Name: shuttle box assay as an associative learning tool for cognitive assessment in learning and memory studies using adult zebrafish
Uploaded: 2021-07-12T13:00:00
Description: Watch this Scientific Journal Video about Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish at JoVE.com

作者要感谢海德实验室成员的深思熟虑的讨论和弗雷曼生命科学中心斑马鱼护理和饲养的技术人员。这项工作得到了圣母大学斑马鱼研究中心、圣母大学干细胞和再生医学中心以及国家眼科研究所NIH R01-EY018417（DRH）、国家科学基金会研究生研究奖学金计划（JTH）、圣母大学LTC尼尔·海兰研究金（JTH） 的资助。 自由奖学金（JTH）和帕特·蒂尔曼奖学金（JTH）的哨兵。图1用 BioRender.com 制作的。

斑马鱼是在弗雷曼生命科学中心的圣母院斑马鱼设施中饲养和饲养的。本手稿中描述的方法已得到圣母院大学动物护理和使用委员会（动物福利保障编号A3093-01）的批准。
1. 穿梭箱学习范式 （图1A）
注：学习范式提供了对关联学习认知的快速评估。
<ol><li>通过修改一个 30.5 x 19 x 7.5 厘米的凝胶盒，在两侧以 45° 角添加 5 x 19 厘米的?...

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认知障碍会对生活质量产生显著和负面影响。由于整个人群的能见度和脑震荡和创伤性脑损伤的发生增加，了解它们如何导致认知障碍以及如何将损害降到最低或逆转非常重要。由于这些原因，可以测试认知衰退的模型生物在这些研究中起着至关重要的作用。长期以来，啮齿动物一直是研究神经行为和认知的主要模型，然而，斑马鱼已成为一个有用的模型，具有许多不同的行为，以调查一系列发?...

学习和记忆经常被用作认知障碍的指标，这些障碍是由于衰老、神经退行性疾病或损伤造成的。创伤性脑损伤 （TBIs） 是导致认知缺陷的最常见损伤。TBI越来越受到关注，因为它们与几个神经退行性疾病有关，如前脑痴呆症和帕金森病1，2。此外，在一些TBI患者中观察到的β-淀粉样蛋白聚合增加表明，它可能也与阿尔茨海默病3，4的发展有关。TBI通常是钝力创伤的结果，跨越一系列的分离5，轻度脑损伤（miTBI）是最常见的。然而，miTBI往往没有报告和误诊，因为它们只导致轻微的认知障碍很短的时间，受伤的人通常完全恢复6。相比之下，反复发生的miTBI事件越来越受到关注，因为它在年轻人和中年人中非常普遍，可以随着时间的推移积累7，会损害认知发展，并加剧神经退行性疾病1，2，3，4，5，类似于经历中度或重度TBI8的人。
斑马鱼（Danio rerio）是探索神经科学中各种主题的有用模型，包括在整个中枢神经系统9、10、11、12、13中重建丢失或受损神经元的能力。神经再生也表现在脑脊膜中，它包含在正交内区域的原型。这个神经解剖区域类似于海马，可能是在鱼类和人类14，15，16的短时间记忆的认知所必需的。此外，斑马鱼的行为已被广泛的特点和编目17。学习已经通过各种技术，包括习惯到惊人的反应18，它可以代表一种快速形式的非关联学习时，执行短块和注意快速衰变时间19。更复杂的关联学习测试，如T盒，加迷宫，和视觉歧视20，21使用，但往往很费时，需要几天或几周的准备，并依靠浅滩或积极的强化。在这里，我们描述了一个快速的范式来评估关联学习以及即时或延迟记忆。这种穿梭盒检测使用逆向刺激和负强化调理来评估钝力TBI之后的认知缺陷和恢复。我们证明，未受损的控制成年斑马鱼（8-24个月）可重复学习避免在航天飞机箱中的20个试验（<20分钟的评估）内闯红灯，观察者之间高度一致。此外，使用穿梭盒，我们证明成人（8-24 个月大）的学习和记忆能力是一致的，并且对于在不同的 TBI 分离或重复 TBI 之间具有显著障碍的怀疑认知是有用的。此外，这种方法可以迅速用作衡量标准，以跟踪影响成人斑马鱼认知维持或恢复的药物干预措施的各种疾病进展或疗效。
在这里，我们提供了快速认知评估的教学概述，可以检查复杂的关联学习（第1节）和记忆在即时和延迟记忆方面。此范式提供了对学习的关联认知任务的短期和长期记忆的评估（第 2 节）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Flashlight</td>
			<td>Ultrafire</td>
			<td>9145</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Ocean</td>
			<td>Instant Ocean</td>
			<td>SS15-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Large DNA Gel Box</td>
			<td>Fisher Scientific</td>
			<td>FB-SB-1316</td>
			<td>Shuttle Box</td>
		</tr>
		<tr>
			<td>Power Supply</td>
			<td>Fisher Scientific</td>
			<td>FB-105</td>
			<td></td>
		</tr>
	</tbody>
</table>

