Ultrasonic vocalization-based behavioral detection method and application of information attention bias in mice

Abstract

The present invention discloses a method and application of behavioral detection of mouse information attention bias based on ultrasonic sounding, which combines social information contained in ultrasound with detection of attention bias, takes mice as objects receiving social information, uses mouse ultrasound representing different meanings as sound stimulation of social information, uses pure sound as control sound stimulation, compares the attention bias of mice to different sound stimulations in experiments, thereby detecting abnormalities in mice receiving social information, and providing important technical means for studying the neural mechanism of social information attention; based on the cue-target paradigm, objectively and impartially quantifies and compares the attention bias of mice to sound stimulation containing social information; and is used to study the attention bias of mice to ultrasound containing different types of social information, with clear training indicators and corresponding judgment methods from training to formal testing stages, and the obtained results of the attention bias of mice to social information have high reliability and validity.

Description

Technical Field

[0001] The invention belongs to the technical field of neurobiology, and in particular relates to a method and application of detecting information attention bias behavior of mice based on ultrasonic sounding. Background Art

[0002] Social behavior is a kind of behavior with important significance in many social animals including humans. The process of individuals forming a personality adapted to society and mastering socially recognized behaviors is called socialization. Humans rely on various social information received in the process of socialization, among which social information with language as the medium of communication accounts for a large proportion and plays an important role in human social life. In the process of transmitting social information, individuals need to maintain a certain degree of attention in order to extract social information from the numerous and complex stimuli in the external environment. Therefore, attention to social information is one of the key functions required in the process of individual socialization.

[0003] Mice are currently the most commonly used model animals and are crucial for studying the neural mechanisms of social behavior. Testing of mouse social behavior is still mainly focused on active social behavior detection. For example, the three-box experiment and the home cage free social experiment, all of these experiments use the test mice as the subject of social information, but ignore their role as the receiver of social information. However, social information is a two-way process, and its reception is equally important. Compared with humans, mice have underdeveloped vision, so it is not the main way for them to transmit social information. Although mice have a developed sense of smell, odors are easy to diffuse, making it difficult to implement quantitative stimulation, and there are high requirements for experimental equipment, so they are not the first choice for social information media in experiments. Mice can emit ultrasonic sounds of a unique frequency to transmit social information, which can be simply understood as the "language" of mice. Sound transmission has the advantages of good directionality and easy quantification of various parameters such as stimulus intensity, and is a potential medium for social information transmission. At present, the detection of attention to social information in animal experiments is mainly based on the reaction time of a single visual stimulus. There is no mature behavioral paradigm to detect the attention bias of more complex auditory stimuli with social information attributes.

[0004] Mouse ultrasonic vocalization refers to the sound emitted by mice with a frequency greater than 20KHz, which is usually emitted in specific social situations and under stimulation. Young mice can emit ultrasound when separated from their mothers, when subjected to low temperature stimulation and tactile stimulation, thus inducing maternal behaviors such as exploration, breastfeeding and nesting in mother mice. Adult mice emit ultrasound during courtship, territorial social and hierarchical social behaviors. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a behavioral detection method for mouse information attention bias based on ultrasonic sounding, which combines the social information contained in ultrasound with the detection of attention bias, takes mice as the object of receiving social information, uses mouse ultrasound representing different meanings as sound stimulation of social information, and uses pure tone as control sound stimulation. The attention bias of mice to different sound stimuli in the experiment is compared, thereby detecting the abnormality of mice in receiving social information, and providing an important technical means for studying the neural mechanism of social information attention.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present invention is: a method for detecting the behavioral information attention bias of mice based on ultrasonic sounding, and a device for detecting the social information attention bias, comprising an opaque acrylic operation box, in which an ordinary speaker, two speakers that can play ultrasound and pure sound, two signal lights, two nose touch sensors and a food bowl are arranged, the signal lights and the nose touch sensors are arranged one by one, the ordinary speaker and the two speakers that can play ultrasound and pure sound are connected to the output end of the playback device, and the two signal lights are provided with a control switch; comprising the following processes:

[0007] During the food restriction phase, the experimental mice were housed in single cages with limited food but unlimited water. The weight of the mice was recorded every day and controlled at 80% to 90% of the original weight.

