Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

Conditioned Lick Suppression: Assessing Contextual, Cued, and Context-cue Compound Fear Responses Independently of Locomotor Activity in Mice

条件性舔抑制：评估独立于小鼠运动活动的情境、线索和情境线索复合恐惧反应

YB Youcef Bouchekioua
NN Naoya Nishitani
YO Yu Ohmura

发布: 2022年12月05日第12卷第23期 DOI: 10.21769/BioProtoc.4568 浏览次数: 1839

评审: Edgar Soria-GomezNanci WinkeAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Translational Psychiatry, featuring study using the protocol.

Feb 2022

Advertisement

“科研可重复性与创新”系列网络研讨会
Special Issue on Protocols for Organoid Research
Technologies for RNA Detection
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

利用新物体识别实验评估小鼠学习和记忆的昼夜节律依赖性方案

利用新物体识别实验评估小鼠学习和记忆的昼夜节律依赖性方案

Jordan Mar [...] Isabella Farhy-Tselnicker

2025年09月20日 2990 阅读

基于 rAAV-α-Syn 与 α-Syn 预成纤维共同构建的帕金森病一体化小鼠模型

基于 rAAV-α-Syn 与 α-Syn 预成纤维共同构建的帕金森病一体化小鼠模型

Santhosh Kumar Subramanya [...] Poonam Thakur

2025年12月05日 2106 阅读

利用光遗传学 LTP 操控和数学建模研究小鼠指导性信号的价值可塑性

利用光遗传学 LTP 操控和数学建模研究小鼠指导性信号的价值可塑性

Takashi Nagashima [...] Ayako M. Watabe

2026年06月05日 209 阅读

Abstract

Pavlovian fear conditioning is a widely used procedure to assess learning and memory processes that has also been extensively used as a model of post-traumatic stress disorder (PTSD). Freezing, the absence of movement except for respiratory-related movements, is commonly used as a measure of fear response in non-human animals. However, this measure of fear responses can be affected by a different baseline of locomotor activity between groups and/or conditions. Moreover, fear conditioning procedures are usually restricted to a single conditioned stimulus (e.g., a tone cue, the context, etc.) and thus do not depict the complexity of real-life situations where traumatic memories are composed of a complex set of stimuli associated with the same aversive event. To overcome this issue, we use a conditioned lick suppression paradigm where water-deprived mice are presented with a single conditioned stimulus (CS, a tone cue or the context) previously paired with an unconditioned stimulus (US, a foot shock) while consuming water. We use the ratio of number of licks before and during the CS presentation as a fear measure, thereby neutralizing the potential effect of locomotor activity in fear responses. We further implemented the conditioned lick suppression ratio to assess the effect of cue competition using a compound of contextual and tone cue conditioned stimuli that were extinguished separately. This paradigm should prove useful in assessing potential therapeutics and/or behavioral therapies in PTSD, while neutralizing potential confounding effects between locomotor activity and fear responses on one side, and by considering potential cue-competition effects on the other side.


Graphical abstract



Schematic representation of the compound context-cue condition lick suppression procedure. Illustration reproduced from Bouchekioua et al. (2022).


Keywords: Conditioned suppression (条件抑制) search Fear conditioning (恐惧条件反射) search Context (情境) search Cue (线索) search Locomotor activity (运动活动) search Rodents (啮齿动物) search Pavlovian conditioning (巴甫洛夫条件反射) search

Background

Pharmacological treatments for PTSD, such as the 5-HT2CR antagonist SB242084 (Browne et al., 2017), increase locomotor activity and could alternatively explain the reduction of fear responses when measuring the time spent in freezing. The potential locomotor activity confounding variable has thus to be neutralized before drawing any conclusion regarding the role of a treatment in fear responses.


Exposure therapies developed to treat PTSD consist of gradually confronting patients to stimuli that are identified as triggers of the symptoms; yet patients do not necessarily have conscious access to these triggers. Moreover, according to associative learning theories, the intensity of fear responses to stimuli can be affected in situations where multiple conditioned stimuli (CSs), previously paired with the same aversive event, interact (Kamin, 1969; Rescorla and Wagner, 1972; Mackintosh, 1974; Pearce and Hall, 1980). It has been shown that a reduction in fear responding to a cue is associated with sustained contextual fear responses when cue-specific and contextual information are combined during conditioning, but tested separately (Grillon et al., 2002; Baas et al., 2008; Baas, 2013). Underestimating cue-competition effects may thus lead to an incomplete evaluation of potential therapeutics.


To overcome these limitations and improve the screening efficiency of candidate drugs in fear conditioning, we propose a condition lick suppression procedure to assess contextual, cued, and context-cue compound fear responses.

