Method, device and application for detecting social rank behavior of mice

Abstract

The application discloses a method and device for detecting social rank behavior of mice and application thereof, and comprises the following steps: placing the mice to be detected in a low-temperature environment for precooling treatment; transferring the mice after precooling treatment to a test environment, wherein a heating plate is arranged at a central position in the test environment, and the size of the heating plate is only suitable for one mouse; recording the cumulative time of each mouse occupying the heating plate in the test environment; and arranging the social rank according to the length of the cumulative time, and the longer the cumulative time is, the higher the social rank is. By using the cold plate device, the environmental temperature can be accurately maintained at 0 DEG C, and the temperature fluctuation problem caused by the ice box in the traditional hot area experiment is avoided. The stable low-temperature stimulation ensures that the mice have the motivation to find the hot area, avoids the interference of excessive stress on the behavior result, and can complete the social rank detection of multiple mice in a cage in a short time.

Description

Technical Field

[0001] This invention belongs to the field of neurobiology, specifically relating to a method, apparatus, and application for detecting social hierarchy behavior in mice. Background Technology

[0002] Social hierarchy is the fundamental organizational mechanism of most animal societies. It determines the acquisition of resources and the distribution of social influence, and has a profound impact on various behaviors such as survival, health, and reproduction. Once established, social hierarchies are relatively stable, minimizing intense competition among group members and thus improving overall group efficiency. Higher-ranking individuals typically have priority access to food, mates, and territory, but may also bear higher physiological costs; lower-ranking individuals, with limited resources, can avoid direct confrontation through strategic behaviors (such as avoidance and alliances), thereby maintaining survival to some extent. To explore the neural mechanisms and behavioral basis behind social hierarchy, a robust, reliable, and standardized behavioral testing method is crucial.

[0003] Currently, the tube-drilling test is the primary method for detecting social hierarchy in mice, with some studies also using the hot zone test to further validate the results. The tube-drilling test, based on mice's use of spatial resources, simulates a common right-of-way confrontation in the wild: two mice, after training, enter from opposite ends of a tube and meet in the middle. The mouse that pushes its opponent out of the tube has a higher social hierarchy, while the mouse that exits first has a lower social hierarchy. Based on the transmissibility and stability of social hierarchy, pairwise testing can determine the social hierarchy of mice in the same cage, and dominant behavior indicators (such as pushing time and retreat distance) can be used to quantify the social hierarchy. However, it has the following drawbacks: First, it requires a high level of adaptation and training to the experimental environment, typically requiring at least 5 days for mice to reduce stress and master the rules; second, it takes a long time to obtain a stable social hierarchy, potentially 4-14 days for male mice, increasing the experimental period; third, due to the use of a round-robin pairing system, the number of pairings required increases exponentially (n×(n-1) / 2 times) when the number of mice in the same cage increases (e.g., n>4), significantly increasing the workload. Traditional hot zone experiments utilize animals' natural desire to stay warm by having mice in the same cage compete for a small warm corner in a cold environment. The longer a mouse occupies a warm corner, the higher its social status. This experiment introduces a cold environment as a negative stimulus, resulting in smaller fluctuations in mouse social status during the testing process, a shorter testing time, and the ability to simultaneously assess the social status of all mice in the same cage. Therefore, the number of mice tested does not affect the testing time—a significant advantage over the tube-drilling experiment. However, traditional hot zone experiments have the following key drawbacks: First, they lack a clear experimental procedure and specialized equipment. There is no systematic experimental data to support whether environmental acclimatization is necessary before the formal experiment, or for how long. The cold environment is often temporarily constructed using ordinary mouse cages placed on ice, making precise temperature control impossible and leading to unstable cold stimulus intensity, affecting the reproducibility of the results. Second, the location of the warm zone (heat source) is often poorly designed. Traditional designs place the warm zone in a corner, allowing the mouse occupying the warm zone to use the side walls for support, increasing its stability and making it less likely to be pushed off by competitors. This may overestimate its true social rank. Furthermore, corner locations sometimes allow multiple mice to crowd together and share the warm zone, creating a "shared warm corner" phenomenon, further obscuring the true reflection of rank differences. Existing patent application CN118556616A discloses a device and method for detecting social rank and social behavior in mice. Multiple mice are placed in an experimental chamber, providing a semi-natural environment.At the group level, based on the radio frequency identification (RFID) chip fixed to the mouse and the RFID position tracking system placed at the bottom of the experimental box, the activities or movements of each mouse in the group in a semi-natural environment are identified and tracked in real time. The focus is on how to identify the identity, location and complex social behavior of multiple mice with the same appearance in a group breeding condition that is close to the natural environment in a long-term, automatic and accurate manner, so as to stably and reliably quantify their social status. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method, apparatus, and application for detecting social hierarchy behavior in mice. Based on the traditional hot zone occupancy test, it provides a novel mouse social hierarchy detection device and standardized experimental procedure—including a precisely temperature-controlled cold plate, a small-sized heating plate in the center, and a clear adaptation and testing scheme—to more efficiently and accurately detect the social hierarchy of mice. This behavioral paradigm can be used to explore the neural circuit mechanisms of social hierarchy, the possible pathological causes of related diseases (such as autism and schizophrenia), and drug screening and verification.

