A method for evaluating the spatial learning and memory ability of tree shrews using a cheese board maze

Abstract

A method for evaluating the spatial learning and memory ability of tree shrews using a cheese board maze, which includes three steps: experimental preparation, experimental training, and determination of modeling indicators. In experimental preparation, various parameters such as the cheese board maze device, experimental layout, and environmental settings are optimized and designed. In experimental training, training is carried out before and after hippocampal lesion. A behavioral paradigm for evaluating the spatial memory of tree shrews is successfully established, and the modeling criteria and indicators are reasonably formulated. The successful establishment of the spatial learning and memory tree shrew model of the present invention provides a behavioral experimental method applicable to tree shrews for subsequent neuroscience research such as constructing AD models, in vivo electrophysiological recordings, and optical recordings, and can also provide more powerful scientific data for researchers in the research of multiple disciplines and fields such as spatial coding, learning and memory, new drug development, screening, and evaluation.

Description

Technical Field

[0001] The invention relates to a method suitable for evaluating the spatial learning and memory ability of tree shrews in the field of life sciences, in particular to a method for evaluating the spatial learning and memory ability of tree shrews by using a cheese board maze. Background Art

[0002] Spatial learning and memory is one of the brain's advanced neurophysiological activities. It is a very complex neurophysiological activity process. At the same time, it is also a behavioral pattern that can evaluate the cognitive level of animals and humans. It is a cognitive process in which organisms form internal representations of the external environment. Spatial memory is divided into reference memory and working memory. Reference memory can be acquired through repeated training. For example, experimental animals search for food rewards in a maze based on reference memory. The hippocampus of experimental animals participates in learning, spatial memory, anxiety, stress and other processes, and performs spatial cognition and memory functions. Existing studies have shown that experimental rats with hippocampal damage cannot effectively combine their spatial positioning with past experience, which destroys the memory and retrieval of spatial cues in the short term. Therefore, the function of the hippocampal structure largely determines the animal's spatial learning and memory ability.

[0003] Tree shrews are small, semi-arboreal insectivorous mammals that live in mountain forests. They are agile and good at climbing. They mainly move, forage, play and rest on the ground. Tree shrews are close relatives of primates. They have the characteristics of short breeding cycles and low breeding costs. They have gradually been used in the study of related diseases such as infection, cancer, metabolism and immunity. In the field of neurocognitive research, tree shrews have the advantage of being close to non-human primates, so they are an ideal experimental animal. Spatial positioning is an important skill for tree shrews in the wild. They must obtain memory information from spatial clues and the environment in order to accurately locate their own position, the position of prey or enemies, and thus formulate the best attack or escape path.

[0004] In spatial learning and memory experiments, food rewards are often used to train experimental animals. Food rewards are placed in different predefined locations according to experimental requirements. When learning to distinguish the spatial locations of reward wells and non-reward wells, different numbers and locations of reward wells can be set to perform training of different task difficulties. This is a task that requires reliance on environmental clues to locate rewards. In summary, using the natural habit of tree shrews foraging on the ground and training them to test spatial position and orientation learning and memory by finding food reward wells is an effective method to evaluate the spatial learning and memory ability of tree shrews. However, due to the characteristics of tree shrews being timid, easily startled, and highly reactive to external stimuli, it is difficult to establish a cognitive behavioral paradigm for tree shrews, so there is currently no relevant experimental research. Summary of the invention

[0005] The present invention establishes a method for evaluating the spatial learning and memory ability of tree shrews using a cheese board maze, aiming to evaluate the spatial learning and memory ability of tree shrews and establish a paradigm for the cognitive ethology of tree shrews. In addition, in order to test the degree to which the execution of this maze task depends on the animal's spatial memory, a self-control experiment with the same training after hippocampal lesion of tree shrews is added. The spatial learning and memory experiment of the tree shrew cheese board maze includes three steps: experimental preparation, experimental training, and determination of the modeling index.

