷却诱导的皮层失活, 以评估顶叶皮层贡献的记忆引导障碍运动在猫。小的冷却回路, 被称为 cryoloops, 是特殊的形状, 以关闭分散的兴趣区域, 以评估其贡献的公开行为。这种方法已被用来阐明的作用, 5 在记忆引导的障碍, 避免在猫的顶叶区。

该协议已成功地用于检查顶叶皮层贡献的障碍记忆在步行猫19。在这项研究中, cryoloops 在5和7在三成人 (> 6 月龄) 雌性猫 (图 5A) 上被植入了双侧壁区。在没有冷却 (温暖、控制条件) 的情况下, 或者当5或7区被双边钝化时, 在触觉障碍记忆模式中对动物进行了评估。
该研究的结果表明, 当5区被双边冷却时, hindleg 阶梯在障碍试验中明显衰减 (图 5D, 蓝色)。在温暖条件下, 前导和尾随后腿的平均峰值台阶高度分别为9.5 ±2.2 cm 和8.0 ±2.1 cm。单向多变量方差分析显示, 当5区被冷却时, 前导和尾随后腿的峰值步高度分别显著降低到4.3 ±2.2 cm (p < 0.0001) 和3.4 ±1.4 cm (p < 0.0001)。在障碍试验中前肢的峰值台阶高度或无障碍试验中的任何腿部, 不受5区失活的影响。同样地, 在障碍物或无障碍试验中, 任何一条腿的峰值台阶高度与7区停用时的温暖条件没有区别。
此外, 在5区停用时, hindleg 步隙也受到同样的影响。与温暖和区域7冷却的情况相比, 步隙减少到4.7 ±2.2 cm 在主导的 hindleg 步 (p < 0.0001;图 5G)和−5.6 ±1.4 厘米在尾随 hindleg 步骤 (p < 0.0001)。此外, 尾随 hindleg 的步进轨迹受到区域5失活的影响, 因为峰值发生在障碍物之前, 不同于在温区和区域7冷却条件下的步进 (图 5G)。
总之, 在5区停用时, 峰值台阶高度、台阶间隙和台阶轨迹的变化表明了深刻的障碍记忆缺陷。重要的是, 由于5区的失活只改变了 hindleg 步在障碍试验中的特性, 并没有削弱进行步进运动的能力, 这些观察到的运动变化反映了记忆, 而不是运动缺陷。此外, 在行为测试结束时进行的热成像证实, 当每个半球单独冷却时 (图 6), 冷却仅限于区域5或7。因此, 总的来说, 这些结果证明了顶骨区5对记忆制导障碍在猫的运动的贡献。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56196/56196fig1.jpg"> 图 1: 描述摄像机、冷却设备和用于评估猫的障碍记忆的行走装置的图示.一个2.43 米长, 29 厘米宽的走道是封闭的18厘米高清晰有机玻璃墙。沿走道的一半, 一个25.8 厘米宽的3毫米厚的障碍, 可以提高在人行道上通过一个狭窄的插槽使用杠杆安装在人行道下面。对于每一个试验, 动物在人行道起始区的障碍物上放置几步。食物被放置在一个小的高架平台上 (23 厘米长的 x 23 厘米宽 x 16 厘米高), 位于障碍物相对于起始区域的一侧。所有试验都是通过安装在三脚架上的以太网摄像头记录的, 并保存在笔记本电脑上。此图已从黄et al.中修改19<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56196/56196fig1large.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/56196/56196fig2.jpg"> 图 2: 描述视觉和触觉障碍记忆任务的图示和用于评估行走猫的障碍记忆的步骤测量.(A) 评估视觉障碍记忆, 当动物接近食物平台时, 障碍物会被抬高到人行道上。只用前腿踩过障碍物后, 动物就可以从站台上进食, 因为障碍物被悄悄地与人行道的表面齐平。在可变的延迟期间, 食物被移动批转鼓励动物恢复步行。(B) 评估触觉障碍记忆, 当动物接近食物平台时, 障碍不会在人行道上升起。当动物进食时, 障碍物在食物平台的正下方的走道上默默地升起。食物被移动批转导致动物的前腿接触障碍, 在跨步之前它。这种动物可以继续在食物平台上进食, 同时跨越前腿和后腿之间的障碍物。在这段时间里, 障碍物悄悄地从人行道上降下。食物再次向前移动, 以鼓励动物恢复行走。测量 Hindleg 步骤以评估障碍记忆。(C) 通过测量峰值台阶高度、台阶间隙和每个台阶的峰值与障碍物之间的水平距离来评估视觉和触觉障碍记忆范例中的步进。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56196/56196fig2large.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/56196/56196fig3.jpg"> 图 3: cryoloop 的示意图.cryoloop 包括一个保护帽, 它适合于入口和出口管。这些管子通过一个螺纹的柱子运行, 形成与皮层表面直接接触的回路。microthermocouple 是焊接在环的联合, 以测量 cryoloop 温度。它的电线通过热收缩管 (也包裹不锈钢油管) 运行, 并连接到连接器上。整个大会是固定的头骨与牙科丙烯酸。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56196/56196fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56196/56196fig4.jpg"> 图 4: 冷却电路.该冷却电路由甲醇储罐、往复活塞泵、冰浴、温度计和 cryoloop。为了冷却, 泵通过吸入管 (1.6 毫米的身份) 从油层中抽取甲醇。甲醇通过聚四氟乙烯管子 (0.5 毫米身分) 出口泵浦通过到干冰浴, 流动甲醇在管材被冷却到-75 ° c。冷冻甲醇然后退出冰浴和运行通过附着的 cryoloop, 然后返回到甲醇水库。这 cryoloop 可能是一个假的循环 (不植入) 在初始设置中使用, 或可能是一个植入 cryoloop 在测试动物。cryoloop 还连接到数字温度计, 以记录整个行为测试中的环路温度。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56196/56196fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56196/56196fig5.jpg"> 图 5: 可逆、冷却导致的顶叶区失活5导致障碍记忆缺陷.(A) 猫大脑右半球的侧视图, 显示 cryoloops 直接移植在顶骨 5 (蓝色) 和 7 (绿色) 在黄et al.19 D: 背, A: 前面。(B-E)条形图描绘平均阶梯高度± SD 为障碍礼物 (B, d) 和障碍缺席试验 (C, E) 为前腿 (B, C) 和后腿 (D, e) 为温暖 (红色), 区域5冷却 (蓝色) 和区域7冷却的情况 (绿色)。在5区停用的障碍试验中, 主要和尾部后腿的台阶高度明显降低。(F) 条形图描绘了每个冷却条件下的平均 hindleg 步间隙± SD。5区失活导致了前导和尾随 hindleg 步骤的间隙减少。(G) 条形图, 描述每个步骤的峰值与每个冷却条件的障碍物之间的平均水平距离。当区域5被冷却时, 步轨是更加易变的并且与温暖和区域7冷却的情况显著不同。*p < 0.005, **p < 0.0001, 生理盐水: 不重要。此图已从黄et al.中修改19<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56196/56196fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56196/56196fig6.jpg"> 图 6: 热成像用于确认冷却过程中5或7区域的限制停用.(A) 照片, 描述 cryoloops 与右半球5和7的顶叶区域的接触。顶部是背部, 右前方。虚线表示顶骨区5和7之间的边界。(B-C)当 cryoloop 区 5 (B) 或面积 7 (C) 被冷却到3° c 时, 被拍照的顶叶皮层表面的热图像。此图已从黄et al.中修改19<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56196/56196fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat at JoVE.com

