本协议适用于我们实验室中以前的工作 (黄et )。8). 这里描述的所有方法都得到了贝勒医学院动物护理和使用委员会 (ACUC) 的批准。它得到了 NINDS 医疗机构的支持, R01NS078294 B.R.A.、NIH IDDRC 赠款 U54HD083092、NIDDK 赠款 F30DK112571 到就业选配和 NINDS F31NS092435。

所有老鼠都是根据国立卫生研究院的标准, 由贝勒医学院动物保育和使用委员会批准的一项议定书使用的。在本议定书中使用的老鼠都是成年小鼠 (> 6 周龄) 的 C57BL6/j 背景, 包括雄性和雌性小鼠。在训练/分期任务后, 老鼠被送回自己的笼子里。
1. 使用操作性学习框的构造和一般规则 (图 1B、C)
<ol><li>用房间地板组装一个小鼠室。保持训练箱在低流量, 昏暗的灯光区域, 以避免分心。</li>
	<li>钻每个水口, 上面有一个小孔, 允许18口径的针头在港口内分配水。</li>
	<li>填充玻璃瓶与气味溶解在矿物油 (更换每周), 并安全收紧瓶盖。</li>
	<li>将18口径针连接到硅胶油管。皮尔斯玻璃瓶帽与18口径针, 并连接其他端的硅胶管的入口的气味端口。</li>
	<li>将每个硅胶管放入一个气味阀。</li>
	<li>将真空线连接到异味端口。</li>
	<li>将两个10毫升注射器连接到金属夹杆上, 并将油管与注射器相连。将油管的另一侧连接到18克针。将针放入鼠标室的鼻戳口钻孔。将油管的另一端连接到水阀。</li>
	<li>用啮齿动物的饮用水填满这两个10毫升注射器。</li>
	<li>将空气流量计与进气口连接, 并保持3–5升/分钟的气流。</li>
	<li>连接2个阀门, 2 个气味阀门, 2 个水端口, 气味端口, 电源到 USB 接口系统。将所有阀门连接到 "输出" 端口、所有的气味和水端口到 "输入" 端口。将所有设备连接到 usb 接口框中的电源输出, 最后将 usb 接口框连接到电源。</li>
	<li>调整真空吸力, 避免试验之间的异味交叉污染。</li>
	<li>使用气味特定的油管连接气味瓶到房间。</li>
</ol>2. 鼠标准备: 1–3天
<ol><li>把老鼠分成3组: 控制 (不需要嗅觉训练), 伪训练 (随机接受奖励或惩罚的老鼠) 和训练有素的团体。将伪训练的小鼠暴露在训练箱和气味的传递上, 但不提供嗅觉训练, 因为奖励与惩罚的结果是随机关联的气味传递。 注: 伪训练组将在 "Pseudotraining" 阶段完成训练范式。训练有素的老鼠将完成所有训练阶段。Pseudotraining 是可选的, 如果实验的目的是检测气味歧视或学习的行为差异。这里提供的协议增加了这个小组, 如果实验者想探索在训练前后的神经元差异。pseudotrained 组将控制被动气味暴露和非嗅觉相关训练。</li>
	<li>开始在小鼠的水限制到40毫升/公斤/天。避免体重损失大于20% 的动物的基线重量, 以避免遇险 (图 2A)。</li>
	<li>每天对老鼠进行称量, 以确保它们比基线重量高80% 以上。如果鼠标低于此阈值, 请将鼠标从研究中移除, 并提供免费的水访问。</li>
	<li>在整个协议中保持所有环境因素恒定, 包括温度、噪音和杂散气味 (包括个人身体和香水/科隆/除臭气味)。 注意: 与所有动物行为测试一样, 小环境变化会对结果产生很大影响。</li>
</ol>3. 所有阶段的指示
<ol><li>下面每个阶段的代码培训软件。在行为软件上运行软件。 注意: 所有阶段的编码都包含在补充编码文件中。20条路径的数据被分组为单个块, 并且跨块显示鼠标性能。此外, 每个阶段都可以在鼠标上重复数天, 直到达到完成标准。</li>
	<li>不要把老鼠放在行为盒里超过60分钟/天。</li>
	<li>在每只老鼠被转移到笼子之前, 从笼子里清理老鼠废物。喷雾和擦拭房间与70% 乙醇, 以减少鼠标气味分心。</li>