[0006] S01: Experimental preparation

[0007] S011: Prepare the cheese board maze device

[0008] The cheese board maze is a circular arena. The main board is a circular white polypropylene plate evenly distributed with 120 holes, and the distance between the centers of two adjacent holes is equal. The main board is divided into 4 sector plates for easy disassembly and cleaning. A bottom board is installed below the main board, and the main board and the bottom board form a blind hole maze containing 120 food wells;

[0009] There is an iron sheet cylindrical fence around the cheese board maze, and clue prompts of different shapes are pasted on the fence;

[0010] The top of the cheese board maze is covered with a nylon net to prevent tree shrews from escaping from the maze.

[0011] S012: Conduct experimental layout for the cheese board maze

[0012] Select any position against the wall on the side of the cheese board maze as the starting area of the maze, and place an acrylic transparent three-dimensional rest box. Each time a tree shrew starts training, it goes out from the three-dimensional rest box to look for rewards. Eating three food rewards within 2 minutes is considered to complete one training. Then, after using food to lure it back to the three-dimensional rest box, the experimenter places the food reward for the next training in the food well.

[0013] S013: Environmental setting during the experimental period

[0014] For the experiments before and after hippocampal lesion, the environmental temperature of the cheese board maze room is maintained at 20°C - 26°C, and the humidity is 40% - 70%. Hint cards of different shapes and colors are set on the inner wall and surrounding walls of the maze. The brightness of the maze is uniform during the experimental period, and the furnishings, light sources, and other environmental conditions in the room remain unchanged during the training period, and the environment is kept quiet.

[0015] S02: Experimental training

[0016] S021: Preparation before the experiment

[0017] Before each experiment every day, weigh and record the body weight of each tree shrew, and determine the limited food feeding amount during the experimental period in combination with the body weight change and the state and performance of the tree shrew during training.

[0018] S022: Training before hippocampal lesion

[0019] ④. Familiarize with the environment: Food is scattered on the surface and in the holes of the maze. When the tree shrew can independently forage for food in the well in the maze, its footprints are evenly distributed, and there are fewer behaviors caused by emotions such as nervousness, anxiety, and inattention, it can enter the next stage of training.

[0020] ⑤. Change the position of the food well: Conduct training for 3 days. Select any three wells to place food every day. The position of the food well changes every day, and the area of the food well composed of food wells increases successively with the number of training days. That is, the areas of the food wells on the 3 days are 1, 4, and 8 units respectively. After completing this stage, enter the formal training period.

[0021] ⑥. Formal training: Conduct training for 6 days. Change the position of the food well every day. During the formal training period, the areas of the three food wells are 11 - 13 units. Statistically analyze the data during the training period.

[0022] S023: Lesion of the hippocampus of tree shrews with the neurotoxin colchicine

[0023] ③. Select the method of local injection of the neurotoxin (colchicine) to lesion the hippocampus. Inject three points into each of the left and right brains to lesion the dorsal, middle, and ventral hippocampi of the animal respectively. The lesion coordinates are inferred from the tree shrew brain atlas.

[0024] ④. The tree shrews after lesion can undergo the cheese board maze experiment after 3 weeks of rest.

[0025] S024: Experimental training after hippocampal lesion

[0026] ③. Familiarize with the environment: The training method and standards are the same as those in step S022 before hippocampal lesion.

[0027] ④. Formal training: The number of training days and times is the same as that in step S022 before hippocampal lesion. The position of the food well is symmetric about the Y-axis with that before lesion and corresponds one by one with the first to sixth days before lesion, so as to avoid affecting the experimental results due to different difficulty settings of the reward wells before and after lesion.

[0028] S025: Precautions for the experiment

[0029] ①. Clean the food and animal residues in the maze in time after each experiment. After each animal finishes, the maze and the rest box need to be thoroughly cleaned.

