Experimental animal learning and memory impairment model, construction method, and use

Abstract

A construction method for an experimental animal learning and memory impairment model, comprising: individually housing a CD-1 mouse to allow the CD-1 mouse to develop territorial awareness; subjecting an experimental mouse and the CD-1 mouse to physical confrontation in a cage for a preset duration; and once the experimental mouse has developed acute violent stress, placing the experimental mouse into a Skinner box apparatus to perform a learning ability test, after a preset duration, performing a memory ability test, and after the tests, obtaining the experimental animal learning and memory impairment model. The method assesses animal learning and memory abilities by means of a Skinner box, expands assessment means related to learning and memory, and provides new data support for the construction and assessment of an animal model involving learning and memory impairment induced by acute violent stress.

Description

Laboratory animal learning and memory impairment models, construction methods, and applications Technical Field

[0001] This application pertains to an animal model, specifically relating to an experimental animal model of learning and memory impairment, its construction method, and its application. Background Technology

[0002] Stress has a dual effect on the body: on the one hand, stress can alter an animal's learning and memory abilities, but the effect varies depending on the intensity of the stress. Moderate stress can improve an animal's learning and memory abilities, while excessive stress has the opposite effect. Studies have found that when humans or animals are under intense acute stress or prolonged chronic stress, their learning and memory abilities are significantly affected, resulting in decreased learning and memory capacity. Currently, research models related to learning and memory often employ active avoidance responses and spatial learning and memory experiments.

[0003] Compared to common behavioral systems, Skinner box experiments offer richer assessment methods, more diverse training approaches, and the ability to analyze various indicators such as animal learning motivation. However, there is still a lack of systematic experimental paradigms and assessment methods for evaluating changes in learning and memory abilities after stress using Skinner boxes, and relevant parameters are also scarce. Technical issues

[0004] This application addresses the lack of systematic experimental paradigms and evaluation methods for Skinner box experiment data assessment, as well as the lack of relevant parameters, by providing a model, construction method, and application for experimental animal learning and memory impairment. Technical solutions

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] Firstly, this application proposes a method for constructing a model of learning and memory impairment in experimental animals, comprising the following steps:

[0007] CD-1 mice were housed individually to develop territorial awareness.

[0008] Experimental mice and CD-1 mice were subjected to physical confrontation in cages for a predetermined period of time.

[0009] Once the experimental mice have developed acute violent stress, they are placed in a Skinner box for learning ability testing. After a preset time, their memory ability is then tested. After passing the tests, an acute violent stress model of experimental animals is obtained.

[0010] Furthermore, after inducing territorial awareness in CD-1 mice, the process also includes:

[0011] Before the end of the single-cage rearing of CD-1 mice, non-experimental mice were used to test the aggression of the single-cage CD-1 mice, and CD-1 mice with an aggression frequency lower than the preset requirement were removed.

[0012] Furthermore, the physical confrontation lasting for a preset time includes:

[0013] Engage in 10-20 minutes of physical contact.

[0014] Furthermore, the 10-20 minute physical confrontation includes:

[0015] The physical confrontation is conducted continuously and periodically over a period of 10-20 minutes, with at least one round of physical confrontation per minute and each round lasting 5-10 seconds.

[0016] Furthermore, during the learning ability test and the memory ability test after a preset time, the evaluation parameters include the number of effective lever presses, accuracy rate, and immobility latency.

[0017] Furthermore, the learning ability test includes:

[0018] The experiment was conducted once a day, with each experiment lasting 60 minutes, for a total of 5 days.

[0019] Furthermore, the memory ability test is conducted after a preset time, including a one-day memory ability test after three weeks.

[0020] Secondly, this application proposes an experimental animal learning and memory disorder model prepared using the above-mentioned method for constructing an experimental animal learning and memory disorder model.

[0021] Furthermore, the experimental mice used were C57BL / 6J mice.

[0022] Thirdly, this application proposes the application of the above-mentioned experimental animal learning and memory impairment model in acute violent stress-induced learning and memory impairment. Beneficial effects

[0023] Compared with the prior art, this application has the following beneficial effects:

[0024] This application proposes a method for constructing an experimental animal model of learning and memory impairment. By optimizing the experimental procedure of a traditional chronic social frustration model, a stable learning and memory impairment model was induced in mice, and the obtained experimental animals exhibited a clear learning and memory impairment phenotype, enriching the diversity of animal models of stress-related disorders. The Skinner box was used to assess the animals' learning and memory abilities, expanding the means of assessment related to learning and memory, and providing new data support for the construction and evaluation of animal models of learning and memory impairment induced by acute violent stress.