[0005] To achieve the above objectives, firstly, the present invention provides a method for detecting social hierarchy behavior in mice, comprising the following steps: The mice to be tested were pre-cooled in a low-temperature environment. The pre-cooled mice were transferred to a test environment containing a centrally located heating plate, the size of which was only large enough for one mouse to occupy. Record the cumulative time each mouse occupies the heating plate in the test environment; The social ranks are sorted according to the length of the accumulated time, with a longer accumulated time indicating a higher social rank.

[0006] Furthermore, the temperature of the low-temperature environment is 0°C; the temperature of the heating plate is 34°C.

[0007] Furthermore, an adaptation training step is included before the formal testing: a single mouse is placed in a low-temperature environment for pre-cooling, and then transferred to the test environment to observe whether it finds and occupies the heating plate. If it fails, the operation is repeated until it succeeds.

[0008] Furthermore, the adaptation training was conducted one day before the formal test, with each pre-cooling and test lasting 20 minutes.

[0009] Furthermore, all mice in the same cage were pre-cooled simultaneously for 20 minutes; all pre-cooled mice were then transferred to the test environment simultaneously for 20 minutes.

[0010] Furthermore, continuous testing was conducted for several days, with tests repeated daily. The final social rank was determined by combining the results from multiple days. If the rank of each mouse was completely consistent across multiple days, that rank was taken as the final social rank of that cage of mice. If the rank of mice fluctuated during the testing process, the final social rank was determined using a weighted scoring method based on the results from multiple days, with the weight coefficient gradually increasing from the first day to the last day. The daily rank of each mouse was converted into a numerical value, multiplied by the corresponding weight for each day, and summed. The smaller the resulting weighted value, the higher the social rank.

[0011] Meanwhile, the present invention also provides a device for detecting social hierarchy behavior in mice, used to assist in implementing the method for detecting social hierarchy behavior in mice as described above, comprising a combined box and at least one cold plate box; the sides of the combined box and the cold plate box are transparent plates; a camera is disposed above the combined box. The bottom plate of the cold plate enclosure is made of metal or ceramic plate, and the cold plate enclosure is used to provide a low-temperature environment; The combined chamber is used to provide the testing environment. A heating plate is located at the bottom of the combined chamber and is located in the center of the chamber. The size of the heating plate is only large enough for one mouse to occupy. The heating plate is connected to a heating power source and contains a temperature controller.

[0012] Furthermore, the cold plate temperature of the cold plate box is set to 0℃, the heating plate temperature is set to 34℃, and the heating plate is a circular plate with a radius of 2cm and a height of 1cm.

[0013] Furthermore, the modular enclosure is composed of two cold-plate enclosures, and a camera is also installed on the side of the modular enclosure.