[0030] ②. After the daily training of each tree shrew, restrict the supply of dog food to the tree shrew. The specific food restriction needs to be carried out on the premise of maintaining the body weight not less than 80% of the body weight before the experiment and not decreasing too fast.

[0031] During the food restriction period, the body weight of the experimental tree shrews should decrease smoothly.

[0032] S03: Determine the molding index

[0033] ③. Use the average route coefficient of the group of tree shrews during the formal training before and after hippocampal lesion to evaluate the spatial memory ability of tree shrews. The calculation formula of the route coefficient is as follows:

[0034] Route coefficient = sum of the straight-line distances in the order of passing through the food wells ÷ sum of the actual travel lengths passed, that is

[0035] Among them, D1 is the straight-line distance between the tree shrew from the rest box to the first reward well, D2 is the straight-line distance between the tree shrew from the first reward well to the next reward well, and D3 is the straight-line distance between the tree shrew from the second reward well to the third reward well; P1 is the actual travel length of the tree shrew from the rest box to the first reward well, P2 is the actual travel length of the tree shrew from the first reward well to the next reward well, and P3 is the actual travel length of the tree shrew from the second reward well to the third reward well; the larger the route coefficient, the better the foraging route planned by the tree shrew.

[0036] ④. Select the five times with the largest route coefficients in the 20 trainings per day for analysis, which represents the best level that the tree shrew can achieve in the training on the same day.

[0037] Among them, during the process of the experimenter placing food in the food well in step S012, the three-dimensional rest box is covered with a towel to prevent the tree shrew from seeing the experimenter placing food.

[0038] Among them, in step S012, a camera is fixed directly above the cheese board maze device and connected to a computer to record the behavior of the tree shrew.

[0039] Among them, during the experiment of familiarizing with the environment in step S022, the tree shrew can be artificially induced to fully explore the maze until the tree shrew can forage independently and the footprints are evenly distributed throughout the maze, then it can enter the next stage of training.

[0040] Among them, the calculation formula for the area of the food well in step S022: (a×h)×1 / 2; the area of the triangular food well composed of three adjacent food wells is 0.5 unit.

[0041] A method for evaluating the spatial learning and memory ability of tree shrews using a cheese board maze provided by the present invention. This method uses the natural nature of tree shrews to be good at searching for hidden food on land for experiments. The main experimental method is carried out under normal lighting conditions. There is an adaptation stage for familiarizing with the environment, without aversion stimuli, and there is no need to use stimulation means such as high-intensity light sources, water stimulation, loud noises or wind to complete this task. Therefore, the cheese board maze tree shrew experiment is a spatial memory evaluation method with very little stress on experimental animals, especially tree shrews, and it is an effective and innovative method.

[0042] The experimental method of the cheese board maze for tree shrews provided by the present invention includes the equipment of the experimental device, and the optimization settings for various parameters such as environmental temperature, humidity, room layout, and maze environment. The training of tree shrews in the experimental operation steps has been repeatedly demonstrated through multiple experiments in terms of training steps, times, time, frequency, and reward settings. A behavioral paradigm for evaluating the spatial memory of tree shrews has been successfully established, and the modeling criteria and modeling indicators are reasonably formulated. At the same time, the tree shrew model of the cheese board maze for spatial learning and memory has an advantage more similar to humans than the mouse and rat models. Therefore, the successful establishment of the tree shrew model of spatial learning and memory provides a behavioral experimental method applicable to tree shrews for subsequent neuroscience research such as constructing AD models, in vivo electrophysiological recordings, and optical recordings, and can also provide more powerful scientific data for researchers in the research of multiple disciplines and fields such as spatial coding, learning and memory, new drug development, screening, and evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a diagram of the cheese board maze;

[0044] Figure 2 It is a three-dimensional diagram of the cheese board maze;

[0045] Figure 3 It is a diagram for calculating the route coefficient;

