An experimental method for evaluating the spatial learning and memory ability of tree shrews using the eight-arm maze
Through the experimental method of evaluation of spatial learning and memory ability of tree shrews in the eight-arm maze of tree shrews, the behavior of tree shrews was screened, training and evaluation, which solved the problem that tree shrews had difficulty in establishing spatial learning and memory models, and effectively evaluated the spatial learning and memory ability of tree shrews.
Patent Information
- Application Number
- CN202310278749.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-21
- Publication Date
- 2025-04-22
- Estimated Expiration
- 2043-03-21
AI Technical Summary
At present, no technology can effectively use tree shrews to determine the spatial learning and memory ability of eight-arm mazes, mainly because tree shrews are short in domestication time, timid, frightened, and difficult to establish a spatial learning and memory model.
An experimental method for evaluating the spatial learning and memory ability of the tree shrew's eight-arm maze was established, including screening of animals before the experiment, preparation of experimental equipment, experimental operation, and setting of mold formation indicators. This method gradually helps the tree shrew establish a spatial learning memory learning model by optimizing environment settings, training steps and reward settings.
Through this experimental method, a spatial learning and memory model of the eight-arm maze of tree shrews was successfully established. The number of times the tree shrews entered the rewardless arm during the training process was significantly reduced, and the number of times the repeated entry of the reward arm was also significantly reduced. The spatial learning and memory ability of the tree shrews was effectively evaluated.
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Abstract
Description
Technical Field
[0001] The invention relates to the determination of spatial learning and memory ability of experimental tree shrews, in particular to an experimental method for evaluating the spatial learning and memory ability of tree shrews in an eight-arm maze. Background Art
[0002] The eight-arm maze is a maze model for studying animal spatial memory. It consists of eight arms that radiate from a central platform, so it is also called a radial maze. In the experiment, several arms are selected manually and food is placed at the end as a reward. Driven by the food, hungry animals will explore each arm of the maze. After a certain period of training, the animals can remember the specific spatial position of the food arm in the maze and go to the food arm first. According to the animal's feeding strategy, this method can simultaneously measure the animal's working memory and reference memory. The eight-arm maze is an important means of quantitatively measuring changes in animal behavior. It is mainly used for research on animal learning, memory, spatial cognition, foraging strategies, and other aspects.
[0003] Tree shrews are potentially ideal model animals for studying spatial cognition and memory because of their closer relationship to primates, diurnal nature, and excellent vision. Although the methods for measuring the spatial learning and memory abilities of rats and mice using the eight-arm maze are relatively mature, tree shrews are quite different from rats and mice. Currently, no technicians have used tree shrews to conduct eight-arm maze experiments. Therefore, establishing a method for evaluating the spatial learning and memory abilities of tree shrews using the eight-arm maze is an issue that needs to be resolved in behavioral research. Summary of the invention
[0004] In view of the current demand for the development of an emerging experimental tree shrew model of spatial learning and memory in an eight-arm maze, and in view of the difficulties of currently artificially bred tree shrews such as short taming time, timidity, easy to be frightened, and difficulty in establishing a spatial learning and memory model, the present invention establishes an experimental method for evaluating the spatial learning and memory ability of tree shrews in an eight-arm maze, which includes four steps: screening of animals before the experiment, preparation of experimental equipment, experimental operation, and modeling indicators.
[0005] S01: Screening of animals before experiment
[0006] The candidate tree shrews are transferred to the maze. Within one week, if the candidate tree shrews have basically no abnormal behaviors caused by obvious anxiety or fear, they can be used as formal experimental tree shrews and enter the formal training stage.
[0007] S02: Experimental equipment preparation
[0008] S0201: Environment settings during the experiment period
[0009] The environment of the eight-arm maze room is maintained at a temperature of 22℃-24℃ and a humidity of 50%-70%. Reminder cards of different shapes and colors are set around the maze. During the experimental period, there are no obvious shadows in the seven experimental arms and the lighting is uniform. The furnishings, light source and other environmental conditions in the room remain unchanged, and the environment must be kept quiet during the experimental period.
[0010] S0202: Pre-experimental arrangements
[0011] Before the experiment, bread crumbs were placed outside the long hole at the end of the arm, 7 white food boxes were placed at the end of the experimental arm, and 3 arms were randomly selected and food was placed in the food boxes at their ends.
