Series article:

Morris Water Maze Experiment Guidelines (1)

From the reading biology forum:

Third, the hardware guidelines

1. Pool and platform: For the size of the pool, there is a problem of artificial selection. It seems that the standard should be unified. It is not easy. But there is a principle that the pool should not be too small and the platform should not be too big. Morris originally (1981) used a rat pool of 1.32 meters in diameter and 0.6 meters in height, and the depth of the pool was 0.40 meters; but later (1984) changed to a diameter of 2.14 meters and a height of 0.4 meters, and the depth of the pool was 0.25. Meter. For experimental pools in mice, the labyrinth diameters described in the literature vary widely, from as small as 0.6 meters to as large as 2.0 meters. In general, too small a pool increases the chance of a mouse climbing onto a platform and reduces the difficulty of the task. An oversized pool will prolong the swimming path of the mouse, consume too much physical strength, and reduce the chance of climbing the platform. Most of the labyrinth pools used in the domestic literature are mostly 1.6m or 1.8m in rats, while mice are generally 1m or 1.2m and 0.5m high. The platform is generally circular, and the diameter can be divided into 12 cm in rats and 6 cm in mice. The surface is rough and the height is generally 30 cm. The labyrinth pools used in foreign literature are mostly 1.8m or 2.0m in rats, 0.6m in height and 10cm in diameter. The mice are generally 1m to 1.5m in height and 0.3m in height and 5cm in height. Can domestic size be unified? How to set the standard?

2. Water temperature: The temperature of the water requires constant temperature during the experiment. The temperature range of rats is generally 25 ± 1 °C, and the mice may be lower at 18-22 °C. It has been reported in the literature that the effect of water temperature on maze performance was observed in mice. The results showed that the mice in the 27~28°C group had the worst scores, while the scores in the 17~18°C group and the 21-22°C group did not differ. Therefore, we recommend the experiment of mouse water maze with 21~22 °C water. The water temperature has a great influence on the incubation period, especially in rats. If the temperature is too high, the rat gradually adapts to the labyrinth environment after a few swims. It will use the maze as a "bathtub". After being put in, it will float. If the water temperature is too low, the body temperature of the rat will drop rapidly, and the physical strength will be dissipated too much. The swimming ability of the rat can not be maintained for a long time, and there will be a sudden increase in the incubation period. Even some mice have a more obvious stress response, such as cramps and diarrhea. The water temperature is generally maintained at about 25 degrees. In addition, the temperature will cause the water temperature to deviate too much. The water temperature should be adjusted artificially. Therefore, it is required to have a heating rod or other heating device at the bottom of the pool that can constantly maintain the water temperature.

3. Light source: To ensure that there is no light and shadow on the water surface of the pool, it is mainly to avoid the reflection of light on the water surface, so as to avoid the shadow of the light left on the water surface being confused by the software collection system with the shadow of the mouse; if it is a white pool background, You can use two strips of divergent fluorescent light above the pool to reduce the back of the reflected light on the surface of the water; if it is a black pool background, place four 60-watt bulbs or 25 watts above the four quadrants of the pool. Tube lights, no need for power lamps. The height of the bulb should be high enough to be captured by the camera. In addition, you can also install a curtain around the pool (the color is generally black, light blue or white), which can block the shadow of light and maintain a certain degree of transparency. At the same time, it can also reduce the presence of people outside the pool or walk around, forming an indefinite spatial reference for the rats.

4. Space cues (reference objects): The pool should be placed in a larger room with more than two objects hanging on the wall as close-range visual reference objects, and a variety of long-distance visual reference objects in the room outside the pool. For example, geometric figures (squares, triangles, circles, etc.) hang from the wall outside the pool. The height is the range that must be seen when the big mouse is swimming, so it should be above its horizon, these geometric figures can be Made of 5cm to 15cm wide plastic sheets, the height must be higher than the platform, and can be hung around the pool with a 1m line; these geometric shapes are generally painted black, because rodents are more sensitive to black; these geometric figures are generally not less than 3 In addition, the laboratory environment is a very important factor. The location of the laboratory equipment, instruments, workbench, chairs, doors and windows, and lamps and the position where the experimenter is standing during the experimental operation may be seen by the big mouse. Will affect the experimental results. Because animals sometimes use the environment inherent in the laboratory as a reference for their search targets.

The Morris water maze experiment mainly measures spatial learning and memory. The main experimental contents include hidden platform test, space exploration test, reverse test and visual platform test. Here we mainly introduce the process guidelines for the hidden platform test and space exploration test which are mainly carried out in the literature.

Covert platform test:

When rodents swim for the first time, they generally do not find invisible platforms hidden under the water. If the animal fails to find the platform in the pool or fails to climb the platform within 60 to 90 seconds of entering the water, the experimenter can place the animal guide on the platform for 10 to 30 seconds, so that the animal feels standing on the platform. In this way, from the second time, the academic performance can be improved rapidly. After the animals climbed onto the platform, let the animals stand on the platform for 5 to 10 seconds. The rats were taken off the platform and rested for 30 to 60 seconds before the next training. Under normal circumstances, normal rats quickly learn to search for the exact position of the platform with the fastest and best trajectory after 4 to 6 experimental days of training.