[0046] Figure 4 It is a diagram of the maximum five-time comparison of the population average route coefficient before and after hippocampal damage in tree shrews. D1 is the straight-line distance between the rest box and the first reward well for the tree shrew, D2 is the straight-line distance between the first reward well and the next reward well for the tree shrew, and D3 is the straight-line distance between the second reward well and the third reward well for the tree shrew; P1 is the actual travel length of the tree shrew from the rest box to the first reward well, P2 is the actual travel length of the tree shrew from the first reward well to the next reward well, and P3 is the actual travel length of the tree shrew from the second reward well to the third reward well;

[0047] 101 - main board; 102 - food well; 103 - bottom board; 104 - the area of the food well is 0.5 unit, a - the base length of the triangle formed by the three food wells; h - the height of the triangle formed by the three food wells. 201 - three-dimensional rest box; 202 - iron sheet fence; 203 - nylon net; 204 - bottom board; DETAILED DESCRIPTION OF THE EMBODIMENTS

[0048] The following will combine specific embodiments and the attached Figures 1-4 to detail the implementation manner of the present invention, so as to fully understand how the present invention applies technical means to solve technical problems and achieve the realization process of technical effects and implement accordingly.

[0049] The present invention provides a method for evaluating the spatial learning and memory ability of tree shrews using a cheese board maze, comprising the following steps:

[0050] S01: Experimental preparation

[0051] S011: Prepare the cheese board maze device

[0052] The cheese board maze is a circular arena. The main board 101 is a circular white polypropylene board evenly distributed with 120 holes, and the distance between the centers of two adjacent holes is equal. The main board 101 is divided into 4 sector-shaped boards for easy disassembly and cleaning. A bottom board 103 is installed below the main board 101, and the main board 101 and the bottom board 103 form a blind hole maze containing 120 food wells; there is an iron sheet cylindrical fence 202 around the cheese board maze, and clue prompts of different shapes are pasted on the fence; a nylon net 203 is covered on the top of the maze to prevent the tree shrews from escaping from the maze.

[0053] S012: Experimentally arrange the cheese board maze

[0054] Select any position against the wall on the side of the cheese board maze as the starting area of the maze, and place an acrylic transparent three-dimensional rest box 201. Each time a tree shrew starts training, it goes out from the three-dimensional rest box 201 to look for rewards. Eating three food rewards within 2 minutes is considered to complete one training. Then, after luring it back to the three-dimensional rest box 201 with food, the experimenter places the food rewards for the next training in the holes.

[0055] S013: Environmental setting during the experimental period

[0056] Before and after the hippocampal lesion experiment, the environmental temperature of the cheese board maze room is maintained at 22°C - 24°C, and the humidity is 50% - 70%. Hint cards of different shapes and colors are set on the inner wall and surrounding walls of the maze. Before starting the experiment, the lights need to be adjusted to ensure uniform illumination, no reflection and no shadow in the maze, so as to avoid the influence of light differences on the preferences of tree shrews. The furnishings, light sources and other environmental conditions in the room remain unchanged throughout the experimental period, and the environment needs to be kept quiet during the experiment to reduce interference to the animals.

[0057] S02: Experimental training

[0058] S021: Preparation before the experiment

[0059] Select 11 tree shrews for the cheese board maze experiment. Before each experiment day, weigh and record the weight of each tree shrew, and determine the feeding amount after training on that day by combining the weight changes and the state and performance of the tree shrews during the training period.

[0060] S022: Experimental training of tree shrews before hippocampal lesion

[0061] ①. Familiarize with the environment: Food is distributed on the surface and in the holes of the maze. The experimental tree shrews are randomly generated every day. The tree shrews are gently driven from the breeding box into the three-dimensional rest box 201, and then transferred to the starting area of the maze. A 1-minute cooling-off period is given. After the cooling-off period, the gate of the three-dimensional rest box 201 is opened. Wait for the tree shrew to enter the maze to look for food rewards, and at the same time operate the computer to start recording its trajectory. When the tree shrew enters the maze and eats 3 foods or reaches the 2-minute cut-off time, stop recording. Induce the tree shrew to enter the three-dimensional rest box 201 and give a bread reward. Tree shrews with uniform footprints and those that have learned to forage in the holes will enter the next stage of training.