[0012] S03: Experimental Operation
[0013] S0301: Preparation before experiment
[0014] ① Before each experiment, weigh and record the weight of each tree shrew. Combined with the weight changes and the state and performance of the tree shrew during behavioral training, determine the restricted feeding amount during training and testing.
[0015] S0302: Spatial learning and memory experiment
[0016] ① The order of experimental tree shrews was randomly generated every day. The experimental tree shrews were transferred to the end of the starting arm through the transparent nest box, and the tree shrews entered the starting arm and were given a 1-min cooling-off period. After the cooling-off period, the gate of the starting arm was opened and closed after the tree shrews entered the central platform. At the same time, the computer was operated to start recording their trajectory.
[0017] ② After the experimental tree shrew enters the maze, the recording stops until it finishes eating the food in the three food arms or reaches the cut-off time. The tree shrew is induced to enter the starting arm and given a bread reward. After a 1-minute rest, the next experiment is conducted. When the tree shrew's behavior matures, each can be tested 20 times a day, and the experimental steps are the same as above.
[0018] S0303: Precautions during the experiment
[0019] ① After each experiment, the remaining marks of animals in the maze must be cleaned up. After each animal is finished, the maze and transparent nest box must be thoroughly cleaned to prevent odor interference to the tree shrews to be tested later.
[0020] ② After each tree shrew is trained on the same day, it is given limited dog food. The specific food restriction must meet the conditions of ensuring the training effect of the tree shrew on the same day while maintaining a body weight of no less than 80% of the body weight before the experiment and not decreasing too quickly.
[0021] ③. Initial stage of the experiment: Spread the cakes evenly in various areas of the maze to induce the tree shrews to fully explore the maze and familiarize themselves with the environment as quickly as possible. When the tree shrews can explore all areas of the maze independently and have fewer behaviors caused by emotions such as tension, anxiety, and inattention, they can enter the formal training stage.
[0022] S04: Modeling index
[0023] During the initial training of the eight-arm maze, food was placed in the food box of each experimental arm. If the tree shrews were able to eat the food in the seven arms within 5 minutes in a total of three experiments, and there was no abnormal behavior caused by obvious anxiety, the data from the next day would be included in the formal experiment.
[0024] In the formal reference memory experiment stage, the tree shrews did not make working memory errors and reference memory errors for 5 consecutive times in the valid trials of the day's training, which was used as the standard for judging that the experimental tree shrews had established a spatial learning memory learning model in the eight-arm maze experiment.
[0025] The total number of arm entries, working memory error rate, and reference memory error rate of each tree shrew in each experiment were calculated based on the trajectory of each tree shrew during the formal training phase. After excluding the affected trials, the effective training number was guaranteed to be about 20 for each tree shrew based on its status on that day.
[0026] Among them, the working memory error rate was used to evaluate the short-term spatial memory ability of tree shrews, that is, whether they could remember the food arm they had just entered, that is, whether the food arm was entered only once, and repeated entry into the food arm was defined as a working memory error;
[0027] Working memory error rate = number of working memory errors ÷ total number of arm entries.
[0028] The long-term spatial memory ability of tree shrews was evaluated by the reference memory error rate: that is, the number of times the animal entered the arm without food, and entering the arm without food was defined as a reference memory error;
[0029] Reference memory error rate = number of reference memory errors in the arms ÷ number of experimental arms.
[0030] Furthermore, in the step S0302, after all experiments on the experimental tree shrew are completed on that day, the tree shrew is transferred from the starting arm to the permanent nest box using a transparent nest box.
[0031] Furthermore, in the food restriction operation in step S0303, because tree shrews have large individual differences in physiology and personality, the specific degree of food restriction needs to be adjusted multiple times according to their health status and training effect to achieve the optimal level.
[0032] The present invention discloses the specific steps of all the features in establishing the above-mentioned eight-arm maze spatial learning and memory model of tree shrews. The method comprises the following steps: screening tree shrews before the experiment; optimizing the settings of various parameters such as ambient temperature, humidity, room interior, maze environment, etc. in the preparation of experimental equipment; training tree shrews in the experimental operation steps; the training steps, number of times, time, frequency, and reward settings are repeatedly demonstrated through multiple experiments; and finally, the study finds that the eight-arm maze experiment can be used as a behavioral paradigm for studying spatial learning and memory of tree shrews.
