Summary

对大鼠使用扩展的恐惧调节协议进行恐惧孵化

Published: August 22, 2020
doi:

Summary

我们描述了一种扩展的恐惧调节协议,在大鼠中产生过度训练和恐惧孵化。该协议包括一次训练,包括25个音震配对(即过度训练),以及比较在背景和提示测试期间的条件冻结反应48小时(短期)和训练后6周(长期)。

Abstract

情感记忆主要以恐惧调节范式来研究。恐惧条件是一种学习形式,通过这种学习,个人通过这种学习来学习逆境事件和其他中性刺激之间的关系。研究情感记忆的最广泛使用的程序是老鼠的恐惧调节。在这些任务中,无条件的刺激(美国)是单次或多次在单次或多次会话中呈现的足部冲击,并且条件响应 (CR) 是冻结的。在这些程序(称为”恐惧调理”)的版本中,在训练阶段,音调(条件刺激,CS)与脚蹄(美国)配对。在第一次测试中,动物暴露在训练发生的相同环境中,在没有脚蹄和音调(即上下文测试)的情况下测试冻结反应。在第二次测试中,当上下文发生变化时(例如,通过操纵实验室的气味和墙壁)时测量冻结,在没有脚踏车(即提示测试)的情况下显示音调。大多数恐惧调节程序需要很少的音震配对(例如,在一个会话中进行1-3次试验)。人们越来越关注较不常见的版本,这些版本涉及大量配对(即过度训练),这些配对与称为恐惧孵化的长期影响有关(即,恐惧反应会随着时间的推移而增加,而不会进一步暴露于风险事件或有条件的刺激)。延长的恐惧调节任务是理解恐惧孵化的行为和神经生物学方面的关键,包括它与其他心理现象(例如创伤后应激障碍)的关系。在这里,我们描述了一种扩展的恐惧调节协议,在大鼠中产生过度训练和恐惧孵化。该协议包括一次训练,包括25个音震配对(即过度训练),以及比较在背景和提示测试期间的条件冻结反应48小时(短期)和训练后6周(长期)。

Introduction

记忆是一个心理过程,包括不同的阶段:信息获取、整合(允许获取信息的稳定性)和检索(整合过程的证据)1。在合并阶段,将建立新的突触连接并修改预先存在的连接。这表明有必要在一段时间内,分子和生理事件导致这些变化发生1,1,2。这些生理或分子的变化各不相同,无论检索到的事件是否充满情绪(即情感记忆)。例如,研究表明,横向核和基莫拉特杏仁核复合物与情感记忆,3、4、54特别相关3

情感记忆现象主要研究与恐惧调理范式5,5,6。恐惧条件是一种学习形式,通过它个人学习逆境事件和其他中性刺激之间的关系7。恐惧调理范式在杏仁核中产生分子、细胞和结构变化。此外,恐惧调理在情感记忆的巩固和检索过程中改变海马体的连通性。

研究恐惧记忆最常用的程序之一是大鼠的经典(巴甫洛夫)调理。此过程通常使用脚击 (US) 作为逆刺激,在一次或多次会话中传递一次或多次。接触此程序的大鼠的调节反应(CR)是冷冻(即”由动物骨骼肌肉的一般补性反应引起的普遍不动”,除了呼吸中使用的肌肉外),7)。 此响应可以通过两种类型的测试进行评估:上下文和提示测试。对于上下文测试,该主体在训练过程中会经历一些脚蹄,然后在规定的时间内从实验室中移除。在测试期间,该科目被送回培训进行时所处,在没有脚踏车(例如,冷冻发作的持续时间、百分比或频率)的情况下收集不同的冻结措施,并与培训阶段建立的基线水平进行比较。对于第二种类型的测试,提示测试,刺激(通常是音)在训练阶段与脚踏车配对(即条件刺激,CS)。训练完成后,动物在规定的时间内从训练环境中移走,然后被放置在一个经过修改的上下文中(例如,具有不同的实验室,具有不同形状的墙壁和不同的气味)。然后,给出提示的给定次,对提示的冻结反应进行测量,并与训练期间收集的基线水平进行比较。此范式的最常见版本在一次训练期间使用 1 到 3 个音调冲击配对,然后是数小时或几天后进行的上下文和提示测试。

其他不太频繁的恐惧调节程序涉及大量的休克提示配对(即试验),这通常被称为过度训练程序8。对这些任务的兴趣与它们长期和增加的记忆效应有关,称为恐惧孵化(即,在没有进一步接触逆动事件或有条件刺激的情况下,有条件的恐惧反应会随着时间的推移而增加)9、10、11。9,10,11例如,这种过度训练程序包括100个音震配对的训练阶段,分布在10个会话中,然后是48小时和30天后11、12,的上下文和提示测试。为了避免几天来的广泛训练,Maren(1998年)报告说,在一次训练中可以建立和优化过度训练,有25对8。与训练后48小时测试的老鼠相比,在训练后31天测试的老鼠中,潜伏效应明显更高。延长的恐惧调节任务是理解行为和神经生物学方面的关键,这些方面是恐惧孵化的基础,包括它与其他心理现象(如创伤后应激障碍)的关系(如延迟发作的创伤后应激障碍)11、12、13。11,12,13