[0062] ②. Change the position of the food wells:

[0063] Day 1: Randomly select three food wells 102 with an area of 1 and place food crumbs as the reward wells for that day. Relatively concentrated food rewards can reduce the experimental difficulty and enhance the confidence of the tree shrews in performing tasks. Transfer the experimental tree shrews to the starting area of the maze through the three-dimensional rest box 201. Give a 1-minute cooling-off period. After the cooling-off period, open the gate of the three-dimensional rest box 201. Wait for the tree shrew to enter the maze to look for food rewards, and at the same time operate the computer to start recording its trajectory. When the tree shrew enters the maze and eats 3 foods or reaches the 2-minute cut-off time, stop recording. Use bread crumbs to induce the tree shrew back into the three-dimensional rest box 201. At this time, the experimenter places food in the food wells again. After the tree shrew rests for 1 minute, conduct the next experiment. Each tree shrew is trained 15 times a day, and the training method is the same each time.

[0064] Day 2: Randomly select three food wells 102 with an area of 4 and place food crumbs as the reward wells for that day. The training method and number of times are the same as on the first day.

[0065] Day 3: Randomly select three food wells 102 with an area of 8 and place food crumbs as the reward wells for that day. The training method and number of times are the same as on the first and second days.

[0066] ③. Formal training: Randomly select three food wells 102 with an area of 11 - 13 and place food crumbs as the reward wells during the training period. The position of the food wells is changed every day during the training period. The method is the same as in the training period of changing the position of the food wells, and the number of training times per day is increased to 20 times.

[0067] S023: Destroy the hippocampus on both sides of the tree shrew with colchicine

[0068] ①. After resting for three days, the tree shrews that have completed the cheese board maze training are subjected to hippocampal destruction. After anesthesia with isoflurane, locate the 6 coordinate points of the left and right brains, and then inject colchicine with a micro-injection pump. Suture the wound and observe the state of the tree shrews. Provide careful care after the operation.

[0069] ②. The concentration of colchicine is 0.6 μg / μl, and the volume of each injection site is 1 μl.

[0070] ⑤. The tree shrews after hippocampal lesion can undergo the cheese board maze experiment after 3 weeks of rest.

[0071] S024: Experimental training after hippocampal lesion

[0072] ①. Familiarize with the environment: The training method and criteria are the same as those in the experiment before hippocampal lesion.

[0073] ②. Formal training: The experimenter, the number of training days and times are the same as those in the experiment before lesion. From the first day to the sixth day, the setting of the food wells corresponds one by one to that in the experiment before lesion, and the positions are symmetric about the Y-axis with respect to the experiment before lesion.

[0074] S025: Precautions for the experiment

[0075] ①. After each experiment, the food and animal residues in the maze need to be cleaned up in time. After each animal finishes the experiment, the maze and the three-dimensional rest box need to be thoroughly cleaned to prevent odor interference to the subsequent tree shrews to be tested.

[0076] ②. After the training of each tree shrew on the same day, the tree shrew is given dog food restrictively. The specific food restriction needs to be carried out on the premise of maintaining the body weight not less than 80% of the body weight before the experiment and not decreasing too fast.

[0077] ③. After hippocampal lesion, the experimental training is divided into two stages: familiarizing with the environment and formal training. In the stage without the change of the food well position, because the tree shrews before lesion already knew that the food wells changed every day.

