Construction method and application of pathological social memory generalization mouse model

Abstract

The invention provides a construction method and application of a pathological social memory generalization mouse model. The construction method provided by the invention comprises the following steps: firstly, carrying out acute subthreshold social contusion on normal mice; secondly, putting the mouse subjected to at least one acute subthreshold social contusion into a test box for adaptive test, counting the interaction time of the mouse with the vacant first social cage and the vacant second social cage, and recording the interaction time as A1 and A2; counting social interaction time of the mice with familiar aggressive mice and unfamiliar aggressive mice, and recording the social interaction time as B1 and B2; when B1 / A1 is less than 1 and B2 / A2 is less than 1, the pathological social memory generalization of the mouse occurs, and the pathological social memory generalization mouse model is obtained. According to the mouse model provided by the invention, the blank of memory generalization caused by negative experience of a social environment is filled, and an important basis is provided for exploring the influence of social contusion on social memory generalization.

Description

Technical Field

[0001] The present invention relates to the technical field of mouse model construction, and particularly relates to a method for constructing a mouse model with generalized pathological social memory and its application. Background Art

[0002] Generalization refers to the fact that after an individual has learned a conditioned stimulus for a period of time, they also show similar responses to other stimuli similar to that stimulus. Generalization is a basic learning mechanism. Adaptive generalization can extract the similar attributes of things and integrate them into new memories. This function helps to improve the classification of things and scenarios by individuals, can draw inferences from the mastered experience, and greatly improves the efficiency of learning and work. Normal generalization plays an important role in individual survival and learning memory, while excessive generalization may lead to negative changes in cognition and emotion. For example, the common mental illness post-traumatic stress disorder is caused by excessive generalization. This disease occurs when an individual experiences a major stress event and shows negative emotions such as anxiety and panic in similar traumatic environments or even similar neutral environments, seriously interfering with the normal life of the patient. Excessive generalization has many aspects, but current research is mostly focused on the generalization of contextual fear memory. These studies have fully explored aspects such as the generalization of contextual fear memory in individuals and discovered the relevant neural circuit mechanisms. However, current research on the generalization of social memory is blank.

[0003] However, in real life, compared with simple environmental factors, the negative emotions triggered by the social environment are often more complex and profound. For example, individuals who have suffered social traumas such as beatings and intimidation not only have fear responses when encountering social objects, but this fear response often generalizes to other social situations, leading to the occurrence of psychological disorders such as social phobia. Therefore, compared with traditional research on contextual fear memory, exploring the generalization of social memory is of more important significance for understanding the group pathogenesis mechanism. The study of social memory generalization can be closer to the social challenges faced by individuals in real society, especially in the context of pathological social environments. These environments are often accompanied by negative social experiences of individuals, such as bullying, exclusion, or violence. These experiences not only have a long-term impact on the mental health of individuals, but may also lead to social anxiety disorders such as social phobia. The study of social memory generalization helps to reveal the neural mechanisms of these mental disorders and provides a scientific basis for developing targeted treatment strategies.

[0004] In summary, the study of social memory generalization can not only enrich our understanding of fear memory generalization, but also has important clinical and social value for revealing the pathogenesis of social anxiety disorder and formulating effective prevention and intervention measures. Using pattern mice to construct a model of social memory generalization helps to reveal the pathogenesis of pathological social memory generalization dysfunction, is of great significance for elaborating and supplementing the important biological process of generalization, provides a new mouse paradigm for the study of treating mental diseases caused by insufficient generalization function and overgeneralization, and provides a basis for elaborating general biological function phenomena and exploring treatment methods for social interaction disorders. Summary of the Invention

[0005] The present invention provides a method for constructing a pathological memory generalization mouse model and its application.

[0006] The present invention provides a method for constructing a mouse model, including: S1. Subject normal mice to acute subthreshold social defeat: expose normal mice to aggressive mice and receive attacks from the aggressive mice; after the attack, place the normal mice and the aggressive mice separately at a distance where they can smell each other but cannot make contact. S2. Place the mice that have undergone at least 1 acute subthreshold social defeat into a test chamber for an adaptation test: Place a first social cage and a second social cage at opposite corners of the test chamber, and record the interaction time of the mice with the empty first social cage and the second social cage as A1 and A2; subsequently, place a familiar aggressive mouse and a strange aggressive mouse in the first social cage and the second social cage respectively, and record the social interaction time of the mice with the familiar aggressive mouse and the strange aggressive mouse as B1 and B2. S3. When B1 / A1 < 1 and B2 / A2 < 1, it indicates that the mice have pathological social memory generalization, and a pathological social memory generalization mouse model is obtained.

