Morris water maze & eight-arm maze experimental method strategy

This experiment was established in 1981 by American scientist Richard GM Morris. Originally used to study the regulation of brain structure on learning and memory, and later gradually developed into the most commonly used model for evaluating animal learning and memory. The basis of this experiment is that rodents have a strong incentive to escape from the water in the water and escape the water environment in the quickest and most direct way. The process of learning to escape the water environment reflects the animal's ability to learn; spatial positioning according to the surrounding environment, purposefully swimming to a safe place (platform) in the water, reflecting the animal's spatial memory ability.

(1) Experimental equipment

The experiment was carried out with rats or mice. Rat water maze experimental equipment is manufactured by Shanghai Xinsoft Information Technology Co., Ltd. (), including a gray or black circular pool (diameter 160cm, height 55cm; mouse size reduced by half), a platform (diameter 10cm), a tracking camera and a computer connected to the camera (Figure 25-1). The water in the pool is 50cm deep and the water temperature is 22~24°C. The platform was placed 2 cm below the surface of the water (1 cm for mice). Add water to the water or milk to stir the water to prevent the animal from seeing the underwater platform. The camera is located 200cm above the center of the pool and records the animal's position, swimming distance and time (thus calculating the swimming speed) and swimming path. The pictures on the walls around the room with bright colors and different shapes are used as extra-maze cues.

(2) The experimental method is divided into three processes: acquisition training, exploration and alignment training.

1. The Acquisition phase theoretically divides the pool into four quadrants, with the platform placed in the center of one of the quadrants.

(1) Put the head of the animal (rat or mouse) into the water in the pool, and place it in one of the four starting positions of East, West, South and North. Record the time (s) at which the animal found the underwater platform. In the previous training sessions, if this time exceeds 60s, the animal is guided to the platform. Let the animal stay on the platform for 10s.

(2) Remove and dry the animal. Animals (especially rats) were baked under a 150 W incandescent lamp for 5 min, if necessary, and returned to the cage. Each animal was trained 4 times a day, with 15 to 20 minutes between training sessions and 5 days of continuous training.

2. Probe trial 1 On the second day after the last sexual training session, the platform was removed and the 60s exploration training began. The animals were placed in the water from the opposite side of the original platform quadrant. Record the time spent by the animal in the target quadrant (the quadrant where the platform was originally placed) and the number of times it entered the quadrant as a measure of spatial memory.

3. The working phase of the animal is determined by the random phase. On the second day after the exploratory training, the four-day match training was started. Place the platform in the opposite quadrant of the quadrant where the original platform is located, in the same way as acquired training. Train 4 times a day. Each time you find the platform's time and swimming distance and swimming speed. 4. The second day of the last match training was conducted on the probe trial 2 (probe trial 2). The method is similar to the above exploration training. Record the time spent by the animal in the target quadrant (the second time the platform is located) and the number of times the animal entered the area within 60 s.

(3) Precautions

1. In contrast to food-driven models (such as the radial arm maze), the biggest advantage of the water maze experiment is that animals have greater motivation to escape the water environment. And no need to fast, especially suitable for testing of older animals. Plus it is particularly sensitive to memory loss caused by aging, so water maze is most commonly used in the study of memory in older animals.

2. For example, in the case of mice, the diameter of the platform is also small (7.5 cm) except that the size of the swimming pool is about 50% of that of the rat. The experimental method is similar to that of the rat, but the training period is shorter. General acquired training for 16 days in 3 days (4 times on the first day, 6 times a day in the last two days; interval between the two trainings is 5~10min, the fourth day is the exploration training, and the fifth and sixth days are the alignment) Training, training six times a day, the seventh day for the second exploration training.

3. If observed with the naked eye, the test is always sitting in the same position during all tests, about 60 cm from the nearest edge of the pool.