[0033] Through the test method for evaluating the spatial learning and memory ability of tree shrews in the eight-arm maze provided by the present invention, the eight-arm maze spatial memory tree shrew model was successfully established. In the formal training of the eight-arm maze, the test method provided by the present invention significantly reduced the number of times the tree shrew entered the non-reward arm (reference memory error) with the increase of the number of training days; the number of repeated entries into the reward arm (working memory error) was significantly reduced; the average number of days for the 12 tree shrews experimented with the test method provided by the present invention to reach the modeling standard was basically consistent with the number of days entering the plateau period, and the modeling standard was reasonably formulated. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 This is the reference memory error rate decline curve of tree shrews after training (a total of 12 tree shrews);
[0035] Figure 2 This is the curve showing the decrease in working memory error rate after tree shrew training (12 tree shrews in total). DETAILED DESCRIPTION
[0036] S01: Screening of animals before experiment
[0037] Fifteen experimental tree shrews were initially selected and gently transferred to the maze. In the early stage, due to their nervousness, fear and other emotions, they would circle on the central platform, remain motionless, and only try to enter the arm briefly, and were unwilling to enter the middle and end of the arm. They could be driven away artificially in a moderate manner. Within one week, if the candidate tree shrews had basically no abnormal behaviors caused by obvious anxiety and fear, they could be used as formal experimental tree shrews. Twelve formal experimental tree shrews were selected to enter the formal training stage.
[0038] S02: Experimental equipment preparation
[0039] S0201: Environment settings during the experiment period
[0040] The environment of the eight-arm maze room was maintained at a temperature of 22℃-24℃ and a humidity of 50%-70%. Reminder cards of different shapes and colors were set around the maze. The lighting needed to be adjusted before starting the experiment to ensure that there were no obvious shadows in the seven experimental arms and that the lighting was uniform to avoid the influence of light differences on the preferences of tree shrews. The furnishings, light sources and other environmental conditions in the room remained unchanged throughout the experimental period, and the environment needed to be kept quiet during the experiment to reduce interference with the animals.
[0041] S0202: Pre-experimental arrangements
[0042] Before the experiment, bread crumbs were placed outside the long hole at the end of the arm to evenly spread the smell of food throughout the maze. Seven white food boxes were placed at the end of the experimental arm, and three arms were randomly selected to place food in the food boxes at their ends. After each tree shrew training was completed, the maze, food boxes and transparent nest boxes must be thoroughly cleaned.
[0043] S03: Experimental Operation
[0044] S0301: Preparation before the experiment and precautions during the experiment
[0045] ① Before each experiment, weigh and record the weight of each tree shrew. Combined with the weight changes and the state and performance of the tree shrew during behavioral training, determine the restricted feeding amount during training and testing.
[0046] S0302: Spatial learning and memory experiment
[0047] ① The order of experimental tree shrews was randomly generated every day. The tree shrews to be trained were transferred to the end of the starting arm through the transparent nest box. The nest box door was opened to allow the tree shrews to enter the starting arm. A 1-min cooling-off period was given. After the cooling-off period, the gate of the starting arm was opened and closed after the tree shrews entered the central platform. At the same time, the computer was operated to start recording their trajectory.
[0048] ② After the tree shrew enters the maze, stop recording until it finishes eating the food in the three food arms or 3 minutes (1st to 3rd day of formal training) / 2 minutes (4th to 7th day of formal training), induce the tree shrew to enter the starting arm, give bread rewards after entering, and rest for 1 minute before the next experiment. When the tree shrew's behavior matures, each can be tested about 20 times a day, and the experimental steps are the same as above. After all the experiments on the tree shrew are completed on the day, use a transparent nest box to transfer it from the starting arm to the permanent nest box.
[0049] S0303: Precautions during the experiment
[0050] ① After each experiment, the remaining marks of animals in the eight-arm maze must be cleaned; after each animal is finished, the maze and transparent nest box must be thoroughly cleaned to prevent odor interference to the tree shrews to be tested later.