[0008] In the training phase, mice were trained to adapt to the played audio and to receive sugar pellet rewards by touching their noses according to the signal lights;

[0009] During the attention bias detection stage, the left and right speakers will randomly play sound cues. After the sound cues, the left and right signal lights will light up randomly. The mice can only get sugar pill rewards by touching the corresponding nose pad under the lit signal light. The cue effects of different sound stimuli are calculated through the reactions to valid cues and invalid cues, and the attention bias of mice to sound stimuli related to social information, that is, ultrasound, is compared based on the cue effects.

[0010] A soundproof box is set outside the opaque acrylic operating box.

[0011] The training phase is as follows: first, the mouse is placed in the operating box for adaptation, and then the left and right speakers are controlled to randomly play pure tones or ultrasound clips in the mouse ultrasound library, with the two speakers playing alternately without overlap;

[0012] After the mice adapted to the two speakers playing the sound, they began to learn to receive a sugar pill reward by touching the corresponding nose pad under the signal light according to the instruction of the signal light.

[0013] When the sound and signal light are on the same side, the sound prompt is a valid clue. When the sound and signal light are on the opposite side, the sound prompt is an invalid clue.

[0014] During the training phase, after the mice were placed in the test box, they first adapted to the environment, and then the left and right speakers began to randomly play pure tones of set duration and frequency or ultrasound clips of set duration.

[0015] After the sound adaptation is completed, nose touch training begins. Before the start of each training experiment, the middle speaker will play white noise of a preset length. After 1 second, one of the left and right signal lights will light up randomly for 2 seconds. If the mouse touches the nose touch under the corresponding signal light within 10 seconds after the signal light turns on, the task is successful and a sugar pill reward will be given. If the opposite nose touch is touched, the task fails and no reward is given. Failure to touch the correct nose touch within 10 seconds is considered a task failure and the mouse automatically enters the next training experiment. The interval between each training experiment is 2 minutes, and there are 15 training experiments in total for one training. When the success rate of the mouse reaches the set value, the reaction time window is shortened for intensive training. After reaching the set value again, the reaction time window is shortened again for intensive training. When the mouse can touch the correct nose touch within the 4-s time window to obtain food, the accuracy rate reaches the set value and remains stable for 3 trainings, it enters the testing phase.

[0016] Each playback is randomly generated in the left or right speaker. The randomly played content is pure sound or ultrasound. The playback between the two speakers does not overlap. Pure sound and ultrasound are played m times each, of which m / 2 times are in the right speaker and the other m / 2 times are in the left speaker. The interval between each playback is 30s.

[0017] Each task includes n training experiments. At the beginning of each training experiment, the middle speaker emits a 2-second white noise prompt to start the experiment. After 1 second, the left or right speaker randomly emits a pure tone or ultrasound containing social information, which lasts for 2 seconds. In the last second of the sound prompt, the signal light on either side is randomly turned on for 2 seconds. The mouse needs to choose the corresponding nose touch below and touch it according to the signal light within 4 seconds after the signal light is on to get a sugar pill reward. The time from the signal light turning on to the mouse touching the nose touch is the reaction time. In each task, ultrasound and pure tone stimulation appear n / 2 times on the left and n / 2 times on the right respectively. The signal light and sound stimulation are paired so that half of them are valid cues and half are invalid cues. Cue effect = invalid cue reaction time - valid cue reaction time.

[0018] The larger the cue effect value, the greater the mouse's attention bias towards the cue. When the cue effect of ultrasound containing social information is greater than the cue effect of pure sound, it proves that the mouse has a certain attention bias towards social information.

[0019] The application of the mouse information attention bias behavioral detection method based on ultrasonic sounding of the present invention is to detect the abnormal changes in the social information attention bias of mice under relevant mental illness states based on the detection results of the social information attention bias of mice.