</ol>4. 培训阶段 1: 将水奖励与中心鼻子戳、1–3天联系起来
<ol><li>在这个阶段, 把老鼠和水的奖励联系在水口的勘探上。</li>
	<li>
培训阶段1编程说明
	<ol>
<li>程序此阶段只使用送水端口。让老鼠收到水奖励每个鼻子戳。 注: 该程序将输出试验的时间长短和收到的水奖励的总数量。</li>
	</ol>
</li>
	<li>设置 pseudotraining 阶段1编程与训练阶段1相同。</li>
	<li>
框配置和鼠标设置
	<ol>
<li>将 "行为" 框配置为中心的水口, 并使所有侧面端口无法访问。把一只老鼠放进房间。关闭鼠标室并开始1阶段程序。</li>
	</ol>
</li>
	<li>当鼠标在60分钟内完成100项试验时, 请将此阶段视为完整. 在60分钟或100次试验完成后从会议厅中取出鼠标 (图 2B)。 注意: 由于个体的差异, 一些老鼠自然会避免探索这个盒子。</li>
	<li>如果需要鼓励, 手动将水输送到水口。重复这个阶段长达3天。 注意: 已经训练过的老鼠可以进一步重复以保持奇偶校验, 并使它们保持液体的限制。当组平均达到 100 trials/60 分钟时, 也可以将整个组提升到2级。这将允许所有老鼠在同一天继续训练。</li>
</ol>5. 培训阶段 2: 将侧口水奖励与中心口鼻戳、1–5天关联
<ol><li>在这个阶段, 让老鼠戳他们的鼻子在中心港口, 然后立即收到水奖励的侧面端口。</li>
	<li>
框配置和鼠标设置
	<ol>
<li>配置这个和每一个后续阶段有2个水端口两侧和气味端口在中间。把一只老鼠放进房间。关闭鼠标室并开始2阶段程序。</li>
	</ol>
</li>
	<li>
培训阶段2编程说明
	<ol>
<li>为鼠标提供即时的水奖励双方后, 鼻子戳到中心的气味传递端口。将此阶段的输出参数设置为试验的时间长短、开始的试验次数以及在5秒内收到的水奖赏数量。</li>
	</ol>
</li>
	<li>设置 pseudotraining 阶段2编程与训练阶段2相同。</li>
	<li>考虑到这个阶段是完整的, 当至少有40项试验是在60分钟内进行, 最少有25% 的水奖励在中心口鼻戳的5秒内收到。完成此阶段后移除鼠标 (图 2C)。 注: 培训计划以及时的方式计算收到的水奖励的百分比, 以确定这个阶段的完成情况。虽然小鼠在完成这个阶段的速度不同, 但大多数老鼠在5天内就能达到完成标准。但是, 如果鼠标没有在5天内完成此阶段, 则不要将鼠标推进到下一阶段。此鼠标将从队列中删除。</li>
</ol>6. 培训阶段 3: 将水奖励与特定的气味联系起来, 在特定的时间窗口内, 1–3天
<ol><li>在这个阶段, 让老鼠在中心端口的鼻子戳时收到一股 (S +) 气味。随后, 在5秒内, 当鼻子戳入侧水端口时, 产生水的奖赏。</li>
	<li>
培训阶段3编程说明
	<ol>
<li>在中心气味端口提供 S + 气味。</li>
		<li>提供水奖励, 如果鼠标在 5 s 的气味传递的侧面端口。 注意: 该程序开始时只需要在中心端口100毫秒的鼻子戳, 以获得水奖励。一旦鼠标在80% 的试验中对中心端口进行正确的时间戳, 水奖励所需的鼻腔戳的时间长度将增加50毫秒, 达到400毫秒。</li>
		<li>将输出参数设置为与阶段2相同。</li>
	</ol>
</li>
	<li>设置 pseudotraining 阶段3编程指令与训练阶段3相同。但是, 不要将 s + 气味与气味传递控制器连接, 以确保 s + 气味与水奖励没有关联。</li>
	<li>
框配置和鼠标设置
	<ol>
<li>在阶段2设置相同的配置。</li>
		<li>将 S + 气味连接到气味传递控制器。把一只老鼠放进房间。关闭鼠标室并开始3阶段程序。完成此阶段后, 请移除鼠标。</li>
