[Animal Modeling]-Behavioral Evaluation Method of Alzheimer
动物造模,阿尔茨海默病
z-bo
2022-02-02
419
The benchmark behavior evaluation of the learning and memory abilities of rodent AD models was mainly tested in the Morris water maze experimental system. Below, we introduce the configuration, test procedures and evaluation methods of the water maze system.
One, Morris water maze purchase test
Morris water maze experiment system includes a circulating pool and an automatic image acquisition and processing system. The automatic image acquisition and processing system is mainly composed of a camera, a computer and an image monitor. The monitoring device is activated when the animal enters the water. Record the animal track after the experiment, and automatically analyze and report related parameters.
Morris Water Maze
Morris Water Maze is a round iron bucket with a circular platform with a diameter of 100 cm, a height of 50-60 cm, a depth of 30-40 cm, and a diameter of 9 cm. It is hidden under water at a depth of 2 cm. There must be no signs in the pool. Add an appropriate amount of fresh milk or milk powder to the water to make it look opaque and milky white. On the top of the bucket, set the four marking points east, south, west and north to the same distance as the animal's entry point. Use the projection points of these four entry points on the surface and bottom of the bucket to make the curved surface equal to the bucket. It is divided into four quadrants. The platform can be arbitrarily centered in a specific quadrant according to experimental requirements. Use a suitable thermostat device to keep the water temperature at 23-25°C. The labyrinth pool must be equipped with proper water injection and drainage facilities. After you find the pool, do not change the pool easily, especially when you are doing the same water maze test. The automatic water maze image acquisition and processing system automatically collects the animal's entry into the water level, swimming speed, time required to find the target, running trajectory and search strategy, and other parameters, and collects various collected data. You can perform statistics and analysis on it. Laboratories with limited equipment can still use the manual recording method still used in many laboratories in Western Europe.
1. Experimental methods and procedures The experimental training phase lasts for 3 days. We train four times a day. During the training process, the mouse is placed in the pool from the four entry points facing the pool wall, and the time (in seconds) required for the mouse to enter the water and find a hidden platform standing in the water is expressed in seconds. . Recorded as the incubation period. After the rat finds the platform, place it on the platform for 10 seconds. If the rat cannot find the platform 60 seconds after entering the water, gently drag it from the water onto the platform and stay for 10 seconds before proceeding to the next exercise. In a training session, each rat enters from four water inlet points, and the training interval is 30 seconds.
2. Experimental project
(1) Location navigation: used to measure the ability of rats to learn and remember the water maze. The experiment involves observing and recording the route map and the time it takes for the rat to find and climb onto the platform. That is, the incubation period is recorded.
(2) Spatialprobetest: used to measure the ability of rats to retain the memory of the spatial location of the platform after learning to find the platform. After the positioning and navigation experiment is completed, the platform is removed and placed at the same entrance in the water to measure the time to reach the original platform for the first time and the number of passes through the original platform. (3) Spatial distinguishability experiment: a dual-platform experiment used to measure the spatial distinguishability of rats. We have set up two visible platforms in the water maze, they look the same (except for the "visual difference"), but the location is different (spatial difference). One is unsinkable (safety platform) and the other is unsinkable (pseudo-safety platform). Record the number of correct responses in the rat, that is, the number of times the rat reached the safe platform without touching the pseudo-safe platform in a certain number of experiments. Obviously, the more "correct responses", the higher the spatial discrimination ability.
3. Matters needing attention
(1) When the mouse searches for the target, the environment of the laboratory and the location of the experimenter can be used as a reference. therefore. The location of laboratory equipment and experimenter should be relatively fixed.
(2) The water maze laboratory should be soft, even well-lit and quiet. (Two) Rating the Morris water maze experiment allows experimental animals to learn to find hidden platforms in the water, and to determine their memory function by analyzing the time and path to find the platform is an experimental method. The standard memory experiment is to study spatial learning . The Morris water maze is not only used to study brain function assessment related to spatial learning and memory, but also to assess the effects of related drugs, environment, genes, electromagnetic radiation, etc. on learning and memory. I will. This experiment is simple and reliable.
二. Y-shaped maze experiment
Establishing conditioned reflex is used to test animal behavior and advanced brain functions at home and abroad. The Y maze method can simultaneously observe the ability of animals to avoid conditions and spatial discrimination. It has a simple structure, low price, and is not affected by climate change, and can meet certain special needs. (Implanting electrodes into the head of rats to measure EEG or evoked brain evoked potentials is not suitable for water mazes). Therefore, it is widely used in China. For example, when exploring the etiology of dementia and observing the effects of drugs, it is necessary to determine whether the modeling is successful or whether the drugs are available according to the changes in learning and memory ability. Rats are mainly used as experimental subjects for the V-shaped electrical maze test.
(1) The structure and principle of the Y-shaped maze
The Y-shaped maze is also called a three-part radial maze box. It consists of arms I, II and III of equal length and three connecting areas. At the bottom of the box is a grid arm with a diameter of 0.2 cm, a length of 14 cm, and a spacing of 1 cm. The inner wall is covered with a layer of conductive copper. It is 45 cm long and has 15 watts of stimulating light on the top. The Y-shaped labyrinth operation panel has four buttons: voltage operation button, delay operation button and 4→II→III→0. When you press the I, II or III key respectively, the signal light of the corresponding robot arm will light up. At this time, the safe area (red light area) of the robotic arm is not energized, and the two unilluminated robotic arms and the joint area are energized, becoming a non-safe area (electric shock area). ); When the 0 button is pressed, the three arms are not energized, but the joint area is energized. At the beginning of the experiment, the rat was adapted to the starting area (the arm where the rat started) for 3.5 minutes, and then the position of the safety zone and shock zone was changed by specific rules or random switches, and the animal was placed in the shock zone. Escape and observe safe entry into the regional reaction space. At the beginning of training, under the influence of electric shock (passive avoidance response), the rat may be shocked into the non-safe area, escape from the starting area and eventually enter the safe area, resulting in false reaction indications. After many training sessions, if electrical stimulation is not started, the safe zone lights will light up and the rat will immediately escape to the safe zone. This is to form a discriminative reflection of light and dark (active avoidance response).