[0020] Or based on the social information attention bias test results of mice, evaluate the improvement of specific treatments on abnormal social information attention bias;

[0021] Or based on the detection process, the neural circuit mechanism that regulates the social information attention bias of mice is studied through optical fiber recording, optogenetics or chemical genetics methods.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects:

[0023] The present invention is based on the cue-target paradigm, and objectively and impartially quantifies and compares the attention bias of mice to sound stimulation containing social information; it is used to study the attention bias of mice to ultrasound containing different types of social information; the entire experimental process is standardized, and there are clear training indicators and corresponding judgment methods from training to formal testing stages, and the obtained results of the attention bias of mice to social information have high reliability and validity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the behavioral testing apparatus (left) and experimental flow chart (right).

[0025] Figure 2 Schematic diagram of valid clues (left) and invalid clues (right). DETAILED DESCRIPTION

[0026] refer to Figure 1 and Figure 2 The present invention provides a method for detecting the behavioral information attention bias of mice based on ultrasonic sounding, which is characterized in that the device for detecting the social information attention bias comprises an opaque acrylic operating box, in which an ordinary speaker, two speakers that can play ultrasound and pure sound, two signal lights, two nose touch sensors and a food bowl are arranged, the signal lights and the nose touch sensors are arranged in a one-to-one correspondence, the ordinary speaker and the two speakers that can play ultrasound and pure sound are connected to the output end of the playback device, and the two signal lights are provided with a control switch; the device for detecting the social information attention bias provided by the present invention comprises a soundproof box of 80*80*80cm and an opaque acrylic operating box of 20*20*20cm, in which an ordinary speaker, two speakers that can play ultrasound and pure sound, two signal lights, two nose touch sensors and a food bowl are arranged in the operating box, the ordinary speaker and the two speakers that can play ultrasound and pure sound are connected to the output end of the playback device, and the two signal lights are provided with a control switch.

[0027] The mouse social ultrasound library is a collection of audio file clips of ultrasound emitted by mice in different social situations, including but not limited to ultrasound clips of baby mice separated from their mothers, ultrasound clips of adult female mice socializing with the same sex, ultrasound clips of adult male mice socializing with the same sex, and ultrasound clips of adult mice of the opposite sex socializing.

[0028] The behavioral testing method includes the following procedures:

[0029] 1. Food restriction phase

[0030] During this stage, the experimental mice were housed in single cages with limited food and unlimited water. The weight of the mice was recorded daily and controlled at 80% to 90% of their original weight.

[0031] 2. Training Phase

[0032] This stage is divided into two steps. First, the mouse is placed in the operating box for adaptation. Then the left and right speakers are controlled to randomly play pure tones or ultrasound clips in the mouse ultrasound library. The two speakers play alternately without overlap. After the mouse has adapted to the left and right speakers that play the sound, it begins to learn to touch the corresponding nose touch under the signal light according to the instruction of the signal light to get a sugar pill reward.

[0033] 3. Attention Bias Detection Stage

[0034] After the mice learned to choose the corresponding nose touch according to the indicator light to get the sugar pill reward, the formal attention bias test experiment began: the left and right speakers would randomly play sound prompts, which could be pure tones or ultrasound containing social information. After the sound prompts, the left and right signal lights would light up randomly, and the mice could only get the sugar pill reward by touching the corresponding nose touch under the lighted signal light. When the sound and the signal light are on the same side, the sound prompt is a valid clue, and when the sound and the signal light are on the opposite side, the sound prompt is an invalid clue. The cue effects of different sound stimuli were calculated by the reaction time of the valid cues and invalid cues, and the attention bias of mice to the sound stimuli related to social information, that is, ultrasound, was compared based on the cue effects.

[0035] Specific test steps:

[0036] (1) The food restriction phase was from 3 days before the start of the experiment to day 0. All experimental mice were housed in individual cages and their initial weight was recorded. Food was restricted but water was not. A small amount of mouse chow and sugar pellets were fed at 8:00 a.m. and 8:00 p.m. every day. The weight of the mice was controlled at 80%-90% of the baseline level. The changes in the weight of the experimental mice were recorded throughout the behavioral test.