	</ol>
</li>
	<li>如果在60分钟内有大于60的奖励 (图 2D), 则将此阶段视为完成。</li>
</ol>7. 阶段 4A: 关联不去 (S) 气味和超时处罚, 几天
<ol><li>在这个阶段, 把老鼠介绍给一个不去的气味 (S)。在闻到无异味后, 对不正确进入水口的老鼠提供时间惩罚。</li>
	<li>
培训阶段4A 编程说明
	<ol>
<li>在这个阶段仅交付 S + 气味在开始, 与阶段3相同。在小鼠完成40项试验后, 开始随机传递的气味, 包括 s + 和 s-气味。如果老鼠试图在被提出不去的气味后寻求水的奖励, 计划一个2的时间惩罚。 注意 : 为了帮助区分去与不去 , 红外线侧灯可以纵 , 他们是在气味和关闭期间 , 在不去气味。在最初的训练中, 这些灯作为辅助提示来帮助老鼠。一旦老鼠接受了灯光的训练, 他们就应该在没有灯光的情况下进行训练, 以确认他们已经学会了气味的任务。</li>
		<li>将输出参数设置为试用时间、开始的试验次数、完成的试验次数、% 正确率以及收到的总奖励数。</li>
	</ol>
</li>
	<li>设置 pseudotraining 阶段4A 编程指令与训练阶段 4S, 除 s + 和 s 气味以外从开始随机地被提出。无论任务表现如何, 随机给予水奖励或超时处罚。</li>
	<li>
框配置和鼠标设置
	<ol>
<li>将框配置设置为与上一阶段3相同。</li>
		<li>将 s + 和 s 气味连接到气味传递控制器。把一只老鼠放进房间。关闭鼠标室并开始4A 阶段程序。在小鼠完成40项试验后, 切换程序随机传递气味。完成此阶段后, 请移除鼠标。</li>
	</ol>
</li>
	<li>当有40项试验 > 60% 正确的反应时, 请将这个阶段视为完整的。</li>
</ol>8. 阶段 4B: 关联不去 (S) 气味和时间惩罚, 5–11天
<ol><li>
培训阶段4B 编程说明
	<ol>
<li>使这个阶段相同的阶段 4A, 但时间的惩罚, 试图水奖励后, s 气味是 4 s。</li>
	</ol>
</li>
	<li>设置 pseudotraining 阶段4B 编程指令相同的阶段 4A, 但水奖励和超时处罚随机交付每审判。</li>
	<li>
框配置和鼠标设置
	<ol>
<li>将框配置设置为与上一阶段4A 相同。</li>
		<li>将 s + 和 s 气味连接到气味传递控制器。把一只老鼠放进房间。关闭鼠标室并开始4B 阶段程序。在60分钟内有 > 100 项试验, 精确度 > 85% (图 2E) 时, 请考虑此阶段完成情况。</li>
	</ol>
</li>
	<li>监视每个会话的最大和最小百分比正确, 以跟踪每个鼠标在这个阶段的进度。大约85-90% 的老鼠达到了这个阶段的完成标准。排除没有完成这个阶段的老鼠去/不去测试阶段。</li>
	<li>选)在这个阶段结束时, 应用一个反向训练, 在那里去/不去气味在气味瓶平台上切换。确保小鼠不与另一种刺激相关联, 如与侧面端口相关的阀门的声音。</li>
</ol>9. 去/不去化验任务: 1 天, 每只老鼠每天20分钟
<ol><li>认为这是确定气味联想识别准确性和学习辨别气味对的最后阶段。 注: 新的气味用于 s +/s-以测试动物花多长时间学习新的协会。结构上类似的气味对用于增加任务难度。例子包括 1-丁醇对1戊醇, 乙酸异戊酯和丁酸异戊, + 香芹酮 vs. 香芹酮。</li>
	<li>
编程说明
	<ol>
<li>让训练软件检测鼻子戳的长度。程序 300 ms 鼻子戳在中心气味端口执行任务。</li>
		<li>将输出参数设置为与阶段4B 相同, 但不使用任何光引导提示。</li>
	</ol>
</li>
	<li>
框配置和鼠标设置
	<ol>
<li>将框配置设置为与上一阶段4B 相同。</li>
