Introduction
The conditioned fear experiment is a classic experimental paradigm used to study the association, Xi and memory of specific stimuli and unpleasant experiences in animals (e.g., mice). Such experiments typically involve three key elements: conditioned stimuli (CS, conditioned stimulus), unconditioned stimulus (US, unconditioned stimulus or unpleasant experience), and conditioned response (CR, conditioned response or fear response).
Conditioned stimulus (CS) presentation: In the experiment, a neutral stimulus (CS), such as sound or light, is presented to mice at the same time as an unpleasant stimulus (US). This allowed the mice to begin to associate otherwise neutral CS with unpleasant experiences.
Guidance of unconditioned stimuli (US): Unconditioned stimuli are typically stimuli that cause discomfort or fear in mice, such as electric shocks or shocks.
Determination of Conditioned Response (CR): After experiencing multiple associations of CS and US, mice begin to exhibit a conditioned response to CS, i.e., mice still exhibit fearful and fearful behaviors in the presence of only CS and no US.
Regression phase: When CS is no longer present with US, the conditioned response (CR) of mice gradually weakens, and this stage is called the regression phase. In this process, mice learn to adapt to new environments and conditions and no longer exhibit a strong fear response to CS.
1. Principle analysis:
Fear conditioning: This is the core of the experiment, which refers to the repeated association between CS and US, resulting in CS having the ability to elicit fear responses. This is usually measured by recording behavioral or physiological indicators such as electrical responses in mice.
Extinction stage: Extinction does not mean that the association between CS and US is completely erased, but a new memory is formed, which competitively inhibits the association between CS and US. This is reflected in the fact that mice gradually show a weakened fear response to CS.
Memory storage: In the mouse brain, areas such as the basolateral amygdala (BLA), the anterior part of the prefrontal cortex (PL), and the prefrontal cortex (IL) of the lower shell of the brain play a key role. These areas play different but critical roles in conditioned phobia Xi and extinguishing memory.
Hippocampal input: Hippocampal input provides contextual information that is conducive to the expression of extinguished memories during extinguishing memory recall, influencing the tendency of mice to express conditioned responses in different environments.
Second, the experimental process
2.1 Pre-training of the fear conditioning chamber
Equipment placement: Place the experimental setup in a soundproof or quiet room to reduce external noise during the test;Animals should be kept in separate rooms. This prevents animals that are not participating in the test from hearing sound cues or the cries of the test animals.
Animal calm: The animal should remain calm before the test. If the animal's position is changed prior to the test, a long acclimatization period (60-90 min) should be allowed.
2.2 Delayed cues and situational fear conditionalization
Day 1 - Conditional session.
Acclimatization period: Mice are given 2 min of acclimatization in a conditioned chamber before starting the assay in order to explore their surroundings.
Tone cues (CS) :* tones lasting 15 to 30 seconds (auditory cues). If only the fear of entering the environment is conditionalized, then there is no **tone (cs) in the conditionalization process.
Mild Foot Shock (US): A mild foot shock (US) is applied within the last 2 seconds of the tone (CS)**, ending at the same time as the tone (1-2 seconds).
Interval: After the shock (US), set an interval of 60 to 210 seconds and then perform a second identical trial.
Removal of the animal: After the last shock (US), the mouse should be removed from the conditionalized chamber within 30 to 60 sec to reduce its stress and avoid affecting the memory consolidation process.
Day 2 - Contextual and cue test.
Contextual conditioning: Try to mimic the conditions of the training session (including smell, lighting, test time, etc.). The test chamber should be cleaned as it was during training, with no Tone Cues (CS).
Observe animal behavior: Place the mouse indoors for 3 to 5 min and observe if it exhibits a stiff response.
End test: Immediately after the situational test is completed, move the mouse back to its original cage.
Prompt Conditionalized Test Preparation: Performed approximately 30 minutes after the test interval. If the cue test is carried out in the same conditioned room, the size, lighting intensity, background, floor texture and smell of the room should be changed, and thoroughly cleaned.
Cue conditionalized trial: Let the mice acclimatize indoors for 3 min, and then ** tone (CS) of the same intensity as the training phase for 3 min. After the tone is over, observe for another 1 min and then move the animal out.
2.3. Tracking cues and conditionalization of situational fears
Tracking conditionalized training: Similar to delayed conditioning, but with the difference that the plantar shock (US) is given after the end of the tone (CS) (tracking interval, 2-60 seconds).
More trials: In order for mice to associate conditioned stimuli (CS) with unconditioned stimuli (US), more trials (e.g., 5) are required for tracking conditioning. Varying intervals should be used between trials to reduce expectations for new trials.
Day 2 Testing: Stiff Response Test for Situations and Cues is tested in the same way as Delay Conditionalization.
2.4 Control test
Reverse conditionalization: Use reverse conditionalization as a means of behavior control to verify that the Xi truly reflects latency or tracking conditionalization.
Reverse conditioning (also known as reverse conditioning) is a control method used in classical conditioning experiments to verify that the Xi truly reflects the formation of conditioned reflexes. This method differs from traditional conditionalized experiments in the order in which stimuli are presented.
In reverse conditioning, the unconditioned stimulus (US) precedes the conditioned stimulus (CS). For example, the animal is first subjected to a mild electric shock to the soles of the feet, followed by a **tone. This change in order makes it difficult for animals to make the connection between conditioned and unconditioned stimuli, as conditioned stimuli are no longer reliable signals for unconditioned stimuli.
Steps to reverse conditionalization:
Unconditioned stimulus presentation: The animal is first administered an unconditioned stimulus (US), such as a mild plantar shock.
Conditioned stimulus presentation: Conditioned stimuli (CS) immediately after the unconditioned stimulus, such as pitch.
Repeat experiments: Repeat this pairing process at specific intervals to ensure that the animals receive enough reverse conditionalized training.
Observe behavioral responses: Observe the animal's behavioral responses during and after training, paying particular attention to the presence of a stiff response.
The core of reverse conditionalization is that the conditioned stimulus (CS) appears after the unconditioned stimulus (US), which makes the conditioned stimulus lose the ability to ** the unconditioned stimulus. With this method, researchers can verify the effectiveness of conditioned chemical Xi by assessing how well the animals Xi paired with conditioned and unconditioned stimuli.