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

复杂的运动在自然主义的环境需要仔细的肢体协调涉及顶叶皮层的区域。下面的协议描述了可逆冷却诱导失活的使用, 以证明顶骨面积5的作用, 在记忆制导避障的步行猫。

可逆冷却诱导 Deactivations 对行走猫的障碍记忆的皮层贡献研究

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

reversible-cooling-induced-deactivations-to-study-cortical

Nanyang Technological University

Research

JoVE Journal

Behavior

6.9K 次观看.  University of Western Ontario. 复杂的运动在自然主义的环境需要仔细的肢体协调涉及顶叶皮层的区域。下面的协议描述了可逆冷却诱导失活的使用, 以证明顶骨面积5的作用, 在记忆制导避障的步行猫。

视频: Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

Operant Procedures for Assessing Behavioral Flexibility in Rats

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one&#8217;s environment (i.e., behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer&#8217;s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.

The ability to assess executive functions such as behavioral flexibility in rats is useful for investigating the neurobiology of cognition in both intact animals and disease models. Here we describe automated tasks for assessing strategy shifting and reversal learning, which are particularly sensitive to disruptions in prefrontal cortical networks.

操作性程序，以评估大鼠的行为灵活性

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these ...

科研

行为学

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

量化颅放疗引起的认知递减

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

医学

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 ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

T-迷宫强迫交替和评估工作，并参考在小鼠记忆的左，右的歧视任务

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

神经科学

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 ...

Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor locomotion after surgery. For example, rats who undergo surgical procedures and are subjected to the Morris water maze may not be able to swim, thus voiding the experiment.
New Method: We established a modified behavioral spontaneous alternation T-maze test. The major advantage of the modified T-maze protocol is its relatively simple design that is powerful enough to assess functional learning/memory after ischemia. Additionally, the data analysis is simple and straightforward. We used the T-maze to determine the rats&#39; learning/memory deficits both in the presence or absence of mild to moderate (6 min) asphyxial cardiac arrest (ACA). Rats have a natural tendency for exploration and will explore the alternate arms in the T-maze, whereas hippocampal-lesioned rats tend to adopt a side-preference resulting in decreased spontaneous alternation ratios, revealing the hippocampal-related functional learning/memory in the presence or absence of ACA.
Results: ACA groups have higher side-preference ratios and lower alternations as compared to control.
Comparison with Existing Method(s): The Morris water and Barnes maze are more prominent for assessing learning/memory function. However, the Morris water maze is more stressful than other mazes. The Barnes maze is widely used to measure reference (long-term) memory, while ACA-induced neurocognitive deficits are more closely related to working (short-term) memory.
Conclusions: We have developed a simple, yet effective strategy to delineate working (short-term) memory via the T-maze in our global cerebral ischemia model (ACA).