		<li>将 s + 和 s 气味连接到气味传递控制器。把一只老鼠放进房间。关闭鼠标室, 开始舞台去/不去程序。让那些已经学会嗅觉学习任务的老鼠接触到新的化学相似的气味。 注: 在大约10-20 块或200-400 项试验 (图 3A) 之后, 有足够气味歧视的 Wildtype 小鼠达到 > 85% 的准确性, 气味新的。</li>
	</ol>
</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rats+smell+in+stereo.">Rats smell in stereo.</a> Science. 311 (5761), 666-670 (2006).</li><li>Batista-Brito, R., Close, J., Machold, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distinct+temporal+origins+of+olfactory+bulb+interneuron+subtypes.">The distinct temporal origins of olfactory bulb interneuron subtypes.</a> J Neurosci. 28 (15), 3966-3975 (2008).</li><li>Sakamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+postnatal+neurogenesis+contributes+to+formation+of+the+olfactory+bulb+neural+circuits+and+flexible+olfactory+associative+learning.">Continuous postnatal neurogenesis contributes to formation of the olfactory bulb neural circuits and flexible olfactory associative learning.</a> J Neurosci. 34 (17), 5788-5799 (2014).</li><li>Resendez, S. L., Jennings, J. H., Ung, R. L., Namboodiri, V. M. K., Zhou, Z. C., Otis, J. M., Stuber, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+cortical,+subcortical,+and+deep+brain+neural+circuit+dynamics+during+naturalistic+mammalian+behavior+with+head-mounted+microscopes+and+chronically+implanted+lenses.">Visualization of cortical, subcortical, and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses.</a> Nat Protoc. 11 (3), 566 (2016).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+optogenetics+with+multifunctional+flexible+polymer+fibers.">One-step optogenetics with multifunctional flexible polymer fibers.</a> Nat Neurosci. 20 (4), 612 (2017).</li></ol>

