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Scene fear experiment method
Cued and contextual fear conditioning, also added to the scene fear experiment, was based on Baproov's conditional reflection. It measures the ability of animal learning, the association between an aversive experience and environmental cues. Commonly used sound or light cues as a condition stimulus (CS), and aversive stimuli (such as a foot shock) as an unconditioned stimulus (US). Through this CS-US training, animals not only learn the connection between sound and electric shock, but also know that there is a kind of context between the electric shock and the surrounding environment. The former is auditory cue fear conditioning or cued fear conditioning, and the latter is contextual fear conditioning. Fear is one of the few emotional reactions that are rare in non-primate animals. After the animal experiences conditional fear, when it comes to contact with the conditional stimulus again, it will produce a series of physiological reactions, including autonomic nervous tension, increased secretion of stress hormones, and increased defensive behavior. Freezing is a kind of animal defense behavior. It means that the animal has no other activities except breathing. It is like a thunder in the night when the car is shining under the strong light of the car. It is considered a reliable indicator of the fear of rodents.
The brain structure involved in the conditional learning process is mainly amygdala and hippocampus. The former regulates fear, and the latter regulates learning cognition associated with fearful things. Therefore, this model is mainly used for the memory of the amygdala and hippocampus. Animals do not need water or fasting, and the experimental equipment is relatively simple and the training time is relatively short. Therefore, this model is widely used for behavioral phenotypic studies in transgenic mouse knockout mice.
First, the experimental equipment
Including conditional fear box, soundproof box and associated condition box, researched by Shanghai Xinsoft Information Technology Co., Ltd. hair. The conditional fear box includes a shock generator (which can generate 0.1 to 1.0 mA of various strength shocks) connected to the bottom grille floor, a sound generator (which can generate a wide-band click or low-frequency sound), and is associated with a computer. Union. The soundproof box is equipped with a light, a small fan (for ventilation and background sound) and a loudspeaker connected to the sound generator. There is a cat's eye on the door of the soundproof box for observing the animals in the operating box. Two operating boxes can be placed in one soundproof box so that two animals can be observed at the same time, but the two animals cannot see each other. The associated box is made of clear plexiglass with a movable grille floor and a screened top. To prevent the animal from sliding under the grille, the distance between the grill bars should be short enough. For mice and young rats, the associated condition box is usually 26 cm * 21 cm * 10 cm; the grille floor consists of stainless steel strips (1.5 mm in diameter, 0.5 cm in central-pitch mice, and 1.2 cm in young rats). Adult rats, usually associated box 29cm * 23.5cm * 19.5cm, a stainless steel grid floor comprising bars 16 (diameter 2.5cm, the center spacing of 1.25cm). In addition, there are voltmeters and sound intensity meters to detect the intensity of the stimulus.
First, the experimental method
Divided into two stages of training and testing
(1) Training phase (first day)
1. Debug the instrument to ensure that the grille floor is irritated by current, the loudspeaker is audible, and the current intensity and sound intensity (decibel) are recorded separately.
2. Rats were placed in a conditional fear box for 2 min (phase A) and the freezing time of the animals for the first 2 min was recorded as a baseline.
3. Then add the click sound, 80Db, 30s (B phase).
4. Immediately after the electric shock, 0.35mA, 2s (C phase), complete a round of training. If multiple rounds of training are required, repeat A, B, and C three-phase training.
5. The animal's stagnation time (s) was recorded throughout the training period to measure the unconditioned fear of the animal.
6. Remove the rat from the operating box. Thoroughly clean the box with 70% alcohol or 4% acetic acid for training in the next batch of animals.
(b) The test phase is performed on the second day after training, including context testing, altered context or pre-conditioning-stimulus (pre-CS) testing, and conditioning stimulus (conditioning stimulus, CS) Test. There was no electric shock stimulation during the test phase. There are also no auditory conditional stimuli in the association test and change association test, but the operation box used by each animal should be the same as the training phase.
1. Correlation test The animals are placed in a box and the computer automatically records the animal's stagnation behavior. If observed by hand, the observer (should not know the animal's treatment or genotype) observes the animal's behavior through the cat's eye (small window) on the door of the box with a static sound stopwatch and scores: stagnation is 1 point, activity (moving) It is 0 points (there is also only the stagnation time recorded). Observe every 10 seconds for a total of 5 minutes, so that 30 possible bouts of possible stagnation (ie, a maximum stagnation score of 30) can be recorded. The animal's contextually conditioned fear was measured using the lag time of the animals recorded in the same operating box recorded at this stage. This is the association test. After the observation, the animals were returned to the cage, and the operation box was cleaned for observation of the next animal.
2. Change correlation test and auditory conditional stimulation test The correlation test was performed 1h later. The operation is as follows:
(1) Transfer the computer to the required program.
(2) The operation box is modified as follows: 1 replace the grille floor in the associated condition box with a smooth plastic plate; 2 add a colored plastic plate to the diagonal of the box, so that the operation box of the rectangular parallelepiped becomes a triangular body; Olfactory cues. For example, a cup containing a drop of orange or lemon juice can be placed in the corner of the box, or a drop of juice can be applied directly to the wall. Then start changing the association and auditory conditional stimulation experiments.