[0025] This application also proposes an experimental animal learning and memory disorder model, and the application of this model in acute violent stress-induced learning and memory disorder, which has all the advantages of the above-mentioned experimental animal learning and memory disorder model construction method. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 is a flowchart illustrating one method for constructing an experimental animal learning and memory impairment model according to this application.

[0028] Figure 2 is a comparison of the effective compression latent period during the learning period.

[0029] Figure 3 is a comparison chart of accuracy rates during the learning period.

[0030] Figure 4 is a comparison chart of the effective number of compression strokes during the learning period.

[0031] Figure 5 is a comparison of the effective lever latency during the memory retrieval period.

[0032] Figure 6 shows a comparison of accuracy rates during the memory retrieval period.

[0033] Figure 7 is a comparison of the effective number of lever presses during the memory retrieval period. Embodiments of the present invention

[0034] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0035] Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0036] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0037] In the description of the embodiments of this application, it should be noted that if terms such as "upper," "lower," "horizontal," or "inner" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product of the invention is in use, they are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on this application. In addition, terms such as "first" and "second" are only used to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0038] In previous experimental studies, chronic social defeat stress (CSDS) has been commonly used to construct models of depressive disorders. CSDS is a widely used animal experimental model, primarily used to study the pathogenesis and potential treatments for mental illnesses such as depression and anxiety. This application modifies the CSDS model to construct a learning and memory impairment model induced by acute violence stress (AVS).

[0039] Currently, most research models on learning and memory employ active avoidance response and spatial learning and memory experiments (maze tests, such as the Morris water maze and Y-maze, and light-dark discrimination learning). Active avoidance response is a behavior that animals learn to actively avoid harmful stimuli under specific conditions. In these experiments, animals are exposed to conditioned stimuli (such as a bell) and unconditioned stimuli (such as an electric shock). After repeated training, animals learn to avoid the conditioned stimulus when it appears, thus preventing the subsequent unconditioned stimulus. The water maze experiment utilizes animals' natural aversion to water, forcing them to find hidden platforms in the water where they can stand. Statistical analysis of the time mice spend searching for these platforms assesses their spatial location and orientation memory. The Y-maze model is used to study the spatial recognition and memory abilities of mice. This maze utilizes mice's natural tendency to explore new environments, requiring no rules for self-preservation, and effectively reflects their ability to recognize and remember new environments.

[0040] Compared to common behavioral systems such as the Morris water maze and the Y maze, the Skinner box can also be used for assessment. However, there is still no systematic experimental paradigm, assessment method, or relevant parameters for how to use the Skinner box to assess changes in learning and memory abilities after stress.

[0041] Based on the above, this application proposes an experimental animal learning and memory impairment model, its construction method, and its application. The following is a detailed description of this application in conjunction with embodiments and accompanying drawings.

[0042] As shown in Figure 1, one embodiment of the method for constructing the experimental animal learning and memory impairment model of this application may include the following steps:

[0043] S101, CD-1 mice were housed individually to induce territoriality in them;

[0044] S102, which involves physical confrontation between experimental mice and CD-1 mice in cages for a predetermined time;

[0045] S103. After the experimental mice are subjected to acute violent stress, they are placed in the Skinner box device for learning ability testing. After a preset time, memory ability testing is performed. After passing the test, an experimental animal learning and memory impairment model is obtained.

[0046] It should be noted that CD-1 mice are an inbred strain of mice with high genetic diversity, which makes CD-1 mice highly adaptable to the experimental environment and the experimental results highly reproducible. Therefore, although the construction method of this application uses mice, it is reproducible.

[0047] The Skinner box can assess acute violent stress by evaluating the learning and memory abilities of mice. This application constructs a new paradigm for acute violent stress-induced learning disorders, which can comprehensively assess learning disorders through evaluation parameters within the Skinner box. The Skinner box has significant advantages: ① Compared to other common behavioral systems, the Skinner box offers richer evaluation methods and more training options, making it more conducive to studying projection relationships between multiple brain regions. In addition to obtaining behavioral judgment indicators based on the experimental paradigm, the software-recorded data throughout the process allows for analysis of various indicators such as attention and motivation in animal task performance. ② The Skinner box can meet diverse experimental scenarios. Based on software operation and intelligent control, the Skinner box task paradigm has high time sensitivity and rich extended functions, and can be combined with experimental techniques such as optogenetics, fiber optic recording, and electrophysiology. ③ The Skinner box can serve as an experimental paradigm for assessing animal behavioral motivation.

[0048] This application constructs an experimental animal learning and memory impairment model by subjecting experimental mice to physical confrontation with CD-1 mice that have developed territorial awareness, thereby inducing acute violent stress, and then conducting tests using a Skinner box.