[0007] In the method as described above, the duration of pathological social memory generalization of the pathological social memory generalization mouse model is at least 3 days; specifically, within the duration, the pathological social memory generalization mice are tested using the same method, and the test results are all B1 / A1 < 1 and B2 / A2 < 1.

[0008] In the method as described above, the time for each attack of the normal mice by the aggressive mice is 30 seconds.

[0009] In the method as described above, the number of attacks of the normal mice by the aggressive mice is 4 - 8 times, specifically it can be 4, 5, 6, 7, 8 or any range composed of any two of them. Further, the number of attacks of the normal mice by the aggressive mice is preferably 8 times.

[0010] The method as described above, wherein the normal mice are male C57BL / 6J mice at 8 - 10 weeks of age.

[0011] The method as described above, wherein the aggressive mice are male CD - 1 (ICR) mice at 16 - 20 weeks of age. Aggressive CD - 1 mice refer to: directly placing a normal mouse into the cage of CD - 1 mice for 3 minutes, screening continuously for three days, once a day. If the CD - 1 mice initiate attacks more than 5 times and the attack latency is less than 1 minute, they are considered aggressive. If they are aggressive at least 2 times in 3 days, they are aggressive CD - 1 mice.

[0012] The method as described above, wherein the familiar aggressive mice are the aggressive CD1 mice that attack normal mice; the unfamiliar aggressive mice refer to the unfamiliar aggressive mice with the same strain and age as the familiar aggressive mice.

[0013] The method as described above, after each test, the test chamber needs to be deodorized to eliminate olfactory cues, specifically including cleaning the residues of mice and disinfecting the test chamber with 75% alcohol.

[0014] The method as described above, the social interaction time is located with the head of the normal mouse as the positioning point. When the head positioning point is within the circle with the center of the social cage as the center and a radius of 2 cm outside the radius of the social cage, it is considered to have social interaction with the social cage. This judgment method can accurately capture the social behavior of mice and avoid misjudgment due to accidental contact of other parts of the body.

[0015] In a second aspect, the present invention provides the application of the mouse model constructed by the above - mentioned construction method, and the application is selected from at least one of A1) - A2): A1) Application in developing drugs for alleviating and / or treating pathological social memory generalization; A2) Application in screening drugs for alleviating and / or treating pathological social memory generalization.

[0016] In the above text, "pathological" refers to being different from the normal generalization phenomenon. After an individual suffers social traumas such as being beaten, intimidated, or bullied on campus, in a similar social environment or even a normal social environment, there is still social fear similar to that after social trauma, and obvious avoidance and escape behaviors occur.

[0017] The construction method provided by the present invention includes: first, performing acute subthreshold social defeat on normal mice; secondly, putting the mice that have experienced at least 1 acute subthreshold social defeat into a test box for adaptation testing, and counting the interaction time of the mice with the empty first social cage and the second social cage, denoted as A1 and A2; counting the social interaction time of the mice with familiar aggressive mice and strange aggressive mice respectively, denoted as B1 and B2; when B1 / A1 < 1 and B2 / A2 < 1, it indicates that the mice have pathological social memory generalization, and a pathological social memory generalization mouse model is obtained. The mouse model provided by the present invention fills the gap in the phenomenon of memory generalization caused by negative experiences in the social environment, and provides an important basis for exploring the impact of social defeat on the generalization of social memory and contextual memory. Description of the Drawings [[ID=,4]]

[0018] Figure 1 It is a schematic structural diagram of the square social test box used in the embodiment of the present invention; Figure 2 It is a schematic structural diagram of the social cage used in the embodiment of the present invention; Figure 3 It is the construction process and results of the pathological social memory generalization mouse model; among them, A is the schematic diagram of the treatment of mice in the Normal group; B is the movement trajectory of mice in the Normal group in the test box containing an empty social cage and the test box containing CD1 mice; C is the schematic diagram of the treatment of mice in the Control group; D is the movement trajectory of mice in the Control group in the test box containing an empty social cage and the test box containing CD1 mice; E is the schematic diagram of the treatment of mice in the 30s×1time group; F is the movement trajectory of mice in the 30s×1time group in the test box containing an empty social cage and the test box containing CD1 mice; G is the schematic diagram of the treatment of mice in the 30s×4times group; H is the movement trajectory of mice in the 30s×4times group in the test box containing an empty social cage and the test box containing CD1 mice; I is the schematic diagram of the treatment of mice in the 30s×8times group; J is the movement trajectory of mice in the 30s×8times group in the test box containing an empty social cage and the test box containing CD1 mice; K is the statistical result of the social interaction time of each group of mice with the social cages on both sides; L is the statistical result of the social ratio of each group of mice with the social cages on both sides.