At the beginning of each day, the rats were randomly placed into the water from one of the four quadrants (north, northwest, southeast, southwest), and the platform was placed in the southeast quadrant. Each mouse was swimming for 60 to 90s to find a hidden platform. If the platform is successfully found, the mouse can get a rest time of 10-30s on the platform; if the platform is not successfully found within 60~90s, the experimenter will manually place the mouse on the platform, and also give 10~30s. Break time. Sometimes the mouse may fall off the platform or jump into the water to continue swimming before arriving at intervals of 10 to 30 s. Once this happens, return the mouse to the station and re-time it so that the time interval reaches (10 to 30 s). This ensures that each mouse has equal time to observe and acquire spatial information after each experiment.

Space exploration test:

After 24 hours of the end of the concealed platform test, the platform was removed. Then select one of the same water points to put the rats into the water, record the swimming path of the rats within 60s or 120s, record the stay time of the rats in the original station quadrant and the number of crossing the original station, observe the spatial positioning ability of the rats, and in the space Exploring the changing laws in the process. Mark the original position of the platform with a circular ring on the computer screen, so that the number of times that the original station is located can be recorded.

Experimental process guidelines:

1. Experimental time: The general design days of the hidden platform test are 4 days or 6 days. If it is an old and weak, the number of days can be designed as 4 days. The training time is generally 60s or 90s, and it is also useful for 120s. However, it is generally used for 60s. Some people have done experiments. The difference in the number of times that rats can find hidden platforms between 60 and 120s is only about the total number of times they find the platform. 5%, which is statistically a "small probability event." In other words, if the rat can't find the hidden platform within 60s, it will be difficult to find it if it is extended to 120s. Moreover, 60s can also reduce the standard deviation of each group, which can save the experiment time. However, the problem is not so simple. The incubation period of the hidden platform test should be repeated. Since many studies directly include the maximum latency in statistics, it is clear that the difference in maximum latency will inevitably affect the statistical results. Therefore, it is statistically known how much different maximum latency has an impact on the results of the Morris water maze experiment. How to avoid the systematic errors caused by this problem remains to be further studied. Foreign literature reports that if the diameter of the pool is 2m, a single training time of 120s, a diameter of 1m to 1.5m, and a training time of 60s can be set. In view of this, we believe that the training time of 60s is more appropriate. If the time is too long, the physical exertion of the rat will be too large.

2. Number of experiments: In the hidden platform test, 4 times a day, each quadrant is used only once a day, and the rats must not be placed in the same quadrant continuously, because the rats may rely on position or other non-memory information. (such as turning to the right) to locate the station. Moreover, the order of the next quadrants should change. In the space exploration experiment, the rats were placed in the pool for 120s. This process needs to be repeated once, because in some cases, some abnormal variables are observed in the first experiment. It is possible that some mice lost their direction after changing the experimental conditions. . Calculating the average value of two consecutive experiments can reduce the deviation and provide a higher precision and better control of previous learning and memory. However, if the number of experiments is more than two times, it will result in a decrease in the activity time of the target quadrant, resulting in inaccurate spatial exploration results.

3. Experimental animals: Not all mouse strains are suitable for the Morris maze test. For example, inbred BALB/c mice cannot learn this task (the score does not improve with the increase of the number of days), and the 129/SvJ mice also have deviations in their academic performance. For example, mice with the 129/SvJ strain have difficulty in completing visual-based learning and memory tasks during aging due to age-related visual pathological changes. In C57BL/6 mice, due to severe alopecia, some mice may cause depression, and ulcerative dermatitis may eventually affect the swimming ability of mice and affect the experimental results; immersing animals in water may cause endocrine or other reactions. The effect of the latter, the interaction between the latter and brain damage or pharmacological operations is uncertain; due to too much physical exertion, the body temperature is also too much, some elderly and weak rats are more difficult to complete the task, and even drowning death occurs. In addition, the difference in performance between individuals is also huge. For example, some normal rats are placed in the water and then watch the circle to determine the direction and then swim directly to the stage, indicating that his memory is better. This phenomenon is very important. Rats swim faster, so the incubation period is greater than the dementia group. So you can use other methods to sift the rat first, then run the water maze.

4. Experimental personnel: The experimenter should establish good feelings with the test animals and familiarize the animals with the physical information of the experimenter, such as smell and appearance. The action of capturing animals should be gentle and do not irritate animals. Taboo to grab the tail of the mouse, because it will greatly stimulate the animal. Some people suggested intimate contact with rats two or three days before the start of the experiment. In fact, the sense of smell of the mouse is very sensitive. If the experimenter is a woman, the smell of the perfume on the body will change the mood of the mouse.