[0051] ② After each tree shrew is trained on the same day, it is given limited dog food. The specific food restriction must meet the conditions of maintaining the tree shrew's body weight at no less than 80% of the body weight before the experiment and not decreasing too quickly to ensure the training effect of the tree shrew on the same day. Because tree shrews have large individual differences in physiology and personality, the specific degree of food restriction needs to be adjusted multiple times according to their health status and training effect to achieve the best result.
[0052] ③. At the beginning of the experiment, spread the cakes evenly in various areas of the maze to induce the tree shrews to fully explore the maze and familiarize themselves with the environment as soon as possible. When the tree shrews can explore all areas of the maze independently and have fewer behaviors caused by emotions such as tension, anxiety, and inattention, they can enter the formal training stage.
[0053] ④. During the formal training phase, three food arms were randomly selected and fixed, and a small piece of cake was placed in the food box at the end of the three selected arms. The animals were allowed to explore and forage freely, and their trajectory was recorded by a computer until they finished eating the food in the three food arms or the deadline was reached and recording stopped.
[0054] S04: Modeling index
[0055] During the initial training of the eight-arm maze, food was placed in the food box of each experimental arm. If the tree shrews were able to eat the food in the seven arms within 5 minutes in a total of three experiments, and there was no abnormal behavior caused by obvious anxiety, the data from the next day would be included in the formal experiment.
[0056] In the formal reference memory experiment, the tree shrews did not make working memory errors and reference memory errors for 5 consecutive times in the effective trials of the day's training, which was used as the standard for judging that the experimental tree shrews had established a spatial learning memory learning model in the eight-arm maze experiment. The total number of arm entries, working memory error rate, and reference memory error rate of each tree shrew in each experiment were calculated based on the trajectory of each tree shrew in the formal training phase. After excluding the affected trials, the effective training times for each tree shrew were guaranteed to be about 20 times based on its status on the day.
[0057] Among them, the working memory error rate was used to evaluate the short-term spatial memory ability of tree shrews, that is, whether they could remember the food arm they had just entered, that is, whether the food arm was entered only once, and repeated entry into the food arm was defined as a working memory error;
[0058] Working memory error rate = number of working memory errors ÷ total number of arm entries.
[0059] The long-term spatial memory ability of tree shrews was evaluated by the reference memory error rate: that is, the number of times the animal entered the arm without food, and entering the arm without food was defined as a reference memory error;
[0060] Reference memory error rate = number of reference memory errors in the arms ÷ number of experimental arms.
[0061] The trend of search strategy changes was used as a reference indicator. The search strategy gradually changed from blind random search to accurate target search. Random search was characterized by almost no thinking process before entering the arm, which often occurred in the early stage of training when the animals were nervous or over-restricted, or when the animals were too hungry. Target search was characterized by a short thinking process before entering the arm, and if the choice was wrong, it could be adjusted during the initial entry into the arm, and no longer go deep, but turn to explore other arms.
[0062] Through the experimental method for evaluating the spatial learning and memory ability of tree shrews in an eight-arm maze provided by the present invention, an eight-arm maze spatial memory tree shrew model was successfully established. The experimental results show that Figure 1As shown in the figure, during the 1st to 10th days of formal training in the eight-arm maze, compared with the first day, as the number of training days increased, the number of times the tree shrews entered the non-reward arm (reference memory error) decreased significantly (F(11,9)=55.983, P=6.757E-35), among which the reference memory error rate on the 1st to 7th days decreased significantly (F(11,6)=34.468, P=1.599E-18), and there was no significant difference in the downward trend of the reference memory error rate on the 7th to 10th days; Figure 2 As shown in the figure, the number of repeated entries into the rewarded arm (working memory error) during formal training was significantly reduced (F(11,9)=38.349, P=1.95E-28), among which the working memory error rate showed a significant decrease from day 1 to day 7 (F(11,6)=25.819, P=1.455E-15), and there was no significant difference in the downward trend of the working memory error rate from day 7 to day 10. In the figure, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001. Both the reference memory error rate and the working memory error rate entered the plateau period on day 7, and there was no significant decrease in the error rates of both types from day 7 to day 10; the average number of days for the 12 tree shrews to reach the modeling standard and the number of days to enter the plateau period were basically consistent, and the modeling standard was reasonably established.