4. Test at fixed time every day. The operation is gentle and avoids unnecessary stress irritation.

5. When compared with other similar experiments, it is necessary to note the effects of various factors such as animal sex, strain, pool size and water temperature on the experimental results. In addition, when swimming speed is used as an observation index, it is necessary to consider the weight, age, and skeletal muscle development of the animal, which may affect the swimming speed.

6. When conducting experiments with elderly animals, it should be confirmed that the swimming ability and visual acuity of the animals do not affect the behavioral operation due to the increase in age. The method is as follows: the platform is exposed to the water surface to enable the animal to see the platform. After the animals are placed in the pool, they can swim directly to the platform without any difficulty, indicating that the animal's swimming ability and vision are normal, and the experiment can be started.

7. Swimming is a large stress stimulus for animals that can cause neuroendocrine changes. These changes may interfere with the experimental results. In older animals, cardiovascular disease can be induced in severe cases, leading to stroke or even death. Therefore, if necessary, the animal can be placed in the pool multiple times or the swimming time can be appropriately extended to increase the animal's ability to adapt to swimming.

8. When mixing the water in the pool with milk or milk powder, it is necessary to change the water regularly to prevent the water from spoiling. If white paint is used for the same purpose, it must be ensured that the white paint is not toxic to the animal.

Second, the radial arm maze (eight arm maze)

The eight-arm maze (radial arm maze) experiment is also one of the most commonly used models for evaluating animal learning and memory, and was established by Olton et al in the mid-1970s. The basic basis is that the animals that control eating are driven by food to explore the arms of the maze; after a certain period of training, the animals can remember the spatial position of the food in the maze. The method can simultaneously measure the working memory and reference memory of the animal. Animals used include rats, mice and pigeons. This model is sensitive to brain damage and multiple drugs. The latter include ethanol, East ### (anticholinergic) and MK-801 (NMDA receptor antagonist).

(1) Experimental equipment

Most of the experiments used an eight-arm radiation labyrinth, but also a twelve-arm or twenty-four-arm maze. Here we introduce the eight-armed maze produced by Shanghai Xinsoft Information Technology Co., Ltd. Each arm has a length of 41.9 cm, a width of 11.4 cm, and a height of 10.1 cm; it has a transparent cover thereon and two opposite photocells on each side. The central octagonal area of ​​the labyrinth has a diameter of 27.4 cm; there is a transparent top cover. The entrance to the arms of the central area has a movable door for controlling the animal's access arms. The maze is connected to the computer. The photo tracking system can also be used instead of the photocell to record the animal's activity in the maze. There are some external hints (extramaze cues) in the room where the maze is placed. Animals can see these hints in the maze and use them for spatial localization. The experiment was carried out with rats or mice.

(2) Experimental methods

1. Animals were acclimated to the experimental environment for 1 week, weighed and fasted for 24 hours. Thereafter, after the end of the daily training, the normal foodstuffs (weighted, 16-20 g, and 2-3 g of the mice) were restricted to maintain the body weight of 80% to 85% of the normal fed rats.

2. On the second day, the arms and the center of the labyrinth were separated by food particles (4 to 5 each, about 3 to 4 mm in diameter). Then, place 4 animals in the middle of the maze at the same time (the door leading to each arm opens). Let it freely ingest and explore for 10 minutes.

3. On the third day, repeat the training for the next day. This process allows animals to become familiar with the maze environment without strong stress.

4. On the fourth day, the animals were individually trained: one grain was placed at each of the arms near the outer food box to allow the animals to freely feed. Remove the animals after eating or 10 min.

5. On the fifth day, put the food in the food box and repeat the training of the previous day, twice a day.

6. After the sixth day, randomly select 4 arms, one for each arm; each arm door is closed, and the animal is placed in the middle of the maze; after 30s, the arm door is opened, allowing the animal to move freely in the maze and take in the grain. Until the animal has eaten all 4 arms of the grain. If the food has not been eaten after 10 minutes, the experiment is terminated. Train twice a day, with an interval of more than 1h.