[0019] Figure 4 It is the stability test result of the pathological social memory generalization mouse model; among them, A is the schematic diagram of the test method; B is the statistical result of the social interaction time of the mouse model with the aggressive CD1 mice in the social cages on both sides at 24 hours (24h) and 3 days after the test; C is the statistical result of the social ratio of the mouse model with the aggressive CD1 mice in the social cages on both sides at 24h and 3 days after the test.

[0020] Figures 3 - 4In this study, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and n.s. indicates no significant statistical difference. Detailed implementation manners

[0021] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments, and they should not be construed as limiting the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for the purpose of description and cannot be construed as indicating or implying relative importance.

[0022] Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0023] The structure of the square social test box used in the following embodiments is as Figure 1 shown. The square social test box is a cube box with an open top, and its length A1, height A2, and width A3 are all 40 cm. The square social test box is made of white opaque material, and mice cannot observe the external environment outside the box inside the box.

[0024] The structure of the social cage used in the following embodiments is as Figure 2 shown, and its diameter is 8.5 cm and its height is 14 cm.

[0025] The social defeat cage used in the following embodiments is similar to the structure of a breeding cage, but is internally provided with a detachable transparent partition, which can divide the breeding cage into two compartments of equal volume on the left and right. The mice in the compartments can observe the mice in the adjacent compartments and have olfactory communication, but the two cannot have physical contact.

[0026] The C57BL / 6J mice used in the following embodiments were purchased from SPF (Beijing) Biotechnology Co., Ltd.; in order to allow the C57BL / 6J mice to adapt to and become familiar with the behavioral environment and the odor of the experimenter, the experimenter touched and grasped the mice every day within one week before the experiment started, and placed the mouse breeding cage in the behavior room 2 h before each experiment.

[0027] The CD1 mice used in the following examples were purchased from Spefox (Beijing) Biotechnology Co., Ltd. and screened for aggressiveness using conventional methods. Specifically, 8-10 week-old C57BL / 6J mice were placed directly into a CD1 mouse cage for 3 minutes. Screening was performed once daily for three consecutive days. CD1 mice were considered aggressive if they attacked more than five times with an attack latency of less than 1 minute. If they attacked at least twice in three days, they were considered aggressive mice in the model.

[0028] The experiments involved in the following embodiments were all carried out in a quiet, odorless, dimly lit room.

[0029] The following examples use the Ethovision XT 14.0 software behavior tracking system, which includes computer software and camera equipment.

[0030] In the following examples, the social ratio refers to the ratio of the time (B) that a C57BL / 6J mouse interacts with a CD1 mouse in a social cage to the time (A) that the mouse interacts with an empty social cage during the acclimation phase in the social memory test. Specifically, the social ratio is calculated by dividing the time (B) that the C57BL / 6J mouse interacts with a CD1 mouse in the second phase by the time (A) that the mouse interacts with an empty social cage during the acclimation phase.

[0031] In the following examples, independent sample t-test was used to compare the mean differences between the two groups. A p value of less than 0.05 was considered to be statistically significant. SPSS software was used for statistical analysis, and GraphPad Prism software was used for plotting.

[0032] Example 1: Construction of a mouse model of pathological social memory generalization Step 1. Pre-adaptation phase: During the pre-adaptation phase of the experiment, screened aggressive CD1 mice were transferred from their home cages to social frustration cages, allowing the CD1 mice to develop territorial awareness in the social frustration cages without partitions.

