Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test
September 3rd, 2015
Ultrasonic vocalizations (USVs) in mice differ depending on age, sex, condition, and genetic background. Using two ultrasound emitters broadcasting simultaneously in different locations, this two-choice test can evaluate murine recognition and preference responses to different characteristics of USVs.
The overall goal of this procedure is to determine ultrasonic vocalization or USV preferences in mice using a two choice playback test. This is accomplished by first recording U SVS from a live individual. The second step is to edit and trim recorded us vs accordingly and generate stereophonic sound files.
Next, the ultrasound emitters are calibrated and the USV volume is adjusted. Using the attenuator and amplifiers, the final step is to introduce the subject mouse into the test box and start playing the two sound file simultaneously. Ultimately, behavioral monitoring can reveal if the mouse shows a preference for one sound over the other.
The USV emitters we have developed can help us to answer key questions concerning my spoke communications such as parties, biological significance of different type of us vs. The main advantage of our caustic emitter over existing devices like Pazo electric transducers is that the frequency response has no resonant peaks. The broadband flood Mississippi in the ultrasound vision with no harmony distortions makes it possible to use the emitter for reproducing complicated acoustic communication course.
Through this method can provide insight into the female response towards s bs or males. It can also be applied to other type of social interaction such as between mother and pops, between adult males and between adult females. To begin obtain sexually experienced male mice and virgin female mice, track the esra cycle by taking vaginal smears daily.
Stain the smears with Giza and determine the face of the esra cycle according to standard procedures. To record ultrasonic vocalizations or US vs. Connect an ultrasonic condenser microphone to a PC using a USB ad converter.
Set the gain level on the ad converter to seven record acoustic data in 16 bit. Format files with a sampling rate of 400 kilohertz using audio capture software for sound playback, connect a DA converter to the PC and then connect two amplifiers to the DA converter through an attenuator repair a small cage with a hole in the sidewall that is six centimeters in diameter. Cover the hole with 0.5 centimeter wire mesh.
Avoid using bedding or anything else that may produce rustling noises and contaminate the recording. Place the ultrasound microphone beside the mesh covered hole. When ready.
Place a male mouse and a sexually mature female mouse in either mees or diastrous In the cage. Record us vs. For three to five minutes.
The test box is divided into three compartments, a neutral zone and rooms A and B gates. Separate the neutral zone from the other rooms. Rooms A and B each have a four centimeter hole covered with wire mesh at the end.
Place the ultrasound emitter against the mesh and surround it with rubber plates To prevent sound leakage. Place lead seals on both sides of the wall between rooms A and B for sound insulation to create the playback sounds. Capture a 22nd segment of USB's from the previously recorded file.
Using digital audio editing software, select and capture background noise from this segment and create a 22nd background noise file by repeating the noise segment to play back the two sound files. Simultaneously export them as a single stereo sound file with one file for the left ear and the other file for the right ear. Filter the sound files using a high-pass filter with a cutoff of 40 kilohertz.
Reduce noise using the noise reduction tool. In post-processing software, select and capture a segment of the file that contains no US VS.As the noise reduction profile and run noise reduction with level 40 to calibrate sound levels during playback. First place the microphone 10 centimeters away from the ultrasound emitters.
Begin the playback and measure the sound pressure level in decibels. Use the attenuator and amplifiers to adjust the volume of the US v's. Generated by the ultrasound emitters to the same sound pressure level as the male US vs.
Recorded earlier. Confirm that the two ultrasound emitters exhibit the same sound pressure level using a calibration sound when reproducing a sound file composed of two streams of us vs. Adjust the two generated sound pressure levels to the same level.
To begin the two choice test close the gates to rooms A and B and habituate the female subject to the neutral zone. For 30 minutes, cover the box with acrylic board to prevent escape. After 30 minutes, record the entire procedure using a video camera mounted above the box.
Remove the gate and allow the female to freely explore the rest of the test box, including the sound zones. Once the female has investigated both mesh circles and has returned to the neutral zone, start playing the 22nd sound file prepared earlier. Monitor the animal's behavior for five to 10 minutes while repeatedly playing the sound file.
Switch the location of the ultrasound emitters randomly to avoid creating a side bias between the two rooms. Female mice did not show a preference between rooms A and B when allowed to explore the chambers before sound playback. The number of room entries was similar between chambers containing background noise or male US vs.
However, subjects spent a significantly longer amount of time in the chamber in which us vs were regenerated. Especially in the first five minutes. Exploration of the middle zone was not altered by the sound playback.
Females spent significantly more time within the sound zone and searching the wire mesh in the chamber playing male US vs. Especially in the first five minutes, both B six and bub. Females spent more time investigating the side paired with male s vs from another strain.
After watching this video, you should have a good understanding of how to determine use s spray preference in mice using a two choice flavor test After its development. This technique paved the way for researchers in the field or brand research to explore sound human interaction. USV in must differ depending on age, sex condition, and genetic backgrounds.
