The overall goal of this protocol is to record and analyze ultrasonic vocalizations from mouse pups or adults, and then to make the data publicly available at the mouseTube database. So we previously identified mutations in autism, so now we want to understand the role of these genes. We are using mouse models, and especially, we are looking at the ultrasonic vocalization of mice.
This model can help understand key questions in the neuroscience field. For instance, how genetic mutations affect social communication in mouse model for neuropsychotic disorders such as autism. The main advantage of this protocol is that mouse addressing vocalization are reliably elicited.
It should favor a standardization of context information to improve between study comparison. This method will also boost knowledge on mouse intrasonic communication using the mouseTube database. After preparing the mice and obtaining newborn pups, set up a cage to record their calls.
Use either a soundproof box or a Styrofoam box. First, set up a thermometer to monitor the internal temperature. Next, through a hole in the top of the box, lower a condenser ultrasound microphone so that the membrane of the microphone is about 12 to 15 centimeters above the floor.
Now, connect the microphone to a computer sound card. Put the non-experimental pup into a clean plastic pup holder within the recording box and close it up. Make the peak recording sound use the full available amplitude without distortion.
Do not change this setting during the experiment. Now, for experimental measurements, place the pup as gently and quickly as possible into a clean plastic cup. Then, place the pup under the microphone and close the box as gently and quietly as possible.
Then, make a 16-bit, 300 kilohertz recording for a high quality data set. After up to five minutes of recording, remove the pup. Take note of its paw tattoo and then measure its skin temperature with a digital thermometer.
Now, make a small mark on the pup using water-based ink and return it to its nest. Between tests, wash the cup using 10%ethanol and let it dry out completely. For an adult test cage, use a Plexiglas cage that fits into a soundproof box illuminated with low intensity white light.
Clean the cage with soapy water, dry it, and fill it with two centimeters of fresh bedding. Fix the microphone in one corner of the cage, and adjust the angle of the microphone to capture sounds from the whole cage. Next, position a video camera over the soundproof chamber so it views the surface of the test cage without the microphone obscuring a corner.
Using a non-experimental pair of animals, set the gain of the recording as before. For the experiment, introduce one animal to the cage first to fill the occupant role. Allow it to habituate for 20 minutes.
A baseline recording may be made during this time. Then, start the video and audio recordings, and introduce the second animal of the same sex to fill the newcomer role. Synchronize the recordings by triggering a beep tone as the second touches into the cage.
Record the animals'interactions for as long as needed, such as four minutes. To prepare for this test, early in the morning of the test day, take vaginal smears from each female to determine their status in the estrus cycle. Hold the female by the tail and let her grip the cage grid.
Using a pipette, rinse her vagina several times by injecting and recollecting 20 microliters of PBS. Pay attention to the PBS when it's flushed out of the vagina. When it's slightly cloudy, it contain enough cell.
Do this carefully to avoid creating mucus. Spread the sample on a slide. Up to four can be examined at once.
Let the samples dry out before proceeding. Under a fume hood, incubate the slides in a bath of 0.1 molar May-Grunwald solution in PBS for three minutes. Then, transfer the slides to a PBS bath for a minute.
Then, transfer the slides to a bath of 5%weight by volume Giemsa R in PBS. After 10 minutes, rinse the slides in fresh PBS for another minute. Then, examine the stained slides.
If the sample shows only large, cornified epithelial cells that have no nuclei, and stain blue, the female is in full estrus. Only such females can be used for the test. Now, set up the test cage with the male as the occupant.
Allow him to habituate for 10 minutes and take a baseline audio recording if needed. Then, start the video and audio recordings and introduce the female in full estrus. Synchronize the records as before using a beep.
Then, let the animals interact for as long as needed, such as a four-minute period. When the recording is completed, return the mice to their respective cages. To upload data to the mouseTube website at the Institut Pasteur, first log in.
Check whether the mouse strain recorded from exists in the mouseTube database by clicking on the Strains button. If not, ask the administrators to create it. Next, create animal subjects.
Enter the identification codes of the animals recorded. Gather them in groups to ease later retrieval of data. Then, provide a description of the protocol used.
Now, create an experiment for each recording session. Specify the protocol, the group of individuals which has been recorded, the hardware and software used, and their specificities. The experiment gathers all the meta data corresponding to the vocalization files.
Next, create the link to the vocalization files. First, select the experiment within the list. Then, copy and paste the URL of the vocalization file into the corresponding field of the Files to Link column.
It is possible that the link you're putting to this database isn't actually well. Make sure that the server hosting your vocalization files is accessible from outside your institution for the members of the community. At Details, in the Notes section, to provide as needed.
For instance, specify that a link to a video file was entered that accompanies an audio file. Lastly, after making the entries, validate them. It is not necessary to fill each box for each file.
As necessary, modify the links entered. Mice lacking expression of Shank2, a gene associated with autism spectrum disorders, were characterized. These mice showed atypical vocal communication first observable as a decreased call rate in female interactions.
Next, the standard repertoire of five calls that change from puphood to adulthood was assessed. Measurements were first made with P2 pups. The vocal repertoire did not change in P6 pups.
At P10, the vocal repertoire was still the same. Up to this point, there was not much of a deficit shown by the Shank2 null mice. However, in adulthood, genotype related differences became apparent.
When males interacted with females in estrus, there was a notable increase of the proportion of short and unstructured calls made by mutant males. In call patterns heard between females, Shank2 null females emitted more short and unstructured calls, as well as less complex calls and less frequency jump calls when paired with normal females. Paired Shank2 null mice, male or female, vocalized with a lower peak frequency compared to pairs of wild-type litter mates.
Synchronizing the analysis of behavioral events and vocalization emission allowed identification of impairments in the context of emission of ultrasonic vocalizations in mutant mice. Shank2 null mice vocalized less often when in physical contact with newcomers, and more specifically when sniffing the anogenital region. More vocalizations were heard when the newcomers were in the visual field of the Shank2 null mice.
After watching this video, you should have a good understanding of how to record mouse ultrasonic vocalizations, and to upload data to the mouseTube database. While attempting this procedure, it's important to remember to record enough individual preconditioned given the height and internal viability in mouse vocal barrier.