[0049] As an embodiment of the method for constructing the experimental animal learning and memory impairment model of this application, it may include:

[0050] C57BL / 6J mice were used as experimental mice. CD-1 mice were housed individually to establish territoriality. On the last day of individual housing, the aggression of CD-1 mice was tested using other C57BL / 6J mice not involved in the experiment. CD-1 mice that attacked less than 3 times within 10 minutes were culled. The tested C57BL / 6J mice were then placed directly into the selected CD-1 mouse cages for 10-20 minutes of physical confrontation; this process can be termed direct contact stress. Each CD-1 mouse may face multiple C57BL / 6J mice per day, depending on the number of animals in each group.

[0051] To further ensure the stability of the acute violent stress induced by CD-1 mice in experimental mice, close observation during the experiment is necessary to ensure the normal occurrence of high-quality aggressive behavior. Verification has confirmed that high-quality aggressive behavior meets the following conditions: the aggressive behavior must be continuous and periodic within 10-20 minutes of the experiment; there must be at least one round of attack per minute, with each round lasting approximately 5-10 seconds. If the two animals are observed grooming each other, this stage of the experiment is considered a failure.

[0052] It should be noted that in this application, CD-1 male mice can be selected as the attacking mouse (host), and C57BL / 6J mice can be selected as the experimental mice (intruders). Utilizing the biological instincts of CD-1 male mice—that is, the territorial instincts of male animals—when an intruding C57BL / 6J mouse invades, the host CD-1 mouse will spontaneously attack the intruding mouse. This ensures the smooth implementation of the method in this application and the effective occurrence of the attack.

[0053] Selected CD-1 mice were housed individually (usually 10 different selected CD-1 mice) as the stressor for the acute violent stress experiment in the C57BL / 6J mouse experiment. After 10-20 minutes of physical confrontation between C57BL / 6J mice and CD-1 mice in the experimental cage, the test animals were subjected to psychological and physiological stress. Immediately afterwards, the C57BL / 6J experimental mice were placed in a Skinner box device, and their learning ability was tested according to pre-set experimental parameters. Memory ability was tested 3 weeks later.

[0054] When using the Skinner box to assess learning disabilities, relevant parameters that can be used include: ① Number of effective lever presses: the number of times the mouse obtains sugar water (or other rewards) by pressing the lever; ② Accuracy rate: the percentage of effective lever presses in the total number of lever presses; ③ Immobility latency: the time from the start of the experiment to the first effective lever press.

[0055] This application is the first to explore the impact of acute violent stress on learning and memory abilities, and connects the acute violent stress model with the behavioral paradigm of Skinner box learning and memory ability testing.

[0056] In other embodiments of this application, the evaluation parameters may vary depending on the experimental purpose, animal type, equipment, and specific laboratory requirements, and this application does not impose any limitations.

[0057] It should be noted that in practical applications, a control group can also be set up: select two C57BL / 6J mice of the same age and conduct the experiment in the same manner as the experimental group (in the control group, the CD-1 mice in the experimental group can be replaced with control C57BL / 6J mice).

[0058] For the aforementioned experimental and control groups, the specific method of the Skinner box experiment may include:

[0059] ① Preparation: Check the Skinner box apparatus to ensure all components (such as troughs, levers, lights, and sounds) are functioning properly. Depending on the experimental objectives, reinforcement or punishment conditions can be set, such as food reinforcement or electrical stimulation punishment. Select appropriate experimental animals (such as mice of similar age, sex, and strain).

[0060] ② Animal Adaptation: One week prior to the experiment, mice were allowed to acclimatize to the laboratory environment. Temperature, humidity, and light cycle were controlled. Water intake was restricted for 24 hours before the experiment to increase sensitivity to water reinforcement. It should be noted that animal welfare was ensured during the experiment to avoid excessive thirsting of the mice.

[0061] ③ Experiment Start: Place the mouse in the Skinner box and allow it to acclimatize for approximately 10-15 minutes. Set the experimental conditions, such as reinforcement or punishment, and begin the experiment. For example, provide sugar water as reinforcement when the mouse presses the lever; or provide electrical stimulation as punishment for a specified period of time.

[0062] ④ Data Recording: Use the Skinner box's built-in data recording system or manually record the mouse's behavior, such as the number of times the lever was pressed, the number of times reinforcement or punishment was given, etc. Record other relevant behaviors, such as activity level and exploratory behavior, depending on the experimental objective.

[0063] ⑤ Experimental cycle: The experiment was conducted once a day, with each experiment lasting 60 minutes. The experiment was conducted for 5 consecutive days to test the learning level of the mice. After 3 weeks, a memory ability test was performed for 1 day.