Materials and Reagents

  1. Black opaque acrylic or plastic sheet (height: 18 cm; width: 6 cm; thickness: 2 mm)

  2. Triangular-shaped ceiling made with two transparent acrylic sheets (21.5 × 13 cm, 1 mm thick) (YSTIME, catalog number: B08NPMVJGQ)

  3. Transparent acrylic hinges (IVYTOHO, catalog number: B07SMSJJK2)

  4. 0.5 mm thick high-density white polyethylene plastic sheet (Polymersan, catalog number: 0714038308181)

  5. 40 × 40 × 40 mm urethan rubber block (Tokyu Hands, catalog number: 4989606010889)

  6. Solder wire (Editone, Adafruit, catalog number: SAC305)

  7. Hook-up wire spool set, 22AWG solid core, 10 × 25 ft (Adafruit, catalog number: 3174)

  8. Momentary push button switch NO NC AC 5 A/120 V (DIYhz, catalog number: B079D923V9)

  9. DB9 D-SUB RS232 Adapter 9 pin signals terminal breakout plastic cover 2 row (male with screw) (Daier, catalog number: B01C2V2E8BU)

  10. IR break beam sensors with premium wire header ends, 3 mm LEDs (Adafruit, catalog number: 2167)

  11. MPR121 12 key capacitive touch sensor breakout (Adafruit, catalog number: B0778WYR5D) [Alternative: Adafruit 12 × capacitive touch shield for Arduino – MPR121 (Adafruit, catalog number: 2024)]

  12. Adafruit LED Sequins [luminous intensity: minimum = 250 mcd and maximum = 350 mcd with IF (Forward Current) = 20 mA; luminous intensity measurement allowance is ± 10%], warm white (Adafruit, catalog number: 1758)

  13. Speaker 8Ω8W (Akizukidenshi, catalog number: F00805)

  14. Amazon Basics USB 2.0 printer cable – A-Male to B-Male cord (Amazon Basics, catalog number: B00NH11KIK)

  15. White cotton/polyester knit gloves (Setaria Viridis, catalog number: B08QHLCZ11)

  16. Double sided tape (3M, catalog number: 0888519024546)

  17. Alligator to Dupont wire (Oiyagai, catalog number: B07CXLMBY7)

  18. Adult mice (8-week-old to 18-week-old)

  19. 70% ethanol solution

Equipment

  1. Modular chamber (Lab-hacks, model: STCHK0001)

  2. Watering bottle with rubber stopper and water tube (CLEA Japan, catalog number: CL-0917)

  3. Sound attenuating cubicle for mouse (Med associates, model: MED-OFA-022)

  4. Standalone aversive stimulator/scrambler (115V/60Hz) (Med associates, model/catalog number: ENV-414S) [Alternative: Precision animal shocker (Coulbourne Instruments, Harvard Apparatus, catalog number: H13-15)]

  5. Arduino Mega2560 Rev3 (Arduino, catalog number: A000067)

  6. Raspberry Pi 3 model B+ [Raspberry Pi, catalog number: (Digi-Key)2648-RASPBERRYPI3MODELB+-ND]

  7. Digital Genuine Hakko with a standard T18-D16 tip (Hakko, catalog number: FX-888D)

Software

  1. Arduino IDE (Arduino, https://www.arduino.cc/en/software)

  2. Anaconda (Anaconda, https://www.anaconda.com/)

  3. Spyder IDE (Included in Anaconda, https://www.spyder-ide.org/)

  4. Python 3 (https://www.python.org/downloads/)

  5. Python libraries: pyserial 3.5 (https://pyserial.readthedocs.io/en/latest/pyserial.html); csv, time, and os modules are part of Python’s standard libraries

  6. Excel (Microsoft, https://www.microsoft.com/fr-ww/microsoft-365/excel)

  7. Prism 9 (GraphPad Software, https://www.graphpad.com/company/)

  8. SPSS 23.0 (IBM, https://www.ibm.com/analytics/spss-statistics-software)

  9. Arduino and Python scripts (https://github.com/YuYuB/Bio-Protocol-ConditionedLickSuppression)

Procedure

English
中文翻译

文章信息

版权信息

© 2022 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Bouchekioua, Y., Nishitani, N. and Ohmura, Y. (2022). Conditioned Lick Suppression: Assessing Contextual, Cued, and Context-cue Compound Fear Responses Independently of Locomotor Activity in Mice. Bio-protocol 12(23): e4568. DOI: 10.21769/BioProtoc.4568.

ris ris Download Citation in RIS Format

分类

神经科学 > 行为神经科学 > 学习和记忆

神经科学 > 神经系统疾病 > 动物模型

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件