作者声明没有利益冲突, 也没有竞争利益。

啮齿动物嗅觉系统为研究感官依赖性可塑性提供了一个独特的模型。在这里, 我们提出了一个嗅觉学习范式, 训练小鼠将气味对与奖励或惩罚联系起来。通过这项学习任务, 可以在后续实验 (电生理学,体内神经元成像,等) 中研究下游电路的变化。完成后, 小鼠将学会执行一个简单的气味提示任务, 使水奖励与一种气味, 以及超时处罚与另一种气味。
由于这是一种行为分析, 我们建议在实验和控制动物群中利用年龄和性别的平等分布。老鼠之间的条件必须尽可能保持恒定。例如, 确保在整个培训过程中, 鼠标处理程序和光照条件保持不变。如果许多鼠标不按预期执行任务, 请确保满足以下条件: (1) 使行为设置尽可能安静。(2) 水对老鼠的剥夺足够。我们发现, 即使在整整一天的缺水之后, 许多老鼠也不能充分饮用1级的中心端口, 达到完成标准。(3) 在每一个新的鼠标开始, 检查气味/水阀门和管道, 以确保适当的放置。(4) 气味的波动性不同, 因此有些蒸发速度比其他的快。每天用更高的蒸汽压力取代更挥发性的气味, 而不是每周, 特别是如果使用较低浓度。
该协议可根据实验目的进行修改。如果在4B 阶段的学习时间是重要的, 那么最好在这个阶段去掉任何气味。这确保了任何嗅觉学习开始在4B 阶段。为了做到这一点, 我们已经完成了这个协议, 消除了3阶段的气味, 并完全跳过阶段4A。老鼠将有一个更困难的时间达到完成标准使用这种方法, 但这也提供了有价值的信息, 如何快速的老鼠学习这个任务的第一次。
这个协议的一个限制是, 老鼠必须完成不同的阶段, 直到嗅觉测试可以执行。因此, 虽然我们的实验室没有看到, 操纵可能会阻碍认知, 所以阶段4B 始终没有达到。我们试图通过确保每一个阶段足够长, 使大多数老鼠能够毕业到下一步的训练中来否定这个问题。但是, 如果在整个训练期后, 鼠标不能达到某个阶段的完成标准, 我们就从队列中删除该鼠标。这样, 我们就可以在不等待任何单个鼠标的情况下继续进行研究。另一个限制是, 我们还没有把这个议定书扩展到其他对嗅觉领域重要的动物模型。例如, 老鼠在 OB11、12中揭示神经元功能方面发挥了重要作用。由于他们的智能, 老鼠的学习时间也比鼠标13,14快。尽管有这些限制, 我们选择了此协议的鼠标, 因为它们的基因驯良执行单元格类型特定的操作和记录15。
多数现有的协议使用气味嗅探的区别或在气味旁边花费的时间近似歧视或学习4,6,9,10,16。本议定书可以通过试用的方式直接衡量对审判的歧视。此外, 这可以测量每个鼠标需要学习一个关联的确切数量。这种完全自动化的方法消除了数据分析中的任何人为偏见。食物匮乏的动物也被成功地用于嗅觉学习。选择缺水小鼠的原因是, 老鼠可以用水在食物上执行的试验次数增加16。
由于植入式活动监视器 (如分级索引 (咧嘴) 透镜和多单元电极) 继续改进, 我们可能很快就能够将这些技术与此协议17、18结合起来。通过在嗅觉学习过程中记录相关的大脑区域, 可以探讨在联想学习过程中神经元活动的变化。这可能有助于揭示基本的神经科学问题, 例如神经元在学习过程中如何对信息进行不同的编码。

老鼠 OB, 气味信息首先进入中枢神经系统 (CNS), 提供了一个很好的模型来研究经验依赖的结构变化。OB 迂回以活动依赖性的方式不断整合成人出生的神经元。成人出生的神经元前体分裂从祖细胞, 线的脑室下层区相邻的侧脑室 1.当迁移到 OB, 这些神经元前体要么生存, 分化, 并整合为抑制颗粒细胞, 或经历凋亡2。细胞命运的选择受嗅觉活动的影响, 包括嗅觉学习3,4,5,6。整合后, 学习诱导的突触变化发生在颗粒细胞在两个星期的关键期间7,8。因此, 嗅觉学习的化验对于研究经验依赖性可塑性如何影响成熟脑回路的结构和功能重组是有用的。
该协议提供了一种嗅觉训练的方法, 使用一个操作的调节范式。在这项任务中, 缺水的老鼠被训练成将一种气味 ("去" 气味) 与水奖励和另一种气味 ("不去" 气味) 联系在一起试用超时处罚。在2-4 周的过程中, 小鼠通过一系列分级训练阶段取得进展。当训练完成后, 老鼠会用离散的、相应的行为 (在试验中寻求水奖励, 而不寻求不去试验的水奖励) 对去或不去的气味作出反应 (图 1a)。训练完成后, 老鼠可以进一步挑战与化学相似的气味对测试歧视或转变为研究如何嗅觉学习改变的结构或功能的 OB。虽然以前已经描述过气味识别任务, 但大多数依赖于主观测量, 例如两个气味9、10之间的嗅探次数。此外, 人类对这类任务的评分也需要耗费时间。这个协议中描述的去/不去嗅觉学习任务提供了一种无偏见, 直接测量气味歧视和嗅觉学习。