[0014] Furthermore, the present invention also provides an application of the above-described method for detecting social hierarchy behavior in mice, in at least one of the following uses: Extended to rat and guinea pig animal models for cross-species comparative studies of social behavior; This study analyzed the differences in emotional cognition, motor social interaction, immunity, and endocrine function among mice of different social ranks, revealing the impact of social rank on individual physiological and psychological states. Used for research on the mechanisms of diseases related to social class disorder and for drug screening; By combining fiber optic recording, optogenetics / chemogenetics, and two-photon calcium imaging, we will study the neural circuits, cell type-specific activities, and related molecular mechanisms involved in the formation of social hierarchies.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: The device of this invention has a simple structure and standardized experimental procedure, requiring no complex training or manual intervention. Compared with the tube drilling experiment, which requires several days of training and numerous pairing tests, this invention can complete the social rank detection of multiple mice in the same cage in a shorter time, significantly improving experimental efficiency. By using a cold plate device, the ambient temperature can be precisely maintained at 0°C, avoiding the temperature fluctuation problem caused by using ice boxes to build a cold environment in traditional hot zone experiments. Stable low-temperature stimulation ensures that mice are motivated to seek hot zones while avoiding excessive stress from interfering with behavioral results. This invention places the heating plate in the center of the combined box, and the size of the heating plate is only large enough for one adult mouse to occupy. This design overcomes two drawbacks of placing the warm zone in a corner of the traditional hot zone experiment: first, mice cannot use the wall for support to stabilize themselves, and individuals with higher social ranks must actively compete to occupy the hot zone, thus making the occupation time more realistically reflect their dominance; second, it avoids the ambiguity of rank caused by multiple mice crowding the warm zone at the same time, ensuring that only one mouse can occupy the hot zone at a time, improving the distinguishability and accuracy of the results. This invention has determined specific adaptation conditions and testing schemes through systematic experimental research. These standardized parameters fill the gaps in the operation procedures of traditional hot zone experiments. Attached Figure Description

[0016] Figure 1a Schematic diagram of a single cold plate housing Figure 1b This is a schematic diagram of the combined box structure.

[0017] Figure 2a This is a diagram showing the actual test results during the adaptation phase of the cold plate enclosure.

[0018] Figure 2b This is a diagram showing the actual test results during the adaptation phase of the combined enclosure.

[0019] Figure 3a This is a diagram showing the actual testing results of the social grade detection in the cold plate enclosure.

[0020] Figure 3b This is a diagram showing the actual testing results of the combined enclosure's social grade inspection.

[0021] Figure 4 This is a statistical result of the time mice of different social ranks occupied hot zones in a single experiment. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] The mouse social rank detection device provided by this invention comprises the following components: (1) A separate, temperature-adjustable cold plate chamber for pre-cooling mice, such as Figure 1a As shown; (2) A combined box consisting of two cold plates, used for formal social rank testing, such as Figure 1b As shown; (3) A heating plate located at the center of the combined box is used to simulate the hot zone (warm corner), see reference. Figure 1b ; (4) An adjustable power supply for precise control of the heating plate temperature, reference Figure 1b .

[0024] The cold plate temperature of the cold plate enclosure is set to 0℃ to ensure sufficient but non-injurious cold stimulation. The heating plate temperature is set to 34℃ via an adjustable power supply, a temperature close to the mouse's body surface temperature and naturally attractive to the mice. The heating plate is designed as a circular plate with a radius of 2cm and a height of 1cm, with an area only large enough for one adult mouse to stand or curl up, preventing multiple mice from occupying the warm area simultaneously. The heating plate is located in the central area of ​​the combined enclosure to prevent mice occupying the warm area from being pushed off by supports. A camera is also installed above the combined enclosure.

[0025] Furthermore, the cold-plate enclosure and the combined enclosure can be mounted on the same base, making the two enclosures and accessories an integrated structure.

[0026] A camera can also be installed on the side of the modular enclosure.

[0027] The bottom of the cold plate enclosure can be made of metal or ceramic plates, making it easy to achieve a low-temperature environment.