[0078] ④. Due to the different personalities of each tree shrew, the number of days to familiarize with the maze is also different. The timid and easily frightened tree shrews need more time to adapt to the environment. The tree shrews can be artificially induced to fully explore the maze until the tree shrews can forage independently and the footprints are evenly distributed throughout the maze, then they can enter the next stage of training. The tree shrews after lesion are more familiar with the maze environment and spend less time in the stage of familiarizing with the environment.

[0079] S03: Determine the modeling index

[0080] Use DeepLabCut (DLC) to model and analyze the route coefficient during the formal training period. The analysis method before and after lesion is the same. Take the average value of the five largest route coefficients of each tree shrew during the training on the same day. After averaging the 11 tree shrews, the average route coefficient of the group on that day is obtained. Plot the data of the group before and after lesion with the training days as the horizontal axis and the average route coefficient as the vertical axis to obtain the comparison chart of the average route coefficients of the group before and after lesion.

[0081] Route coefficient = sum of the straight-line distances in the order of passing through the food wells ÷ sum of the actual travel lengths passed through, that is The larger the route coefficient, the better the foraging route planned by the tree shrew.

[0082] Wherein: D1 is the straight-line distance between the tree shrew reaching the first reward well from the rest box, D2 is the straight-line distance between the first reward well and the next reward well for the tree shrew, and D3 is the straight-line distance between the second reward well and the third reward well for the tree shrew; P1 is the actual travel length of the tree shrew from the rest box to the first reward well, P2 is the actual travel length of the tree shrew from the first reward well to the next reward well, and P3 is the actual travel length of the tree shrew from the second reward well to the third reward well; the greater the route coefficient, the better the foraging route planned by the tree shrew.

[0083] The results showed that: ① There were significant differences in the top 5 times with the largest group average route coefficient before and after hippocampal lesion in the cheese board maze tree shrew experiment method (P = 0.001), indicating that the spatial memory ability of tree shrews was impaired when performing the cheese board maze task after hippocampal lesion. ② A behavioral paradigm for evaluating the spatial memory of tree shrews was successfully established, and the modeling criteria and indicators were reasonably formulated.