<table><tbody><tr><td>Glass vial</td><td>Qorpak</td><td>GLC-01016</td><td></td></tr><tr><td>Silicon Tubing</td><td>Thermo Scientific</td><td>86000030</td><td></td></tr><tr><td>18 gauge needles</td><td>BD</td><td>305196</td><td></td></tr><tr><td>1-Butanol</td><td>Sigma Aldrich</td><td>437603</td><td></td></tr><tr><td>Propionic Acid</td><td>Sigma Aldrich</td><td>402907</td><td></td></tr><tr><td>Mouse Chamber</td><td>Med Associates</td><td>ENV-307W</td><td></td></tr><tr><td>Chamber Floor</td><td>Med Associates</td><td>ENV-307W-GFW</td><td></td></tr><tr><td>Water Port</td><td>Med Associates</td><td>ENV-313W</td><td>Need two</td></tr><tr><td>Odor stimulus</td><td>Med Associates</td><td>ENV-275</td><td>Contain 2 valves to gate odor delivery&amp;nbsp;</td></tr><tr><td>Odor Port</td><td>Med Associates</td><td>ENV-375W-NPP</td><td></td></tr><tr><td>USB Interface</td><td>Med Associates</td><td>DIG-703A-USB</td><td></td></tr><tr><td>Desktop Computer with Windows 2000, XP, Vista, or 7</td><td></td><td></td><td></td></tr><tr><td>Flow meter</td><td>VWR</td><td>97004-952</td><td></td></tr><tr><td>Behavioral software</td><td>Med Associates</td><td>SOF-735</td><td>This software, which runs each training stage, has now been replaced with Med-PC V</td></tr><tr><td>Data Transfer software</td><td>Med Associates</td><td>SOF-731</td><td>This software formats the data to Excel</td></tr><tr><td>Training Software</td><td>Med Associates</td><td>DIG-703A-USB</td><td>This software is used to program each training stage</td></tr><tr><td>Water Valve</td><td>Neptune Research</td><td>225P012-11</td><td>This valve is used to gate the water delivery. Need Two</td></tr><tr><td>Odor Valve</td><td>Neptune Research</td><td>360P012-42</td><td>This valve is used to gate the odor delivery. Need Two</td></tr></tbody></table>

an objective and reproducible test of olfactory learning and discrimination in mice

嗅觉是小鼠主要的感官形态, 影响着许多重要的行为, 包括觅食、捕食者的检测、交配和养育。重要的是, 老鼠可以被训练, 以联想到新的气味与特定的行为反应, 以提供洞察嗅觉电路功能。本协议详细介绍了训练小鼠进行去/不去操作学习任务的过程。在这种方法中, 老鼠每天都接受数以百计的自动试验的训练, 2–4周, 然后可以测试新的去/不去气味对评估嗅觉歧视, 或用于研究气味学习如何改变嗅觉的结构或功能电路.此外, 鼠标嗅球 (OB) 的特点是不断整合成人出生的神经元。有趣的是, 嗅觉学习增加了这些成年神经元的生存和突触连接。因此, 该协议可以结合其他生物化学, 电生理和成像技术, 以研究学习和活动相关的因素, 调解神经元的生存和可塑性。