[0028] Adaptation phase Before the formal experiment began, each mouse underwent 20 minutes of acclimatization training to eliminate environmental stress and ensure it could effectively locate heat zones. This included the following steps: First, each mouse was pre-cooled for 20 minutes in a single metal cold plate chamber pre-set to 0°C. Figure 2a As shown, the mouse was made to experience a distinct cold sensation. Then, it was gently transferred to a combination chamber containing a 34°C heating plate, as indicated. Figure 2bAs shown, the mouse was recorded whether it found and stayed on the heating plate within 20 minutes. Generally, after 20 minutes of exploration, the mouse was able to instinctively find and occupy the warm heating plate. If the mouse still hadn't found the heating plate after 20 minutes, it was returned to its original cage to rest for 1 hour, and the above procedure was repeated until the mouse could successfully find and stay on the warm area. By comparing the effects of no adaptation, adaptation for 1 day, and adaptation for 2 days, it was determined that adaptation training 1 day before the formal experiment achieved the ideal effect, meaning that the mouse could quickly find and stay on the warm heating plate after entering the cold plate combination box.

[0029] Experimental phase In the formal experiment, the mice to be tested were first individually marked (e.g., by marking their tails or backs with numbers using a non-toxic marker) to ensure that each mouse could be clearly distinguished during video recording. Then, all the mice in the same cage were simultaneously placed in a single, pre-cooled cold plate chamber set to 0°C for 20 minutes, as per reference. Figure 3a After pre-cooling, all mice were gently transferred to a combination chamber equipped with a 34°C heating plate, as per [reference needed]. Figure 3b Simultaneously, video recording was initiated and continued for 20 minutes. To ensure the stability and reliability of the social ranking results, the formal test should be conducted for four consecutive days, repeating the above procedure each day. The final social ranking of the mice was determined based on the combined results of the four days of testing.

[0030] Social ranking evaluation stage After the experiment, the recorded videos were analyzed to calculate the cumulative time each mouse spent occupying the heated plate (hot zone) within 20 minutes. Longer occupation time indicated a higher social rank for the mouse, while shorter occupation time indicated a lower social rank. Based on the daily occupation time, each mouse received a provisional social rank ranking each day. Finally, the final social rank ranking for each mouse was determined using a weighted or unweighted method, based on the combined results of the four days of testing.

[0031] The specific test includes the following steps: Preparations before social level test First, mice of similar weight and age were housed together for at least three weeks to establish a relatively stable social hierarchy. During this period, environmental conditions (noise, light, temperature, humidity) were strictly controlled to avoid external interference affecting the formation of the hierarchy.

[0032] Two to three days before the formal experiment begins, the operator needs to gently handle each mouse daily, ensuring the mouse is relaxed in the operator's hand. The mouse's condition can be assessed by indicators such as breathing rate, whether it curls up into a ball, and whether it produces excrement. After handling, the mouse is returned to its original cage. This helps reduce the mouse's fear and stress response to the experimenter, improving the stability of subsequent experiments.

[0033] Each mouse was individually tagged with a number on its tail or back skin using a non-toxic marker to ensure that each mouse could be clearly distinguished under video recording conditions.

[0034] Social Rank Adaptation Stage The day before the formal testing, each mouse underwent individual acclimatization training. The specific procedure was as follows: each mouse was placed in a pre-cooled metal cooling chamber set to 0°C for 20 minutes. (Refer to...) Figure 2a Then, gently transfer it to a cold plate assembly box containing a 34°C heating plate, for reference. Figure 2b Observe for 20 minutes. If the mouse can find and stay on the heating plate for a long time within 20 minutes, the adaptation is complete; if the mouse still cannot find the hot area at the end of 20 minutes, put it back in its original cage to rest for 1 hour, and then repeat the above operation until successful.

[0035] All mice to be tested for social status must complete the above adaptation process before the formal experiment to ensure that the mice are familiar with the location of the hot zone in advance and reduce the interference of exploration time in the formal experiment.