This protocol describes the use of a modified T-maze to evaluate functional learning/memory in asphyxia cardiac arrest-induced cerebral ischemia.

应用改良 T 迷宫评估心脏骤停后的功能记忆结果

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor ...

A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; O&#x2019;Keefe and Nadel, 1978; Mittelstaedt and Mittelstaedt, 1980; Landeau et al., 1984; Etienne, 1987; Gallistel, 1990;
Maurer and S&#233;guinot, 1995). Piloting requires the use of the relationships between relatively stable external (visual, olfactory, auditory) cues, whereas dead reckoning requires the integration of cues generated by self-movement. Animals obtain self-movement information from vestibular receptors, and possibly muscle and joint receptors, and efference copy of commands that generate movement. An animal may also use the flows of visual, auditory, and olfactory stimuli caused by its movements. Using a piloting strategy an animal can use geometrical calculations to determine directions and distances to places in its environment, whereas using an dead reckoning strategy it can integrate cues generated by its previous movements to return to a just left location. Dead reckoning is colloquially called "sense of direction" and "sense of distance."

Although there is considerable evidence that the hippocampus is involved in piloting (O&#x2019;Keefe and Nadel, 1978; O&#x2019;Keefe and Speakman, 1987), there is also evidence from behavioral (Whishaw et al., 1997; Whishaw and Maaswinkel, 1998; Maaswinkel and Whishaw, 1999), modeling (Samsonovich and McNaughton, 1997), and electrophysiological (O&#x2019;Mare et al., 1994; Sharp et al., 1995; Taube and Burton, 1995; Blair and Sharp, 1996; McNaughton et al., 1996; Wiener, 1996; Golob and Taube, 1997) studies that the hippocampal formation is involved in dead reckoning. The relative contribution of the hippocampus to the two forms of navigation is still uncertain, however. Ordinarily, it is difficult to be certain that an animal is using a piloting versus a dead reckoning strategy because animals are very flexible in their use of strategies and cues (Etienne et al., 1996; Dudchenko et al., 1997; Martin et al., 1997; Maaswinkel and Whishaw, 1999). The objective of the present video demonstrations was to solve the problem of cue specification in order to examine the relative contribution of the hippocampus in the use of these strategies. The rats were trained in a new task in which they followed linear or polygon scented trails to obtain a large food pellet hidden on an open field. Because rats have a proclivity to carry the food back to the refuge, accuracy and the cues used to return to the home base were dependent variables (Whishaw and Tomie, 1997). To force an animal to use a a dead reckoning strategy to reach its refuge with the food, the rats were tested when blindfolded or under infrared light, a spectral wavelength in which they cannot see, and in some experiments the scent trail was additionally removed once an animal reached the food. To examine the relative contribution of the hippocampus, fimbria&#x2013;fornix (FF) lesions, which disrupt information flow in the hippocampal formation (Bland, 1986), impair memory (Gaffan and Gaffan, 1991), and produce spatial deficits (Whishaw and Jarrard, 1995), were used.

In a piloting scent tracking task, the ability of the rats to return to a refuge with food using visual an odor trail or using dead reckoning in infrared light, the integrated record of previous movements, demonstrates that the hippocampus is necessary for dead reckoning.

录像示范保存鼠气味追踪，但经过伞 - 穹窿病变受损的惯性导航试验

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; ...

生物学

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

皮洛卡平诱导癫痫小鼠记忆功能评估

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

An Inescapable Cat Odor Exposure Protocol for Studying Innate and Contextual Threat Conditioning in Rats

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal model with an ethological approach offers a deeper insight into the biological mechanisms underlying threat responses. This paper describes a methodology to measure defensive behaviors toward both innate and learned aversive stimuli in rats. Animals were individually exposed to predator odor in an inescapable chamber to elicit a measurable, sustained, defensive state. The experimental design involved placing a rat in a familiar chamber for 10 min followed by exposure to cat odor for another 10 min in the same context. The next day, the rats were re-exposed for 10 min to the same context chamber where cat odor exposure occurred. Sessions were video-recorded and defensive behaviors were assessed on both days. 
The behavioral test was coupled with reversible functional inactivation and c-Fos immunohistochemistry techniques to determine the role of the interoceptive cortex in threat responses. Rats exposed to cat odor on the first day and re-exposed to the context chamber on the second day displayed higher levels of defensive behaviors, and that cat odor elicited a robust increase in the neural activity of the interoceptive cortex. Moreover, muscimol inactivation of the interoceptive cortex reduced the expression of defensive behaviors in response to cat odor and impaired contextual threat memory. These results show that this behavioral assay is a useful tool for studying neural mechanisms of defensive behaviors and may offer insight into mechanisms that mediate fear in humans and its related disorders.

Cat skin/fur odor exposure provides a reliable method to investigate neural circuits and mechanisms of defensive responses in rodents and may offer insight into mechanisms that mediate fear in humans. Here, we describe a protocol for investigating the role of the interoceptive cortex in threat responses in rats.

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal ...

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 ...