Claims

1. A method for evaluating the spatial learning and memory ability of tree shrews using a cheese board maze, with the specific steps as follows: S01: Experimental preparation S011: Prepare the cheese board maze device The cheese board maze is a circular arena. The main board is a circular white polypropylene plate evenly distributed with 120 holes, and the distance between the centers of two adjacent holes is equal. The main board is divided into 4 sector plates for easy disassembly and cleaning. A bottom board is installed below the main board, and the main board and the bottom board form a blind hole maze containing 120 food wells; There is an iron sheet cylindrical fence around the cheese board maze, and clue prompts of different shapes are pasted on the fence; The top of the cheese board maze is covered with a nylon net to prevent tree shrews from escaping from the maze; S012: Conduct experimental layout for the cheese board maze Select any position against the wall on the side of the cheese board maze as the starting area of the maze, and place an acrylic transparent three-dimensional rest box. Each time a tree shrew starts training, it goes out from the three-dimensional rest box to look for rewards. Eating three food rewards within 2 minutes is considered completing one training. Then, after using food to lure it back to the three-dimensional rest box, the experimenter places the food reward for the next training in the food well; S013: Environmental setting during the experimental period For the experiments before and after hippocampal lesion, the environmental temperature in the cheese board maze room is maintained at 20°C - 26°C, and the humidity is 40% - 70%. Hint cards of different shapes and colors are set on the inner wall and surrounding walls of the maze. The brightness of the maze is uniform during the experimental period, and the furnishings, light sources, and other environmental conditions in the room remain unchanged during the training period, and the environment is kept quiet; S02: Experimental training S021: Preparation before the experiment Before each experiment day, weigh and record the weight of each tree shrew, and determine the limited food feeding amount during the experimental period in combination with the weight change and the state and performance of the tree shrew during training; S022: Training before hippocampal lesion ①. Familiarize with the environment: Food is spread all over the surface and holes of the maze. When the tree shrew can independently forage for food in the food wells in the maze, with evenly distributed footprints and fewer behaviors caused by emotions such as nervousness, anxiety, and inattentiveness, it can enter the next stage of training; ②. Change the position of the food wells: Conduct training for 3 days. Select any three wells to place food every day, and the position of the food wells changes every day. The area of the food wells formed by the food wells increases successively with the number of training days, that is, the areas of the food wells for 3 days are 1, 4, and 8 units respectively. After completing this stage, enter the formal training period; ③. Formal training: Conduct training for 6 days, and change the position of the food wells every day. The area of the three food wells during the formal training period is 11 - 13 units, and the data during the training period is statistically analyzed; S023: Lesion the hippocampus of tree shrews with the neurotoxin colchicine ①. Select the method of local injection of the neurotoxin colchicine to lesion the hippocampus. Inject three points into each of the left and right brains to lesion the dorsal, middle, and ventral hippocampi of the animal respectively. The lesion coordinates are inferred from the tree shrew brain atlas; ②. The tree shrews after lesion can undergo the cheese board maze experiment after 3 weeks of rest; S024: Experimental training after hippocampal lesion ①. Familiarize with the environment: The training method and criteria are the same as those in step S022 before hippocampal lesion; ②. Formal training: The number of training days and times is the same as in step S022 before hippocampal lesion. The positions of the food wells are symmetric about the Y-axis with respect to those before the lesion and correspond one by one to the first to sixth days before the lesion, to avoid affecting the experimental results due to different difficulty settings of the reward wells before and after the lesion. S025: Precautions for the experiment ①. After each experiment, promptly clean the food and animal residues in the maze. After each animal finishes the experiment, thoroughly clean the maze and the rest box. ②. After the daily training of each tree shrew, restrict the supply of dog food to the tree shrew. The specific food restriction should be carried out on the premise of maintaining the body weight not less than 80% of the body weight before the experiment and not decreasing too rapidly. During the food restriction period, the body weight of the experimental tree shrews should decrease smoothly. S03: Determine the modeling index ①. Use the average route coefficient of the group of tree shrews during the formal training before and after hippocampal lesion to evaluate the spatial memory ability of the tree shrews. The calculation formula of the route coefficient is: ； where D1 is the straight-line distance between the tree shrew from the rest box to the first reward well; D2 is the straight-line distance between the tree shrew from the first reward well to the next reward well; D3 is the straight-line distance between the tree shrew from the second reward well to the third reward well; P1 is the actual travel length of the tree shrew from the rest box to the first reward well; P2 is the actual travel length of the tree shrew from the first reward well to the next reward well; P3 is the actual travel length of the tree shrew from the second reward well to the third reward well. ②. Select the five times with the largest route coefficient in 20 daily trainings for analysis, which represents the best level that the tree shrew can achieve in the training on that day.

2. A method for evaluating the spatial learning and memory ability of tree shrews using a cheese board maze according to claim 1, characterized in that during the process of the experimenter placing food in the food well in step S012, cover the three-dimensional rest box with a towel to prevent the tree shrew from seeing the experimenter placing the food.

3. A method for evaluating the spatial learning and memory ability of tree shrews using a cheese board maze according to claim 1, characterized in that fix a camera directly above the cheese board maze device in step S012, which is connected to a computer for recording the behavior of the tree shrew.

4. A method for evaluating the spatial learning and memory ability of tree shrews using a cheese board maze according to claim 1, characterized in that during the experimental process of familiarizing with the environment in step S022, artificially induce the tree shrew to fully explore the maze until the tree shrew can forage independently and the footprints are evenly distributed throughout the maze, then it can enter the next stage of training.

5. A method for evaluating the spatial learning and memory ability of tree shrews using a cheese board maze according to claim 1, characterized in that the calculation formula for the area of the food well in step S022: (a×h)×1 / 2; the area of the triangular food well composed of three adjacent food wells is 0.5 unit.

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