[0036] Social rating testing phase Before the formal testing, first check that the tag number of each mouse is clearly identifiable. After confirming that it is correct, place all the mice to be tested in the same cage (for example, 4 mice) simultaneously in a single metal cold plate box preset to 0℃ for pre-cooling for 20 minutes. Figure 3a .

[0037] After pre-cooling, gently transfer all mice to a combination chamber containing a 34°C heating plate, as per [reference needed]. Figure 3b Immediately after recording, video recording began and lasted for 20 minutes. After recording, all mice were returned to their original cages. The experimental chamber was then thoroughly cleaned with 75% ethanol to remove any residual odor, urine, and feces. Only after the chamber was completely dry and free of any ethanol odor could the next cage of mice be tested.

[0038] The social rank of each cage of mice needs to be monitored for four consecutive days, with the above procedure repeated daily. Monitoring over multiple days can eliminate the influence of random factors (such as fluctuations in the mouse's condition on a given day) on the rank determination, thus improving the reliability of the results.

[0039] Social rating After the video recording ended, the cumulative time each mouse occupied the heated plate (hot zone) within 20 minutes was recorded based on its assigned number. A longer occupation time indicated a higher social status for the mouse; a shorter occupation time indicated a lower social status. Daily monitoring could be used to temporarily rank mice within the same cage based on occupation time.

[0040] The final social rank of mice was determined by a comprehensive analysis of the results from four days of testing, using the following rules: If the ranking of each mouse remained completely consistent across the four days (without fluctuation), that ranking was taken as the final social rank of the mouse in that cage. If the ranking of mice fluctuated during the testing process (e.g., a mouse's ranking differed on different days), a weighted scoring method was used to calculate the final rank. The weighting coefficients were set as follows: Day 1: 0.1, Day 2: 0.2, Day 3: 0.3, Day 4: 0.4. During the calculation, the ranking of each mouse for each day was first converted into a numerical value (e.g., 1st place = 1, 2nd place = 2, and so on), then multiplied by the corresponding weight for each day and summed. The smaller the final weighted value, the higher the social rank of the mouse; the larger the weighted value, the lower the social rank. This weighting scheme considered the trend of mice gradually stabilizing during continuous testing—the later days had a higher weight, better reflecting their true social rank. The time it took for mice to occupy hot zones was also recorded (20 minutes).

[0041] In summary, this invention provides a method and apparatus for detecting social hierarchy behavior in mice. The method includes: first, pre-cooling the mice in a low-temperature environment; then, transferring the pre-cooled mice to a testing environment. A heating plate, sized for only one mouse, is positioned at the center of this testing environment. The cumulative time each mouse occupies the heating plate is recorded, and the social hierarchy is ranked according to the cumulative time, with longer times indicating higher hierarchy. To implement this method, the apparatus includes a cold plate box with transparent sides and a combined box. The cold plate box provides a stable low-temperature environment with a metal or ceramic base. The combined box has a heating plate with a temperature controller and a heating power supply at its bottom center. A camera is positioned above the combined box for behavioral recording. This technical solution avoids interference from traditional hot zone experiments where mice rely on corner support or share space, making the competition results more accurately reflect dominance. Simultaneously, the precisely temperature-controlled cold plate environment ensures the stability of cold stimulation and the reproducibility of the experiment. Combined with standardized procedures, it can efficiently and accurately detect the social hierarchy of multiple mice in the same cage.

[0042] The method and apparatus described in this invention for detecting social hierarchy behavior in mice can be used in the following studies: It can quickly and accurately detect social hierarchy in mice and can be extended to other animal models such as rats and guinea pigs for comparative studies of social behavior across species. It can be used to analyze the differences in emotional cognition, motor socialization, immunity, endocrine and other aspects of mice with different social ranks, and to reveal the impact of social rank on the physiological and psychological state of individuals; It can be used for research on the mechanisms of social hierarchy disorders (such as autism and schizophrenia) and drug screening. It can be combined with technologies such as fiber optic recording, optogenetics / chemogenetics, and two-photon calcium imaging to study the neural circuits, cell type-specific activities, and related molecular mechanisms of social hierarchy formation. This device and method have the advantages of standardization and high repeatability, and can be incorporated into the behavioral testing process as a routine scheme for social grading.