[0033] Step 2, model construction: C57BL / 6J mice raised in a standard environment were divided into 5 groups, namely Normal group, Control group, 30s×1time group, 30s×4times group and 30s×8times group. There were 13 mice in the Normal group and Control group, 10 mice in the 30s×1time group, 13 mice in the 30s×4times group and 11 mice in the 30s×8times group. Figure 3 As shown in Figure A, the C57BL / 6J mice in the Normal group were not subjected to any form of social stress or social frustration, that is, they were kept in the cage normally for 1 hour. Figure 3As shown in C, the Control group of C57BL / 6J mice was placed in a compartment adjacent to the aggressive CD1 mice in a social defeat cage and exposed for 60 min. During this process, there was only olfactory communication between the C57BL / 6J mice and the CD1 mice, without direct physical contact. As Figure 3 As shown in E, the C57BL / 6J mice in the 30s×1time group experienced 1 episode of aggressive stress initiated by the aggressive CD1 mice, with a duration of 30 seconds. After 1 attack, they were placed in a compartment adjacent to the aggressive CD1 mice in a social defeat cage and exposed for 59.5 min, with a total duration of 1 h. As Figure 3 As shown in G, the C57BL / 6J mice in the 30s×4times group experienced 4 episodes of discontinuous aggressive stress initiated by the aggressive CD1 mice, with each attack lasting 30 s. After each attack, they were placed in a compartment adjacent to the aggressive CD1 mice in a social defeat cage and exposed for 14.5 min, with a total duration of 1 h for the whole process. As Figure 3 As shown in I, the C57BL / 6J mice in the 30s×8times group experienced 8 episodes of discontinuous aggressive stress initiated by the aggressive CD1 mice, with each attack lasting 30 s. After each attack, they were placed in a compartment adjacent to the aggressive CD1 mice in a social defeat cage, with a 7-min interval between each exposure, and the total duration of the whole process was 1 h.

[0034] Step 3, Social memory test: The Normal group directly underwent a social behavior test with CD1 mice, while the other groups underwent a social memory behavior test 24 h after social stress. Specifically, provide as Figure 1The square social test box shown, with two empty social cages placed at the diagonal positions of the square test box, named the first social cage and the second social cage respectively; Place C57BL / 6J mice in the central area of the square social test box and allow them to freely explore for 2.5 min. Take out the C57BL / 6J mice and place them in a conventional breeding cage and wait for 1 min, while cleaning the square social test box to eliminate the odor influence. Subsequently, randomly place a strange CD1 mouse (a strange aggressive mouse refers to a strange aggressive mouse with the same strain and age as the familiar aggressive mouse, named Novel CD1) and a familiar aggressive CD1 mouse (i.e., the aggressive CD1 mouse used in the model construction process, named Familiar CD1) in the first social cage and the second social cage, and allow the C57BL / 6J mice to continue to explore the box for 2.5 min. The entire test process uses the Ethovision XT 14.0 software behavior tracking system for identification and recording. For C57BL / 6J mice, the three-point positioning method of the head, torso, and tail is adopted. When the head positioning point is located within a circle with the center of the social cage as the center and a radius greater than 2 cm of the social cage radius, it is regarded as having social interaction with the social cage. After each test, return the mice to their respective home cages, and clean the feces and disinfect with 75% alcohol for the square social test box and the social cages to eliminate olfactory cues.

[0035] Statistically analyze the social interaction time (Time in close interaction) of mice in each group with the first social cage and the second social cage where CD1 mice are placed. The statistical results are as Figure 3 shown in Figure K and Table 1. It can be seen that there is no statistical difference in the social interaction time of mice in the Normal group with CD1 mice in the first social cage and the second social cage, and there is no obvious social preference for different CD1 mice. According to the nature of mice to like the new and dislike the old, it is not found that the social interaction time of mice in the Control group with strange CD1 mice increases significantly, indicating that the mice in this group have not formed social memory. However, there are differences in the social interaction time of C57BL / 6J mice that have been attacked by aggressive CD1 mice with CD1 mice in the first social cage and the second social cage, and with the increase in the number of attacks, the social interaction time with CD1 mice decreases significantly. Statistically analyze the social ratio of mice in each group. The statistical results are as Figure 3As shown in Table 1 and the figure, it can be seen that there is no significant difference in the social interaction ratio between the mice in the Normal group and the Control group and the CD1 mice in the first and second social cages. Mice in the 30s×1time group, 30s×4times group, and 30s×8times group showed social preference. The social interaction time of the mice in the 30s×1time group with unfamiliar CD1 mice increased significantly, and the social interaction ratio with unfamiliar CD1 mice was greater than 1, indicating that the mice in this group had formed pathological social memory. After the mice in the 30s×4times group experienced 4 times of discontinuous social defeat stress, the social interaction time with familiar CD1 mice decreased significantly, forming pathological social memory, and the social interaction ratio of some C57BL / 6J mice with both familiar and unfamiliar CD1 mice was less than 1. After experiencing 8 times of discontinuous acute social defeat, the social interaction time of the mice in the 30s×8times group with both familiar and unfamiliar CD1 mice decreased significantly, and the social interaction ratio of C57BL / 6J mice with both familiar and unfamiliar CD1 mice was less than 1, forming generalization of pathological social memory.