[0037] (2) During the training phase, after placing the mice in the test box, they were allowed to adapt to the environment for 5 minutes, and then the left and right speakers began to randomly play a 2-second, 7500Hz, 75dB pure tone or a 2-second ultrasound segment (the ultrasound segment can be selected from the corresponding ultrasound library according to the experimental requirements). Each playback was randomly generated in the left or right speaker, and the content played randomly was pure tone or ultrasound. The playback between the two speakers did not overlap. The pure tone and ultrasound were played 10 times each, 5 times in the right speaker and 5 times in the left speaker, and the interval between each playback was 30 seconds.

[0038] After the sound adaptation is completed, the nose touch training begins. Before each trial, the middle speaker will play 2s of white noise (1-20KHz, 75dB). After 1s, one of the two signal lights on the left and right will light up randomly for 2s. If the mouse touches the nose touch under the corresponding signal light within 10s after the signal light turns on, the task is successful and a sugar pill reward will be obtained. If the opposite side of the nose touch is touched, the task fails and there is no reward. If the correct nose touch is not touched within 10s, the task is also judged as a failure and the next trial is automatically entered. The interval between each trial is 2min, and a total of 15 trials are trained at one time. When the success rate of the mouse reaches 90%, the reaction time window is adjusted from 10s to 7s for reinforcement training. After reaching 90% again, the reaction time window is adjusted from 7s to 4s for reinforcement training again. When the mouse can touch the correct nose touch within the 4s time window to obtain food with a correct rate of 90% and continues to be stable for 3 trainings, it enters the test phase.

[0039] (3) In the test phase, each session contained 40 trials. At the beginning of each trial, the middle speaker emitted a 2-second white noise to indicate the start of the experiment. One second later, the left or right speaker randomly emitted a pure tone or ultrasound containing social information for 2 seconds. In the last second of the sound prompt, a signal light on either side was randomly turned on for 2 seconds. The mouse had to choose the corresponding nose touch below and touch it within 4 seconds after the signal light was on to receive a sugar pill reward. The time from the signal light turning on to the mouse touching the nose touch was the reaction time. In each session, ultrasound and pure tone stimulation appeared 20 times on the left and 20 times on the right. The signal light and sound stimulation were paired so that half of them were valid cues and half were invalid cues.

[0040] 5.4 Calculation of cue effect

[0041] Cue effect = invalid cue reaction time - valid cue reaction time

[0042] Interpretation of cue effect: The larger the cue effect value, the greater the attention bias of mice to the cue. When the cue effect of ultrasound containing social information is greater than the cue effect of pure sound, it proves that mice have a certain attention bias for social information, and a facilitation effect occurs when ultrasound is used as an effective cue. In some mental illness models, the attention bias of mice to social information may disappear.

[0043] Based on the device and method of the present invention, the following applications can be performed:

[0044] (1) Detect mice's attention bias toward ultrasound with social information;

[0045] (2) In the mother-infant separation experiment, ultrasound of the pups was used as a sound stimulus with social information to detect the maternal nature of the mother rats;

[0046] (3) To study the abnormal changes in social information attention bias in related mental illness states, such as autism model mice, by detecting the social information attention bias of mice;

[0047] (4) Study the neural circuit mechanisms that regulate social information attention bias in mice through fiber optic recording, optogenetics, or chemogenetics;

[0048] (5) To evaluate the effects of specific treatments on improving abnormal social attention bias by detecting the social attention bias in mouse models of related psychiatric disorders.