(3) Place the animals in the modified associated box (starting with the first animal in the associated experiment).
(4) Start the preset experimental procedure and record the animal's stagnation and non-stagnation scores (1 and 0 points, respectively) every 10 seconds for a total of 3 minutes, ie 18 possible stagnation records (stability score of 18).
(5) Adding auditory condition stimulation for 30 seconds. Repeat the above record for 3 minutes.
(6) Return the animals to the cage, clean the operation box, and test the next animal.
Second, the results of calculation and analysis
Because different types of learning have different stagnation times, the most commonly used percentages are:
Association = (stagnation score in association test / 30) * 100%
Change association (before conditional stimulation) = (stagnation score in change association / 18) * 100%
Auditory suggestion (conditional stimulation) = (stagnation score in auditory conditional stimulation / 18) * 100%
Repeated measures one-way analysis of variance (ANOVA) was performed for each of the different stagnation test scores, from which the degree of conditional fear between association and change associations or auditory cues was compared and differences between groups were determined. In general, animals have more stagnation behavior in auditory cues than in association tests. In the change correlation test, since the associated condition box has been modified to be “recognizableâ€, the animal's association with the surrounding environment is almost non-existent during training, so the stagnation behavior should be the least. Associations and changes are very different, and the difference in stagnation scores is greater.
If the animal shows stagnation in the association test and auditory cues test on the next day, and does not show stagnation behavior in the change association test, it can be concluded that the animal's sensory and motor function are normal; remember to pair with the aversive stimulus the day before. A suggestive signal that appears; it can distinguish the suggestive signals that did not appear with the aversive stimulus the previous day, indicating that the animal has normal memory. Any of these tests are augmented or attenuated, suggesting that neuroanatomy, neurotransmitters, and genes that regulate the emotional portion of memory may change.
Third, matters needing attention
1. The response of an animal to an electric shock depends in part on the shock intensity and pain # of the unconditioned stimulus. When animals (especially genetically altered animals, such as transgenic or knockout mice) cannot learn to correlate with conditioned reflexes, they may be caused by insufficient shock intensity or high pain ##. Comparing the degree of conditional response with the shock intensity, the doubt about the electric shock intensity factor can be ruled out; the stimulation current intensity (0.1~0.7mA) and the reaction can be used as a “quantity-effect curve†to evaluate the animal's pain# or electric shock. Sensitivity. In addition, the grille floor can change the electrical stimulation intensity if it is contaminated with animal waste. Therefore, the grille floor should be kept clean and dry.
2. Because of the weakened or even lost hearing of older animals, the auditory suggestive may be unresponsive or unresponsive. In addition, some strains of mice, especially in early adulthood, are particularly responsive to high-frequency stimuli. Therefore, this model should be used with particular caution when measuring older animals or mice of certain strains and avoiding high frequency sonication.
3. In order for an animal to exhibit a stagnant behavior in a correlation test, it must be allowed to have an appropriate time in the operating box. Electrical stimulation should be avoided immediately by placing the animal in the box, as the animal does not have time to establish a connection between the shock and the environment (associated).
4. The observer must quickly and accurately determine whether the animal exhibits stagnation behavior, and should not stare at the animal during the entire 10s observation period to prevent the animal from moving in the 10s interval and causing the score to be inaccurate. Different observers use the same method to score, and the correlation coefficient between the two observers should be above 0.98. Similarly, when scoring is performed using an automatic scoring system, the system should be adjusted so that the correlation coefficient between the two is between 0.98 and 1 compared with the manual method scoring.
5. In this experiment, the association learning is tested first, and then the test conditional response is suggested. There is no auditory suggestive stimulus in the association test, and therefore, the animal's response to the auditory stimulus at the suggestion conditional test may be extinct. In theory, this auditory suggestive response may be diminished. In order to eliminate this experimental bias, half of the animals were tested for auditory suggestive responses, and the associated conditional responses were examined one hour later.
6. The experiment was conducted in two days. Training and testing should be conducted at the same time every day. The test (stagnation score) takes much longer than the training, so the same batch of trained animals should not be too much.
7. Both suggestive and associated fears can show developmental differences and genetic differences. For example, rats aged 18 days have auditory conditional fears, but there are few associated conditional fears; and by 23 days, both association and auditory suggestive conditional fears. Some strains of mice (such as DBA/2 mice) have only weak associated conditional fears, but hearing condition fears are good.
8. For some genotypes, association learning will improve if they are temporarily adapted to the associated conditions. This is generally thought to be related to the enhancement of hippocampal function. This adaptation usually takes place one day before the training and is very short (30~60s).
9. The implied conditional fear is regulated by the lateral amygdala, and the associated conditional fear is mediated by both the amygdala and the hippocampus. Destroying these brain regions can weaken or block the cues that are implied and associated with fear.