Following this procedure, USB profiles of transgenic mass can be measured to determine if sending and the saving USBs are altered.
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Overview
This study investigates ultrasonic vocalizations (USVs) in mice, focusing on their preferences based on various factors such as age, sex, and genetic background. A two-choice playback test is utilized to assess murine recognition and preference responses to different USV characteristics.
Key Study Components
Area of Science
- Neuroscience
- Behavioral Analysis
- Animal Communication
Background
- Ultrasonic vocalizations are important for mouse communication.
- USVs vary with age, sex, and genetic factors.
- Understanding USV preferences can provide insights into mouse behavior.
- Playback tests are a common method for studying animal preferences.
Purpose of Study
- To determine USV preferences in mice.
- To evaluate the impact of different USV characteristics on mouse behavior.
- To enhance understanding of communication in mice.
Methods Used
- Recording USVs from live mice.
- Editing and generating stereophonic sound files.
- Calibrating ultrasound emitters and adjusting volume.
- Conducting a two-choice playback test with behavioral monitoring.
Main Results
- Mice show distinct preferences for certain USVs.
- Behavioral responses vary based on the characteristics of the sounds.
- The two-choice test effectively reveals USV recognition.
- Findings contribute to understanding mouse communication dynamics.
Conclusions
- The study successfully demonstrates USV preference testing in mice.
- Results highlight the influence of various factors on vocalization preferences.
- Further research can expand on the implications of these findings.
Frequently Asked Questions
Executive Industry Relevance
Understanding ultrasonic vocalization (USV) preferences in mice provides a behavioral readout for assessing social communication phenotypes relevant to neuropsychiatric disease models. This two-choice playback test enables quantitative measurement of auditory preference and discrimination, supporting target validation in CNS drug discovery by linking genetic or pharmacological manipulations to measurable changes in social salience processing. The method contributes to mechanistic de-risking by offering a translational assay for evaluating compounds that modulate social motivation, anxiety, or reward pathways.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of hypotheses regarding gene function in social communication circuits by measuring shifts in USV preference behavior.
- Operational Value: Provides a standardized behavioral readout for assessing the impact of genetic variants or pharmacological agents on auditory-guided social approach.
Screening & Assay Development
- Scientific Value: Generates quantifiable preference metrics (time spent in sound zone, mesh investigation) that can serve as dose-responsive endpoints in compound screening.
- Operational Value: Supports assay reproducibility through calibrated ultrasound emitters and controlled sound pressure levels, reducing variability in cross-laboratory studies.
Translational & Preclinical Research
- Scientific Value: Facilitates phenotypic screening of disease-relevant models (e.g., autism, schizophrenia) where USV alterations are predictive of social deficit endophenotypes.
- Operational Value: Enables longitudinal tracking of USV preference changes across developmental stages or treatment regimens to inform therapeutic timing and dosing.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target hypothesis testing through lead identification, offering a behavioral bridge between molecular manipulation and functional output in social behavior domains.
- Discovery Biology: Supports mechanistic de-risking by linking USV preference shifts to specific neural circuits involved in auditory processing and social valuation.
- Screening: Delivers quantitative, preference-based readouts that enable comparison of test compounds against vehicle controls in a counterbalanced design.
- Analytics: Generates analyzable behavioral metrics (entry frequency, duration in sound zone) that inform statistical comparison of experimental conditions.
- Translational Research: Aligns with preclinical continuity by modeling how genetic or pharmacological perturbations affect conserved aspects of mouse social communication.
- Enterprise Reuse: Represents a reusable platform for evaluating diverse genetic backgrounds, strains, or therapeutic interventions affecting social salience.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validity by resolving whether molecular changes alter perceptual or motivational aspects of social sound processing.
- Operational Value: Enhances reproducibility through emitter calibration, background noise controls, and side-bias mitigation via emitter position switching.
- Strategic Value: Improves go/no-go decision-making by identifying early-stage compounds that fail to modulate relevant social communication phenotypes.
- Portfolio Impact: Enables risk-adjusted prioritization of targets based on behavioral rescue of USV preference deficits in disease models.
Implementation Considerations
- Requires expertise in behavioral neuroscience, audio signal processing, and ultrasonic equipment calibration.
- Dependent on specialized instrumentation including ultrasonic condenser microphones, nanocrystalline silicon thermo-acoustic emitters, attenuators, and amplifiers.
- Necessitates cross-team standardization of sound file generation, filtering (high-pass at 40 kHz), noise reduction, and stereo playback protocols.
- Involves adaptation considerations across mouse strains, ages, and hormonal states (e.g., estrous cycle tracking via vaginal smears).
- Practical limitations include potential side bias if emitter placement is not randomized and the need for controlled acoustic environments to prevent rustling noise contamination.