[0064] ⑥ Data Analysis: Analyze the experimental data, such as learning curves and behavior frequencies, according to the experimental objectives. Appropriate statistical methods (such as t-tests or ANOVA) can also be used to compare the differences between the experimental and control groups.

[0065] Stress has a dual effect on the body: it can alter an animal's learning and memory abilities, but the impact varies depending on the intensity of the stress. Moderate stress can improve learning and memory, while excessive stress has the opposite effect. When humans or animals are under intense acute stress or prolonged chronic stress, their learning and memory abilities are significantly affected, resulting in decreased learning and memory capacity.

[0066] Preliminary experiments demonstrated that this application can induce a decline in learning and memory abilities through acute violent stress, and the learning and memory abilities were assessed multidimensionally using multiple parameters of the Skinner box. Based on the above examples, Figure 2 shows a comparison of the effective lever latency during the learning period. Figure 3 shows a comparison of the accuracy during the learning period. Figure 4 shows a comparison of the number of effective lever presses during the learning period. Figure 5 shows a comparison of the effective lever latency during the memory retrieval period. Figure 6 shows a comparison of the accuracy during the memory retrieval period. Figure 7 shows a comparison of the number of effective lever presses during the memory retrieval period. It should be noted that in Figures 2 to 7, the Control group represents the control group, i.e., the mouse group that was not subjected to CD-1 challenge. In the experiment, the total number of mice in each group, N, was equal to 5.

[0067] This application constructs an improved method for assessing changes in learning and memory abilities after acute violent stress. C57BL / 6J mice are challenged using larger CD-1 mice, and their learning and memory abilities are assessed using a Skinner box based on their ability to correctly obtain rewards. This addresses how learning and memory abilities in mice change after acute violent stress and adds more parameters for assessing these abilities.

[0068] Based on the aforementioned method for constructing experimental animal learning and memory impairment models, this application also proposes an experimental animal learning and memory impairment model prepared using the same method, and its application in acute traumatic stress-induced learning and memory impairment.

[0069] It should be noted that specific implementations of the model and application can adopt the implementations of the above construction method, which will not be repeated here.

[0070] The above are merely preferred embodiments of this application and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for constructing a learning and memory disorder model of an experimental animal, characterized by, The method comprises the following steps: The CD-1 mice are singly caged to form a territory awareness; The experimental mice are made to have physical confrontation with the CD-1 mice in the cage for a preset time; After the experimental mice are subjected to acute violent stress, the experimental mice are put into a Skinner box device to detect learning ability, and then memory ability is detected after a preset time, and after the detection, a learning and memory disorder model of experimental animals is obtained.

2. The method of claim 1, wherein the method is characterized by: After the CD-1 mice form the territory awareness, the method further comprises: Before the single caging of the CD-1 mice ends, a non-experimental mouse is used to test the aggressiveness of the singly caged CD-1 mice, and CD-1 mice with an attack frequency less than a preset requirement are removed.

3. The method according to claim 2, wherein the method for constructing a learning and memory disorder model of an experimental animal is characterized by, The physical confrontation for a preset time comprises: The physical confrontation lasts for 10-20 minutes.

4. The method according to claim 3, wherein the method for constructing a learning and memory disorder model of an experimental animal is characterized by, The physical confrontation for 10-20 minutes comprises: The physical confrontation is continuously and periodically performed within 10-20 minutes, and the number of physical confrontation rounds per minute is greater than or equal to 1, and the duration of each round is 5-10 seconds.

5. The method according to claim 4, wherein the method is characterized by, During the detection of learning ability and the detection of memory ability after a preset time, the evaluation parameters include the number of effective pressure rods, the accuracy and the immobile latency.

6. The method according to claim 5, wherein the method for constructing a learning and memory disorder model of an experimental animal is characterized by, The detection of learning ability comprises: The experiment is performed once a day, each experiment lasts for 60 minutes, and the experiment is continuously performed for 5 days.

7. The method according to claim 6, wherein the method is characterized by, The detection of memory ability after a preset time comprises: the detection of memory ability is performed for one day after 3 weeks.

8. A learning and memory disorder model of experimental animals prepared by the method for constructing a learning and memory disorder model of experimental animals according to any one of claims 1 to 7.

9. The learning and memory disorder model of experimental animals according to claim 8, wherein the learning and memory disorder model of experimental animals is a learning and memory disorder model of experimental animals in which the expression of the protein is decreased. The experimental mice are C57BL / 6J mice.

10. The learning and memory disorder model of experimental animals according to claim 8 or 9 is applied to induce learning and memory disorders by acute violent stress.

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