shuttle box assay as an associative learning tool for cognitive assessment in learning and memory studies using adult zebrafish

认知缺陷，包括学习和记忆受损，是各种发育和年龄相关的神经退行性疾病和创伤性脑损伤（TBI）的主要症状。斑马鱼是一个重要的神经科学模型，由于它们在发育过程中的透明度和神经创伤后强大的再生能力。虽然斑马鱼存在各种认知测试，但大多数认知评估都是快速检查非关联性学习的。同时，关联学习分析通常需要多日或数周。在这里，我们描述了一个快速的关联学习测试，它利用了不利的刺激（电击），并且需要最少的准备时间。这里介绍的穿梭箱检测简单，非常适合新手调查员，并且需要最少的设备。我们证明，在TBI之后，这个穿梭盒测试可以重复评估认知缺陷和从年轻到老斑马鱼的恢复。此外，检测适应性强，可检查即时或延迟内存。我们证明，单个 TBI 和重复的 TBI 事件都会对学习和即时记忆产生负面影响，但不会延迟记忆。因此，我们的结论是，穿梭盒检测可重复跟踪认知障碍的进展和恢复。

协议和示意图（图1）中概述的学习范式，对关联学习的认识进行了快速评估。此外，这种模式具有高度的严格性，将学习定义为连续 5 次积极试验的重复和一致显示。此范式也适用于各种年龄和伤害。8个月（年轻成人）、18个月（中年成人）和24个月（成年）未损坏鱼类需要类似数量的试验，以了解避免红灯的行为（未损坏8米：15.28±4.92试验， 18 米： 17.66 ± 5.5 试验， 24 米...

Watch this Scientific Journal Video about Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish at JoVE.com

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

学习和记忆是研究发育、疾病依赖或环境诱发的认知障碍的有力指标。大多数认知评估都需要专门的设备和广泛的时间承诺。然而，穿梭箱检测是一种关联学习工具，利用传统的凝胶盒快速可靠的评估成人斑马鱼认知。