Claims

1. A behavioral detection method for information attention bias in mice based on ultrasonic sounding. It is characterized in that The device for detecting attention bias based on social information includes an opaque acrylic operation box, in which an ordinary speaker, two left and right speakers that can play ultrasound and pure sound, two signal lights, two nose touches and a food bowl are arranged, and the signal lights and the nose touches are arranged one by one. The detection method includes the following process: During the food restriction phase, mice were housed in individual cages with limited food but unlimited water. The weight of the mice was recorded daily and controlled at 80% to 90% of their original weight. During the training phase, mice were trained to adapt to the audio being played, and to receive sugar pill rewards by touching their noses according to the signal lights. First, the mice were placed in the operating box for adaptation, and then the left and right speakers were controlled to randomly play pure tones or ultrasound clips in the mouse ultrasound library, with the left and right speakers playing alternately without overlapping. After the mice adapted to the two speakers playing the sound, they began to learn to receive a sugar pill reward by touching the corresponding nose pad under the signal light according to the instruction of the signal light. When the sound and the signal light are on the same side, the sound prompt is a valid clue; when the sound and the signal light are on the opposite side, the sound prompt is an invalid clue. During the attention bias detection phase, the left and right speakers will randomly play sound prompts, and after the sound prompts, the left and right signal lights will randomly light up. The mice can only get the sugar pill reward by touching the corresponding nose touch under the lighted signal light. The cue effects of different sound stimuli are calculated by the reaction time of effective cues and invalid cues, and the attention bias of mice to sound stimuli related to social information, that is, ultrasound, is compared based on the cue effects. Each detection task includes n training experiments. At the beginning of each training experiment, the middle ordinary speaker emits a 2-second white noise prompt. After 1 second, the left or right speaker randomly emits a pure tone or ultrasound containing social information, which lasts for 2 seconds. In the last second of the sound prompt, the signal light on either side is randomly turned on for 2 seconds. The mouse needs to choose the corresponding nose touch below and touch it according to the signal light within 4 seconds after the signal light is on to get a sugar pill reward. The time from the signal light turning on to the mouse touching the nose touch is the reaction time. In each detection task, ultrasound and pure tone stimulation appear n / 2 times on the left and n / 2 times on the right respectively. The signal light and sound stimulation are paired so that half of them are valid cues and half are invalid cues. Cue effect = invalid cue reaction time - valid cue reaction time.

2. A method for detecting information attention bias behavior of mice based on ultrasonic sounding according to claim 1, It is characterized in that A soundproof box is arranged outside the opaque acrylic operating box; the ordinary speaker and two speakers that can play ultrasound and pure sound are connected to the output end of the playing device, and two signal lights are provided with control switches.

3. The method for detecting information attention bias behavior of mice based on ultrasonic sounding according to claim 1, It is characterized in that After the sound adaptation is completed, nose touch training begins. Before the start of each training experiment, the ordinary speaker in the middle will play white noise of a preset length. After 1 second, one of the left and right signal lights will randomly light up for 2 seconds. If the mouse touches the nose touch under the corresponding signal light within 10 seconds after the signal light lights up, the task is successful and a sugar pill reward will be obtained. If the opposite side of the nose touch is touched, the task fails and no reward is given. Failure to touch the correct nose touch within 10 seconds is considered a task failure and the mouse automatically enters the next training experiment. The interval between each training experiment is 2 minutes, and there are 15 training experiments in total for one training. When the success rate of the mouse reaches the set value, the reaction time window is shortened for intensive training. After reaching the set value again, the reaction time window is shortened again for intensive training. When the mouse can touch the correct nose touch within the 4 second time window to obtain food, the accuracy rate reaches the set value and remains stable for 3 trainings, it enters the detection stage.

4. The method for detecting information attention bias behavior of mice based on ultrasonic sounding according to claim 1, It is characterized in that Each time, the left or right speaker is randomly played. The randomly played content is pure sound or ultrasound. The playback between the left and right speakers does not overlap. Pure sound and ultrasound are played m times each, of which m / 2 times are played in the right speaker and the other m / 2 times are played in the left speaker. The interval between each playback is 30s.

5. The method for detecting information attention bias behavior of mice based on ultrasonic sounding according to claim 1, It is characterized in that The larger the cue effect value, the greater the mouse's attention bias towards the cue. When the cue effect of ultrasound containing social information is greater than the cue effect of pure sound, it proves that the mouse has a certain attention bias towards social information.

6. Use of the method for detecting information attention bias behavior of mice based on ultrasonic sounding according to any one of claims 1 to 5, It is characterized in that Based on the results of social information attention bias detection in mice, we can identify the abnormal changes in social information attention bias in mice under relevant mental illness conditions. Or based on the social information attention bias test results of mice, evaluate the improvement of specific treatments on abnormal social information attention bias; Or based on the detection process, the neural circuit mechanism that regulates the attention bias of mice to social information can be studied through fiber optic recording, optogenetics or chemogenetics methods.

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