一旦老鼠学会了嗅觉学习的任务, 他们现在可以联想到新的气味对与奖励和惩罚。这些受过训练的老鼠通常在去/不去的任务上以大约50% 的精确度开始。该百分比正确可由试用块绘制为新的气味对的学习曲线 (图 3a)。在10块试验中, 大多数小鼠的表现都不到30分钟, 小鼠能够正确辨别出大于85% 精度的气味 (红线)。这表明, 我们的协议已经成功地训练 wildtype 老鼠把一种气味和水奖励联系起来, 另一种是超时惩罚。为了评估两个群体之间的歧视能力, 可以进一步分析这些数据, 以比较达到85% 熟练程度 (图 3B) 或在达到熟练程度后平均百分比正确的试验次数。
一旦老鼠学会了这个任务, 气味对就可以被修改来增加或减少任务难度。例如, 减少气味对浓度会增加任务难度 (图 3C)。该分析可以揭示不同小鼠群的检测阈值。此外, 气味对可以改变成更结构上相似 (即,对映体或单一碳差异)。气味混合物也可以用来增加任务难度 (即, 40/60 混合物与30/70 混合物)。
老鼠也能记住以前学过的气味对。经过7天的学习任务后, 一个召回测试表明, wildtype 老鼠可以很快记住以前学会的气味关联 (图 3D)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57142/57142fig1.jpg"> 图 1: 去/不去培训范例和设备配置.(A) 鼠标在去气味演示过程中获得水或在无异味演示期间不进行正确响应。(B) 配置 "行为" 框, 以便在阶段1中仅可访问单个水口, 并且所有后续阶段都包含中央气味传递端口和两个水口。蓝色圆环: 水口岸。绿色圆环: 气味口岸。(C) 气味传递由一个气味阀门为每个气味封闭。正压进气导致分销商。红色箭头表示正气压线进入准备的气味瓶 (红色圈子)。蓝色箭头表示空气/气味混合物离开气味瓶和通过气味阀门 (淡蓝色圆环) 和进入一个白色空气集成盒。橙色箭头表示正压空气进入中央调节阀 (橙色圆圈) 到集成盒, 以允许正气压将气味/空气混合物从集成箱推入气味传递端口 (底部黑色箭头)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57142/57142fig1large.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/57142/57142fig2.jpg"> 图 2: 培训数据.(A) 在流体限制下, 鼠标的体重百分比不低于80%。红线 = 80% 阈值。N = 8 只老鼠。(B) 阶段1培训结果显示, 中心端口的数量在60分钟内会得到水奖励。N = 8 只老鼠。误差条是平均值的标准误差。(C) 阶段2培训结果显示, 在中心鼻戳的5秒内收到的水分奖励百分比。红线为25% 阈值。平均百分比以灰色突出显示作为标准误差的平均值。N = 8 只老鼠。(D) 阶段3累计奖励显示8个小鼠在1天和2天的训练中收到的奖励数。红线是60奖励阈值。误差线是标准偏差。(E) 阶段4B 精度结果显示对阶段4B 的正确响应百分比。红线是阈值的85%。实心黑线为平均值, 灰度突出显示为平均值的标准误差。N = 4 只老鼠。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57142/57142fig2large.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/57142/57142fig3.jpg"> 图 3:"转/不转" 任务的代表结果.(A) 在对丁香酚 (s +) 和 eucalyptol (s) 进行训练后, 2个新的气味 (s = 醋酸异戊酯/s-戊酯) 的去/不去任务。N = 5 只小鼠, 标准误差为灰色。绿线为50% 精度, 红线为阈值85% 精度。(B) 用于鼠标的 "转/不走" 任务的示例, 对于 hexanol (s +) 和丁酸 (s) 来说, 这是一个快速学习者, 而不是学习迟缓的鼠标。(C) hexanol (s +) 和丁酸 (s) 的不同部分压力的转/不转任务。(D) 小鼠在训练后7天内召回气味对的能力。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57142/57142fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice at JoVE.com

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

在这里, 我们训练老鼠的结合学习任务, 以测试气味歧视。这项议定书还允许对学习诱发的大脑结构变化进行研究。

小鼠嗅觉学习和鉴别的客观和可重复性试验

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

an-objective-reproducible-test-olfactory-learning-discrimination

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.5K 次观看.  Baylor College of Medicine. 在这里, 我们训练老鼠的结合学习任务, 以测试气味歧视。这项议定书还允许对学习诱发的大脑结构变化进行研究。

视频: An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Low-stress Route Learning Using the Lashley III Maze in Mice

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

低应力路线学习，在老鼠身上使用拉什利三迷宫

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

科研

神经科学

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal&#8217;s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. 
In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.

This manuscript describes a protocol to examine the olfactory system of rodents. The olfactory habituation/dishabituation test will allow investigators to determine whether a mouse habituates to a repeatedly presented odor and whether the mouse demonstrates dishabituation when presented a novel odor.

在小鼠试验的气味辨别和习惯化

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the ...

行为学

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

简单和计算机辅助测试嗅觉的小鼠

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

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 ...