Claims
1. An experimental method for evaluating the spatial learning and memory ability of tree shrews in an eight-arm maze, comprising four steps: screening of animals before the experiment, preparation of experimental equipment, experimental operation, and modeling indicators; S01: Screening of animals before experiment The candidate tree shrews are transferred to the maze and observed for a week. If the candidate tree shrews have no abnormal behaviors caused by obvious anxiety or fear, they can be used as formal experimental tree shrews and enter the formal training stage. S02: Experimental equipment preparation S0201: Environment settings during the experiment period The eight-arm maze room environment is maintained at a temperature of 22℃-24℃ and a humidity of 50%-70%. Reminder cards of different shapes and colors are set around the maze. During the experimental period, there is no obvious shadow in the seven experimental arms, and the lighting is uniform. The furnishings, light sources and other environmental conditions in the room remain unchanged, and the environment must be kept quiet during the experimental period. S0202: Pre-experimental arrangements Before the experiment, bread crumbs were placed outside the long hole at the end of the arm, 7 white food boxes were placed at the end of the experimental arm, and 3 arms were randomly selected and food was placed in the food boxes at their ends; S03: Experimental Operation S0301: Preparation before experiment ① Before each experiment, weigh and record the weight of each tree shrew, and determine the restricted feeding amount during training and testing based on the weight change and the state and performance of the tree shrew during behavioral training; S0302: Spatial learning and memory experiment ① The order of experimental tree shrews was randomly generated every day. The experimental tree shrews were transferred to the end of the starting arm through the transparent nest box, and the tree shrews entered the starting arm and were given a 1-minute cooling-off period. After the cooling-off period, the gate of the starting arm was opened and closed after the tree shrews entered the central platform. At the same time, the computer was operated to start recording their trajectory. ② After the experimental tree shrew enters the maze, the recording stops until it finishes eating the food in the three food arms or reaches the cut-off time. The tree shrew is induced to enter the starting arm and given a bread reward. The next experiment is carried out after a 1-minute rest. When the tree shrew's behavior matures, each can be tested 20 times a day, and the experimental steps are the same as above. S0303: Precautions during the experiment ① After each experiment, the remaining marks of animals in the maze must be cleaned up. After each animal is finished, the maze and transparent nest box must be thoroughly cleaned to prevent odor interference to the tree shrews to be tested later; ② After each tree shrew is trained on the same day, it is given limited dog food. The specific food restriction must meet the conditions of maintaining the tree shrew's body weight at no less than 80% of its pre-experimental body weight and not decreasing too quickly to ensure the training effect of the day; ③. Initial stage of the experiment: Spread the cakes evenly in various areas of the maze to induce the tree shrews to fully explore the maze and familiarize themselves with the environment as soon as possible. When the tree shrews can explore all areas of the maze independently and have fewer behaviors caused by emotions such as tension, anxiety, and inattention, they can enter the formal training stage. S04: Modeling index During the initial training of the eight-arm maze, food was placed in the food box of each experimental arm. If the tree shrews were able to eat the food in the seven arms within 5 min in a total of three experiments and had no abnormal behaviors caused by obvious anxiety, the data of the next day would be included in the formal experiment. In the formal reference memory experiment stage, the tree shrews did not make working memory errors and reference memory errors for 5 consecutive times in the effective trials of the day's training, which was used as the standard for judging that the experimental tree shrews had established a spatial learning memory learning model in the eight-arm maze experiment; the total number of arm entries, working memory error rate, and reference memory error rate of each experimental tree shrew were calculated based on the trajectory of each tree shrew in the formal training stage; each tree shrew was trained effectively for about 20 times based on its status on the day, after excluding the affected trials.
2. According to the experimental method for evaluating the spatial learning and memory ability of tree shrews in the eight-arm maze according to claim 1, in the step S0302, after all the experiments on the day of the experimental tree shrew are completed, the tree shrew is transferred from the starting arm to the permanent nest box using a transparent nest box.
3. According to the experimental method for evaluating the spatial learning and memory ability of tree shrews in the eight-arm maze according to claim 1, in the food restriction operation in step S0303, due to the large individual differences in the physiology and personality of tree shrews, the specific degree of food restriction needs to be adjusted multiple times according to their health status and training effect to achieve the optimal level.
Citation Information
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