在这里,我们描述了一个扩展的恐惧调节协议,它诱发过度训练,在大鼠中潜伏恐惧。与其他需要几天培训的范式不同,目前的协议集中在一次训练8。我们使用25个音-休克配对,在训练后6周进行上下文和提示测试时产生更高的条件冻结反应,而训练后48小时进行的测试。

Protocol

以下议定书已获康拉德洛伦茨大学机构动物护理和使用委员会批准。国际动物权利联盟、瑞士日内瓦(1989年)发表的动物权利普遍宣言和国际动物权利联盟发布的动物实验道德原则得到尊重。 1. 主题准备 选择雄性成年威斯塔大鼠(n = 12)。在开始培训和测试程序之前,将他们放在每笼四个笼子里,进行三天的适应。在整个实验中,为老鼠提供免费用水。在 12 h 明暗?…

Representative Results

使用依赖 t 测试(表1)分析所有科目(n = 12)在培训课程不同阶段的冻结时间百分比变化情况。动物活跃,在训练课前三分钟(协议的第一天)探索实验室,在此期间没有音色或冲击(即基线-BL)。如图 2A 所示,在随后的 25 个音震配对中冻结时间百分比(M = 48.88; SE = 4.37)明显高于 BL 期间(M = 14.65; SE = 4….

Discussion

目前的扩展恐惧调节协议是一种有效和有效的方法来评估短期(48小时)和长期(6周)的情绪记忆。因此,该协议允许研究大鼠过度训练和恐惧孵化现象。该协议的不同优点包括:它提供了两种类型的内存测试,即上下文和提示,允许识别两个延迟(48 小时和 6 周)跨上下文和提示操作的差异效应。第二,这项任务需要一次28分钟的训练,这反过来又会产生长期的影响,延长几个星期。考虑到某?…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

孔拉德·洛伦茨大学基金会为这项研究提供了财政支持,赠款号9IN15151。作者要感谢康拉德·洛伦茨大学通信系在录制和编辑视频方面的帮助,特别是娜塔莉亚·里维拉和安德烈斯·塞拉诺(制作人)。此外,妮可·普法勒-萨多夫斯基和卢西亚·梅迪纳对手稿发表了评论,伊比利亚美洲大学学院院长约翰娜·巴雷罗对手稿发表了评论。作者没有利益冲突。

Materials

Acetic acid (ethanoic acid) https://pubchem.ncbi.nlm.nih.gov/compound/acetic_acid
Aversive Stimulation Current Package MED Associates Inc ENV-420 https://www.med-associates.com/product/aversive-stimulation-current-test-package/
Contextual test protocol.pro https://osf.io/4nkfq/?view_only=0640852a88544b239549462f9c21175b.
Cue test protocol.pro https://osf.io/4nkfq/?view_only=0640852a88544b239549462f9c21175b.
Curved Wall Insert MED Associates Inc VFC-008-CWI https://www.med-associates.com/product/curved-wall-insert/
Data processing.zip https://osf.io/4nkfq/?view_only=0640852a88544b239549462f9c21175b.
NIR/White Light Control Box MED Associates Inc NIR-100
Pellets BioServ F0165 http://www.bio-serv.com/pdf/F0165.pdf
Quick Change Floor/Pan Unit for Mouse MED Associates Inc ENV-005FPU-M https://www.med-associates.com/product/quick-change-floorpan-unit-for-mouse/
Small Tabletop Cabinet and Power Supply MED Associates Inc SG-6080D https://www.med-associates.com/product/small-tabletop-cabinet-and-power-supply-120v-60-hz/
Standalone Aversive Stimulator/Scrambler (115 V / 60 Hz) MED Associates Inc ENV-414S https://www.med-associates.com/product/standalone-aversive-stimulatorscrambler-115-v-ac-60-hz/
Standard Fear Conditioning Chamber MED Associates Inc VFC-008 https://www.med-associates.com/product/standard-fear-conditioning-chamber/
Training protocol VFC.pro https://osf.io/4nkfq/?view_only=0640852a88544b239549462f9c21175b.
Video Fear Conditioning Package for Rat MED Associates Inc MED-VFC-SCT-R https://www.med-associates.com/product/nir-video-fear-conditioning-system-for-rat/

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Acevedo-Triana, C., Rico, J. L., Ortega, L. A., Cardenas, M. A. N., Cardenas, F. P., Rojas, M. J., Forigua-Vargas, J. C., Cifuentes, J., Hurtado-Parrado, C. Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats. J. Vis. Exp. (162), e60537, doi:10.3791/60537 (2020).

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