The overall goal of this procedure is to reliably collect and perform an acoustic analysis on an adult male mouse ultrasonic vocalizations or USVs across a variety of social contexts. This method can help answer key questions about vocal communication and associated disorders. Including neuro-psychiatric disorders and autistic disorders using mouse models.

One of the main advantages of this technique is that it provides a means for one to elicit USV's in various contexts. Generally individuals new to this method might struggle to get their mice to behave so you have to handle your mice carefully and gently in order to get them to sing. Standard animal housing is sufficient for this protocol.

Three days before making a recording in neutral cages, expose males to a sexually mature receptive female overnight. In each cage up to three males may be paired to one female. The next day remove the females.

Keep the males housed in their groups for at least two more days before the first recording session. To record USV's from a prepared male gently transfer the animal by the tail to a clean cage without bedding. Place this cage inside a soundproof box.

Then cover the cage with the open wired lid. Close the box and let the animal habituate to it for 15 minutes Meanwhile prepare a stimulant for the assay as described in the following section. To use fresh urine samples collect them within five minutes of the recording session.

First grab a mouse by the scruff as for a drug injection. Then using a cotton tip held by tweezers gently rub and push on the animal's bladder to extract a drop of urine and completely soak it up with the cotton. Then, select a mouse from a different cage and repeat the process using the same cotton tip.

Using a mix of fresh urines is important. Store the cotton tip on a clean dish until it is needed To prepare an alert female as a stimulus, select one that is in the pre-estrus or estrus stage. The mouse will have a wide vaginal opening and pink surrounding tissue.

Isolate such a female until needed. To prepare an anesthetized mouse simply provide anesthesia as usual. Confirm the anesthetized state with a toe pinch and apply ophthalmic ointment.

Until needed, keep the anesthetized animal in a clean cage on a paper towel with a heat pad beneath the cage set to minimun heat. Be sure to regularly check the animals breathing and body temperature. The anesthetized mouse may be used in up to three consecutive sessions.

When ready to record click the record button in the software while simultaneously presenting the stimulus. To present urine or a live animal, simply place either inside the cage. To present an anesthetized animal however, place it on top of the cages lid.

Then quietly close the box and record USV's for five minutes or so. To end a recording, press the stop square in the software. Check on the anesthetized animal at least every 15 minutes.

Until it has regained sternal recumbency. Then it can be returned to its home cage. Between tests clean the test cage with 70%alcohol and distilled water.

Process the acoustic recordings using the mouse song analyzer software. Their syllable, acoustic structure and syntax can thus be analyzed and restructured for playback as needed. First wash the playback apparatus with 70%alcohol followed by distilled water.

Use a black colored, opaque y-maze with 30 centimeter arms. At the extreme ends of two arms have holes drilled at floor level that fit the ultrasound speakers. After washing, towel dry the maze.

Once dried, set up with the playback speakers and mounted overhead video camera. To start the test first place the female in the maze for a 10 minute habituation period. After 10 minutes if the female is not in the starting arm, gently push her back to the starting arm and gently close the plastic separation window.

Then start the video recording and start the audio recordings on looped playback. For example, play a song captured from urinary stimulation in one arm and play song captured from female stimulation in the other arm. Now allow the female to explore the maze for five minutes.

This constitutes one session. After five minutes stop the recording and return the female back into the starting arm. Keep her there for one minute while cleaning using distilled water to remove any urine or excrement.

Do this between all sessions. For the second session, switch recorded playback to the opposite arms and record the female exploring this scenario for five minutes. For the third and fourth sessions repeat the two previous session parameters for a total of 20 minutes of test recordings.

Always stop the video recording at the end of all sessions. A week later, perform another test on the same cohort of animals. But use different song exemplars.

Do this to bolster the reliability of the findings. For analysis measure the time spent by the females in each arm for each session. Using the presented protocol, changes in vocal behavior and syntax of male B6D2F1J mice were characterized.

During a five minute recording session an average male responded very differently to the three stimuli. Males also changed their repertoire across context. For example, in response to fresh urine, males produced a significantly higher fraction of multi-syllable vocalizations in their songs.

Males also changed acoustic features of individual syllables across context. For example, they sang certain syllables at a higher amplitude and bandwidth in the female urine context. And at higher spectral purity in the awake female context.

In a song preference test, females in a y-maze chose to spend more time in the arm with complex songs containing two or more multi-syllable vocalizations. As opposed the the opposite arm where only simple songs were played. After watching this video, you should have a good understanding of how to collect mouse USV's across a variety of social contexts, how to play them back to the mice, and how to do acoustic and sequence analysis of their vocalizations.

Once mastered, behavior recordings can be made within five to 10 minutes per mouse. You should allow several hours for analysis. And be sure to handle mice with care, as to not alter behavioral responses.