[0036]

[0037]

[0038] Note: The social interaction time (S) and the corresponding social interaction ratio of the mice in each group with CD1 mice are listed in Table 1. Among them, the social interaction time and social interaction ratio of the "Normal group" refer to the interaction time and ratio with CD1 mice; while the social interaction time and social interaction ratio of the "Control group", "30s×1time group", "30s×4times group", and "30s×8times group" refer to the interaction time and ratio with Familiar CD1 and Novel CD1.

[0039] Example 2. Test on the duration of generalization of pathological social memory As mentioned above, 90% of the normal mice in the 30s×8times group of C57BL / 6J mice had generalization of pathological social memory after experiencing 8 times of discontinuous acute social defeat. Therefore, in this example, 10 C57BL / 6J mice were reselected and, according to the method shown in Figure 4 A, the social memory test time point of C57BL / 6J mice after previous exposure to CD1 mice was extended from 24 hours to the third day (without any operation on the mice during this period) to evaluate the persistence of social memory.

[0040] The experimental results are as shown in Figure 4As shown in Table 2 of B-C, on the third day, the average social ratio of C57BL / 6J mice to familiar and unfamiliar CD1 mice was less than 1, indicating that the phenomenon of pathological social memory generalization was continuously maintained.

[0041]

[0042] In summary, the present invention constructs for the first time a mouse model of pathological social memory generalization. The experimental results show that acute subthreshold social defeat can induce pathological social memory generalization in mice, which is significantly reflected through social memory tests. The mice in the 30s×8times group not only showed a decrease in social interaction time with familiar CD1 mice but also exhibited social memory generalization towards unfamiliar CD1 mice, which was reflected in the social ratio. At the same time, this pathological social memory generalization can last for at least 3 days, showing a long-term characteristic. Overall, the model provided by the present invention fills the gap in the existing research on the phenomenon of memory generalization caused by negative social experiences. It provides an important basis for exploring the impact of social defeat on social memory and a mouse model foundation for the research and preparation of drugs for social anxiety disorders such as social phobia.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements for some of the technical features. However, such modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Method for constructing a mouse model, characterized in that, Including: S1. Conduct acute subthreshold social defeat on normal mice: expose normal mice to aggressive mice and let them be attacked by the aggressive mice; after the attack, place the normal mice and the aggressive mice separately so that they can smell each other but cannot come into contact; S2. Put the mice that have experienced at least 1 acute subthreshold social defeat into a test box for adaptation testing. Place a first social cage and a second social cage at opposite corners of the test box, and count the interaction time of the mice with the empty first social cage and the second social cage, denoted as A1 and A2; subsequently, place a familiar aggressive mouse and a strange aggressive mouse in the first social cage and the second social cage respectively, and count the social interaction time of the mice with the familiar aggressive mouse and the strange aggressive mouse respectively, denoted as B1 and B2; S3. When B1 / A1 < 1 and B2 / A2 < 1, it indicates that the mouse has pathological social memory generalization, and a pathological social memory generalization mouse model is obtained.

2. The construction method according to claim 1, characterized in that, The duration of pathological social memory generalization of the pathological social memory generalization mouse model is at least 3 days.

3. The construction method according to claim 1 or 2, characterized in that The time for the normal mouse to be attacked by the aggressive mouse each time is 30 seconds.

4. The construction method according to claim 1 or 2, characterized in that, The number of attacks that the normal mouse receives from the aggressive mouse is 4 - 8 times.

5. The construction method according to claim 1 or 2, characterized in that, The normal mouse is a C57BL / 6J mouse.

6. The construction method according to claim 1 or 2, characterized in that, The aggressive mouse is an aggressive CD1 mouse.

7. The construction method according to claim 1 or 2, characterized in that, The statistical method for the social interaction time includes: taking the center point of the social cage as the center of the circle, drawing a circle with a radius of (R + 2), where R is the radius of the social cage in centimeters; counting the time when the mouse's head enters the circle, which is denoted as the social interaction time.

8. Use of the mouse model constructed by the construction method according to any one of claims 1-7, characterized in that, The application is selected from at least one of A1) - A2): A1) Application in developing drugs for alleviating and / or treating pathological social memory generalization; A2) Application in screening drugs for alleviating and / or treating pathological social memory generalization.

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