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

在成年小鼠的嗅觉行为测试

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

生物学

A Low-Cost, Odor-Reward Association Task for Tests of Learning and Memory

Robust and simple behavioral paradigms of appetitive, associative memory are crucial for researchers interested in cellular and molecular mechanisms of memory. In this paper, an effective and low-cost mouse behavioral protocol is described for examining the effects of physiological manipulation (such as the infusion of pharmacological agents) on the learning rate and duration of odor-reward memory. Representative results are provided from a study examining the differential role of tyrosine kinase receptor activity in short-term (STM) and long-term memory (LTM). Male mice were conditioned to associate a reward (sugar pellet) with one of the two odors, and their memory for the association was tested 2 or 48 h later. Immediately prior to the training, a tyrosine kinase (Trk) receptor inhibitor or vehicle infusions were delivered into the olfactory bulb (OB). Although there was no effect of the infusion on the learning rate, blockade of the Trk receptors in the OB selectively impaired LTM (48 h), and not short-term memory (STM; 2 h). The LTM impairment was attributed to the diminished odor selectivity as measured by the length of the digging time. The culmination of the results of this experiment showed that Trk receptor activation in the OB is the key in olfactory memory consolidation.

An odor-reward, associative learning task was used to investigate the differential effects of physiological manipulation on long-term and short-term memory.

Robust and simple behavioral paradigms of appetitive, associative memory are crucial for researchers interested in cellular and molecular mechanisms of ...

评估吸入环境污染物导致小鼠嗅觉损害

Studying the Effects of Inhaled Environmental Pollutants on Olfactory Function in Mice

Olfactory impairment is a significant public health problem and independently predicts the risk of neurodegenerative diseases. Inhaled environmental pollutants exposure may impair olfaction; thereby, there is an urgent need for methods to evaluate the effects of inhaled environmental pollutants exposure on olfaction. Mice are ideal models for olfactory experiments because of their highly developed olfactory system and behavioral characteristics. To assess the effects of inhaled environmental pollutants exposure on olfactory function in mice, a detailed buried food test and social odor discrimination experiment is provided, including the experiment preparation, the selection and construction of experimental facilities, the testing process, and indexes of time. Meanwhile, timekeeping equipment, operational details, and the experimental environment are discussed to ensure the success of the assay. Zinc sulfate is used as the treatment to demonstrate the feasibility of the experimental approach. The protocol provides a simple and clear operational process for assessing the effects of inhaled environmental pollutants on olfactory function in mice.

作者聚焦：评估空气污染物的嗅觉影响 - 埋藏的食物和社会气味测试

This paper provides a detailed description of the buried food test and social odor discrimination experiment to assess the effects of inhaled environmental pollutants exposure on olfactory function in mice.

研究吸入环境污染物对小鼠嗅觉功能的影响

Olfactory impairment is a significant public health problem and independently predicts the risk of neurodegenerative diseases. Inhaled environmental ...

环境科学

Assessment of Mouse Judgment Bias through an Olfactory Digging Task

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This phenomenon has long been observed in humans, with individuals in positive states responding to ambiguity 'optimistically' and those in negative states instead showing 'pessimism'. Researchers aiming to assess animal affect have taken advantage of these differential responses, developing tasks to assess judgment bias as an indicator of affective state. These tasks are becoming increasingly popular across diverse species and fields of research. However, for laboratory mice, the most widely used vertebrates in research and a species heavily relied upon to model affective disorders, only one JB task has been successfully validated as sensitive to changes in affective state. Here, we provide a detailed description of this novel murine JB task, and evidence of its sensitivity to mouse affect. Though refinements are still necessary, assessment of mouse JB opens the door for answering both practical questions regarding mouse welfare, and fundamental questions about the impact of affective state in translational research.

This article provides a detailed description of a novel mouse judgment bias protocol. Evidence of this olfactory digging task's sensitivity to affective state is also demonstrated and its utility across diverse research fields is discussed.

通过嗅觉挖掘任务评估小鼠判断偏差

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This ...

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Subtitles

小鼠嗅觉学习和鉴别的客观和可重复性试验

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此视频中的章节

低应力路线学习，在老鼠身上使用拉什利三迷宫

在小鼠试验的气味辨别和习惯化

简单和计算机辅助测试嗅觉的小鼠

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

在成年小鼠的嗅觉行为测试

A Low-Cost, Odor-Reward Association Task for Tests of Learning and Memory

研究吸入环境污染物对小鼠嗅觉功能的影响

研究吸入环境污染物对小鼠嗅觉功能的影响

研究吸入环境污染物对小鼠嗅觉功能的影响

通过嗅觉挖掘任务评估小鼠判断偏差