穿梭盒测定作为使用成人斑马鱼进行学习和记忆研究认知评估的关联学习工具

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

穿梭盒检测可重复跟踪钝力创伤或斑马鱼任何其他类型的脑损伤后认知损伤的进展和恢复。穿梭盒检测允许相对简单、快速和稳健地测量斑马鱼短期和长期记忆中的关联学习。首先，准备航天飞机盒，修改一个30.5乘19乘7.5厘米的凝胶盒，每边以45度角添加5乘19厘米的水族级有机玻璃。然后划一条线，标记鱼缸的中间点，以评估鱼何时穿过鱼缸中间。在将 800 毫升系统水添加到航天飞机箱中后，将两到三条鱼放入装有系统水的储水箱中。将坦克留在将进行穿梭箱检测的黑暗房间。接下来，在黑暗的房间里，将一条鱼放在航天飞机箱的中心。固定盖子，将电极连接到电源上。鱼应该在黑暗中适应。关掉所有的灯，让鱼适应穿梭箱15分钟。当鱼自由探索鱼缸时，可以考虑成功的适应。在成功适应鱼后，手动照射一个800流明红色镜头手电筒，距离鱼侧面的凝胶盒壁约两厘米。如果鱼在铂金线旁边休息，请不要开始试验。将光线刺激直接照射到鱼身上，并手动跟随鱼的任何横向运动，确保刺激的持续可视化。继续提供光刺激，直到满足成功或失败的试验的以下条件之一。如果鱼在光照射后 15 秒内越过鱼缸的中间点，则考虑试验是否成功。一旦鱼穿过中间点，立即停止光线刺激。如果试验失败，使用电泳电源对一个安培施加 20 毫伏的负冲击刺激，交替两秒钟，关闭两秒钟，最多 15 秒，最多四次冲击，或直到鱼通过盒子的中间点。然后终止光刺激和负刺激。在训练期之前，通过将鱼在黑暗条件下离开15分钟，确保成功适应航天飞机箱。然后手动照射一个800流明红色镜头手电筒，距离鱼所占据的侧面的凝胶盒壁约两厘米。将光线刺激直接照射到鱼身上，并跟随鱼的任何横向运动，确保刺激的持续可视化。当光线照耀在鱼身上时，同时将20毫伏的不良冲击刺激施加到一个安培上，交替两秒钟，两秒钟关闭15秒，最多四次冲击，或直到鱼通过盒子的中间点。一旦实现这一点，终止光和不利的刺激。重复 25 次迭代。完成后，让鱼在黑暗条件下再适应15分钟。在初始测试期间，在航天飞机箱中成功适应 15 分钟，然后仅应用光刺激长达 15 秒并记录响应。如果鱼在启动光刺激后 15 秒内越过穿梭箱的中间点，当鱼穿过中间点时立即停止光刺激，则考虑试验是否成功。如果鱼在启动光刺激后 15 秒没有穿过航天飞机箱的中间点，并在 15 秒后停止光刺激，则将试验视为失败。在初始测试期间，不要在尝试失败后应用不利刺激。在初始测试期后立即执行短期内存测试。诱发严重的脑损伤，等待四个小时后再进行测试。然后在航天飞机箱中将鱼适应15分钟。评估短期记忆，仅应用光刺激长达 15 秒，并记录鱼在灯关闭之前越过框的中间点，被视为通过试验，或未能在被视为失败的试用的 15 秒内穿过中间点。每次试验之间有 30 秒的休息时间，重复上述步骤 25 次，并记录成功和失败的试验次数。在初始测试后四天执行长期内存测试。诱发创伤性脑损伤，等待4小时后再进行测试，然后在航天飞机箱中对鱼进行15分钟的适应。评估长期记忆，仅应用光刺激长达 15 秒，并记录鱼在灯关闭之前越过盒子的中间点，被视为通过试验，或未能在被视为失败的试用的 15 秒内越过中间点。每次试验之间有 30 秒的休息时间重复上述 25 次，并记录成功和失败的试验次数。认知评估的学习和记忆范式的教学概述如下。未损坏的鱼在8个月，年轻的成年人，18个月，中年成人，和24个月，老年成人，需要类似的试验次数，以学习避免红灯的行为。在使用严重的钝力创伤性脑损伤模型（STBI）后，不同年龄的鱼需要类似的试验来掌握受伤后一到五天的检测。在STBI之后的第一天，所有年龄段的鱼都需要同样数量的试验来学习这种行为，这明显大于未受损的对照组。未受损的鱼迅速掌握了航天飞机箱，在大约17次试验中连续进行了5次阳性试验，而在轻度脑损伤或轻度TBI后的一天，鱼显示学习这种行为的试验数量显著增加。在两次轻度 TBI 之后，这一赤字增加，在三次轻度 TBI 受伤后进一步增加。与初始测试期相比，未受损鱼类在即时记忆中成功试验的百分比差异略有增加，并且延迟了记忆。与未受损的鱼相比，单次轻度 TBI 后，鱼类表现出显著且即时的记忆缺陷。这一趋势继续与反复受伤后，两个温和的TBI和三个温和的TBI赤字增加。应用不良刺激的时机至关重要。将不良刺激与光和它们同时去除配对，巩固了它们的关联，并且对于范式至关重要。该检测允许快速评估或复杂的关联学习，可用于调查发育、衰老和环境对认知障碍的影响。

Research

JoVE Journal

Neuroscience

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

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

线虫的积极丁酮学习，短期，长期联想记忆检测

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

科研

神经科学

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

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

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

Mapping Cortical Dynamics Using Simultaneous MEG/EEG and Anatomically-constrained Minimum-norm Estimates: an Auditory Attention Example

同时MEG / EEG和解剖约束的最小模估计皮质动态映射：听觉注意示例

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

Appetitive Associative Olfactory Learning in Drosophila Larvae

欲求联想嗅觉学习<em&gt;果蝇</em&gt;幼虫

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Drosophila Adult Olfactory Shock Learning

Drosophila Adult Olfactory Shock Learning

果蝇嗅觉成人学习的冲击

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

在啮齿动物中枢神经系统的体内光遗传学刺激

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

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

评估空间学习记忆小鳞类爬行动物

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

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Drosophila Courtship Conditioning As a Measure of Learning and Memory

果蝇调理作为学习和记忆的措施

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