[0043] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for detecting social hierarchy behavior in mice, characterized in that, Includes the following steps: The mice to be tested were pre-cooled in a low-temperature environment. The pre-cooled mice were transferred to a test environment containing a centrally located heating plate, the size of which was only large enough for one mouse to occupy. Record the cumulative time each mouse occupies the heating plate in the test environment; The social ranks are sorted according to the length of the accumulated time, with a longer accumulated time indicating a higher social rank.

2. The method for detecting social hierarchy behavior in mice according to claim 1, characterized in that, The temperature of the low-temperature environment is 0°C; the temperature of the heating plate is 34°C.

3. The method for detecting social hierarchy behavior in mice according to claim 1, characterized in that, The formal testing process also includes an adaptation training step: a single mouse is placed in a low-temperature environment for pre-cooling, and then transferred to the test environment to observe whether it finds and occupies the heating plate. If it fails, the operation is repeated until it succeeds.

4. The method for detecting social hierarchy behavior in mice according to claim 3, characterized in that, The adaptation training was conducted one day before the formal test, with each pre-cooling and test lasting 20 minutes.

5. The method for detecting social hierarchy behavior in mice according to claim 1, characterized in that, All mice in the same cage were pre-cooled simultaneously for 20 minutes; all pre-cooled mice were then transferred to the test environment simultaneously for 20 minutes.

6. The method for detecting social hierarchy behavior in mice according to claim 1, characterized in that, The tests were conducted continuously for several days, with repeated tests performed each day. The final social rank was determined by combining the results of the multiple days. If the rank of each mouse was completely consistent over the multiple days, then that rank was taken as the final social rank of the mice in that cage. If the mouse's ranking fluctuates during the testing process, the final social ranking is determined by a weighted scoring method based on the results of multiple days, with the weight coefficient gradually increasing from the first day to the last day. The ranking of each mouse on each day is converted into a numerical value, multiplied by the weight of the corresponding day, and summed. The smaller the weighted value, the higher the social ranking.

7. A device for detecting social hierarchy behavior in mice, characterized in that, The method for assisting in the detection of social hierarchy behavior in mice as described in any one of claims 1-6 includes a combined box and at least one cold plate box; the sides of the combined box and the cold plate box are transparent plates; the sides of the box are transparent plates, and a camera is disposed above the combined box; The bottom plate of the cold plate enclosure is made of metal or ceramic plate, and the cold plate enclosure is used to provide a low-temperature environment; The combined chamber is used to provide the testing environment. A heating plate is located at the bottom of the combined chamber and is located in the center of the chamber. The size of the heating plate is only large enough for one mouse to occupy. The heating plate is connected to a heating power source and contains a temperature controller.

8. The apparatus for detecting social hierarchy behavior in mice according to claim 7, characterized in that, The cold plate temperature of the cold plate box is set to 0℃, and the heating plate temperature is set to 34℃. The heating plate is a circular plate with a radius of 2cm and a height of 1cm.

9. The device for detecting social hierarchy behavior in mice according to claim 7, characterized in that, The modular enclosure is composed of two cold-plate enclosures, and a camera is also installed on the side of the modular enclosure.

10. An application of the method as described in any one of claims 1-6, characterized in that, Application in at least one of the following uses: Extended to rat and guinea pig animal models for cross-species comparative studies of social behavior; This study analyzed the differences in emotional cognition, motor social interaction, immunity, and endocrine function among mice of different social ranks, revealing the impact of social rank on individual physiological and psychological states. Used for research on the mechanisms of diseases related to social class disorder and for drug screening; By combining fiber optic recording, optogenetics / chemogenetics, and two-photon calcium imaging, we will study the neural circuits, cell type-specific activities, and related molecular mechanisms involved in the formation of social hierarchies.

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