(2) Standards for correctness judgment
(1) After shocking the rat, it is the correct response to escape from the starting area to the safe area. Escape to the other arm without light will be recorded as a false reaction.
(2) It is a correct response for a mouse to run to a safe area within 10 seconds after the sole of the foot is powered on, otherwise it is a wrong response. The latter considers the time factor that can reflect the length of the rat's reaction time. In addition, both emphasize "direct" or "one-off". That is, if the mouse escapes to the starting area where it is located, it is classified as an error response.
(three) observation indicators
1. The number of training required to meet the standard (number of electric shocks, number of responses, number of tests). Zoology meets the social standard to represent the learning and memory performance of spatially resolved responses, depending on the number of tests previously required. The less the number of times required means the faster the learning speed or the higher the learning ability, the more the number of times required means the lower the learning ability and memory ability.
2. The number of days required to reach the standard (days) Record the number of days required for each rat to reach the academic standard. The longer it takes to reach the standard on average, the longer it takes to reach the academic standard. Secondly, learning and memory performance is reduced.
3. errornumber (EN) The number of error responses for all reactions completed on the experimental day. 4. Total reaction time (TRT) The reaction time (waiting time) of an animal refers to the time it takes for the rat to escape to the illuminated area for the first time after turning on the signal light. The total daily reaction time is the time required to complete all reactions (including correct and incorrect reactions) on the experimental day (all day). EN reflects the accuracy of the rat's response, and TRT reflects the length of the rat's response time, which can comprehensively evaluate the rat's learning ability and memory ability.
5. After the active escape frequency indicator is turned on but not powered on, the number of times the rat completes the escape response within 5 seconds accounts for the total number of responses. If the safe zone lights were on and electrical stimulation was not started, the rats fled to the safe zone. This indicates that the rat has formed a light and dark discriminative reflex (active avoidance response).
6. Correct response rate (correct operation rate) The ratio of correct response to the total number of tests.
7. The correct ratio for the entire group is the ratio of the number of animals learned to the total number of animals in the group. 8.3d learning rate The percentage of animals meeting the learning standard on the third day of training accounted for the total number of animals in this group.
(4) Eligibility (social) conditions
① The social condition is to answer 9 times or more 10 times (correct answer rate ≥90%). ② Correct electric shock 10 times in a row. ③According to the social standard, correct 18 times or more than 20 times in a row (the correct answer rate of actions ≥90%). (4) Conduct 20 training tests on each experimental day, and use EN ≤ 2 times and TRT ≤ 120 s as the criteria for judging rat learning. The above is the standard for each rat to reach the standard (learning), and the standard for the entire group of rats to reach the standard (learning) is the correct rate of the entire group (the number of learning animals/group). x The total number of animals within 100% is greater than 85%.
(5) Training method
1. Screening (pre-selection)
(1) Place the rat in the Y-maze for 3-5 minutes and apply appropriate power until it explores and enters all three arms. Startled to choose an active sensor that is sensitive to electric shock and can quickly escape and respond to the test. Remove mice that are too late or particularly sensitive.
(2) Pre-selected rats, they get the correct response twice in a row, the number of electric shocks ≤ 3, and is more sensitive to experimental electric shocks. (3) According to the above preliminary screening, the rats that do not meet the (learning) learning standards are eliminated through formal maze training. This is because some rats have learning and memory impairments. However, only the first two screens are difficult to find. Strictly speaking, the third screening method is the most detailed method, but this method can only test the memory retention (reproductive) ability of rats before experimental treatment (modeling or administration). All selected rats passed the learning test.
2. Learning test No matter which of the following methods is used for the learning test, the common point is that the rat is placed in the maze and used for 3-5 minutes before starting the formal experiment.
(1) Random rest method: The safe area changes in an irregular order, and the animals are trained to distinguish between light stimulation and safety in various directions. After the animal escapes to a safe area by electric shock, the light will continue to work for 10 to 15 seconds. After the light goes out, one test ends, and the animal's arm is used as the starting point for the next test. The interval between the two tests is 30 seconds, and the remaining time is one minute before the second test. Repeat this process until the test reaches a continuous 9/10 standard.
(2) Random and uninterrupted method: The safety zone is changed in an irregular order. First, energize the animal's arm and the other arm. After the animal escapes to a bright area after an electric shock, hold it for 10 or 30 seconds in one test, and then use the animal's arm as the starting point for the next test. test.
(3) Sequential method: Use the 3 arm end region as the starting area or safe area of the I-II-III-I arm in turn. The above specific methods can be further divided into two categories: ① Fixed training time per day, usually 20 times, continuous training for 3-4 days, animals generally can meet the learning standard. ②Using non-fixed training time, continuous training in segments. Rest for 1 minute every 10 training sessions, and then continue training until you learn. The training will be completed in one day. Usually, the first method uses more.
(6) Memory retention (reproduction) test
Study and test 24 hours, 48 hours, 7 days, 30 days, 9/10 standard using the same method. The correct response number is set to A, A/10 means memory reservation, the higher the value, the better the memory.