Record the following four indicators: 1 working memory errors, that is, in the same training, the animal re-enters the arm that has eaten the grain; 2 reference memory errors, that is, the animal has never been eaten The arm of the granule; the total number of arms into the arm; the test time, which is the time taken for the animal to eat all the granules. In addition, the computer can record the activity of the animal in the radial arm and in the central area, including the distance of movement and the time of exercise. If the working memory error of the 5 consecutive trainings is zero and the reference memory error is less than 1 time, the drug test or brain core structure damage test can be started. Generally, the solvent (such as physiological saline) is given first, then the memory-impairing drug (such as East MK-801, etc.) is given, and then the drug for enhancing memory is added, and the dose is from low to high.

(iii) Calculation and data analysis

The two indicators are used to evaluate the memory of the animal, that is, the frequency of working memory errors and the frequency of reference memory errors, which are equal to the working memory error or the reference memory error and the total number of input arms. ratio. These two indicators were used to evaluate working memory and reference memory, respectively. At the same time, the average exploration time, that is, the ratio of the test time to the total number of input arms, is an index for evaluating the general exercise activity. The frequency of working memory errors and/or the frequency of reference memory errors is significantly increased depending on the structure of the damaged brain region or the drug given to impair memory, and the memory-enhancing drug or treatment can reduce this frequency of error.

(four) matters needing attention

1. The mouse radial maze device and experimental procedure are similar to those of the rat, but the maze size should be 1/4~1/2 smaller than the rat maze, so as not to increase the difficulty of mouse behavior.

2. This experiment can also be used only to determine working memory. The only difference in the method is that the pellets are placed on all the radial arms instead of just 4 arms.

3. Chronic stress can have an impact on animal maze operations and there are gender differences. After chronic stress, the memory of male rats was weakened, which showed an increase in the frequency of memory errors. The spatial memory of male rats increased, showing a decrease in the frequency of errors.

4. Even under restricted eating conditions, rats should be given a weekly weight gain of 5g to prevent the animal from getting sick due to malnutrition. Remove animals with poor physical condition.

5. Any item around the maze can be used by animals as a sign of space. Removing or moving these markers may make the animal difficult to operate and reduce the accuracy of the labyrinth arm selection.

6. The number of different labyrinth arms may vary depending on the purpose of the experiment, including the 8, 16, 24, 32, 40, and 48 arm maze. The less the labyrinth arm requires the animal to remember that the lesser the arm has been explored, the simpler the animal's behavior will be. Increasing the number of arms increases the requirement for spatial memory in animals on the one hand, and introduces more and more necessary interference factors (such as the impact of past maze learning on current measured memory). Therefore, an 8-arm radiation labyrinth is often used to reduce unnecessary and excessive arm interference while reducing the time spent on training and testing.

7. The food used is usually small, chocolate-flavored (one of the animal's favorite flavors) or a sweet breakfast ring (10 mg each); liquid foods (such as chocolate milk or water) are also available. The latter is particularly useful for testing certain drugs that affect the swallowing of solid foods (such as East ###).

8. There are two main factors that affect animal maze operations: fear of the maze or observer and animal exploration habits and the driving of food known to be placed in the labyrinth arm. Excessive fear factors can prevent animal maze operations, leaving the animal to stay in a certain place in the maze without exploring. Lack of food cravings can produce similar results, increasing the stroking of animals and, if necessary, increasing the side walls of the labyrinth arms, helping to reduce animal fear. If the food is not driven enough, it can reduce the amount of food, but it must monitor both body weight and general physical condition. Usually, the body weight of rats should not be lower than 80% before fasting; for most rats, the body weight can be reduced by 15%.

9. Unlike the water maze, the radial arm maze is suitable for repeated testing or long-term memory testing. It is generally believed that working memory represents short-term memory and reference memory represents long-term memory.