All procedures were approved by the Ethics Committee of Azabu University. All experiments were carried out in a soundproof chamber.

1. Animal Preparation

1. Males for Recording
   1. Obtain sexually mature male mice experienced in mating.
2. Female Subjects
   1. Obtain virgin female mice that have been housed with 2 to 5 littermates per cage (usually 8 – 12 weeks old).
   2. Obtain vaginal smears daily to determine the phase of the estrous cycle before the test, according to McLean²¹. Care should be taken to minimize the vaginal stimulation to avoid pseudopregnancy.
   3. Stain the smears with Giemsa, and determine phase of the estrous cycle based on the presence or absence of leukocytes, cornified epithelial cells, and nucleated epithelial cells, according to Nelson²².

2. Devices (Figure 1)

Figure 1. Schematic of the Two-choice Test Box and Devices. Mice can access rooms A and B through the small gates located between them and the neutral zone. The two-choice box and two ultrasound emitters are placed on the floor of a soundproof chamber. The microphone is suspended inside the soundproof chamber. Abbreviation: PC, personal computer. Please click here to view a larger version of this figure.

1. For Recording
   1. Connect an ultrasonic condenser microphone to a PC using a USB A/D converter.
   2. Set the gain level of the A/D converter to “7”.
   3. Record acoustic data in 16-bit format files with a sampling rate of 400 kHz using audio capture software.
2. For Playback
   1. Connect a D/A converter to the PC using a USB interface.
   2. Connect two amplifiers to the D/A converter through an attenuator. (The latter device serves to prevent overload.)
   3. Connect an ultrasound emitter to each amplifier.

3. USV Recording

1. Place a male mouse in a small- (17 x 10 x 11 cm) or medium-sized (21 x 14 x 13 cm) cage with a 6 cm-diameter hole in the sidewall and the hole covered with a 0.5 cm wire mesh. When using a medium-sized cage, construct a partition across the center of the cage to reduce the area.
2. Do not use bedding in the cage to avoid rustling noises that could contaminate the recordings.
3. Place a microphone beside the mesh.
4. Begin ultrasonic vocalization monitoring with the recording setup shown in step 2.1.
5. Place a sexually mature female mouse in either metestrus or diestrus in the cage with the male mouse.
6. Record USVs for 3 – 5 min.

4. Test Box

1. Build the test box using acrylic board (Figure 1).
   Note: The test box (35 x 20, and x 20 cm high) is divided in three compartments: a neutral zone (15 x 20 cm), and rooms A and B (20 x 10 cm, each). Rooms A and B each have a 4 cm-diameter hole that is covered with a 0.5cm wire mesh. The holes and mesh are located at each end of rooms A and B. Make gates (5 x 5 cm each) between the neutral zone and rooms A and B.
2. To prevent sound leakage insulate each ultrasound emitter, except for the side facing the mesh, using rubber plates, sealing them to the box around the perimeter of each mesh.
3. Place lead seals on both sides of the wall between rooms A and B for sound insulation.

5. Preparing for Sound Playback

1. Creation of Playback Sounds
   1. Capture a 20 sec segment of USV from the recorded file using digital audio editing software.
   2. Select and capture background noise (0.35 sec) from the 20 sec USV segment file. Create a 20 sec background noise file by repeating the noise segment.
   3. 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.”
   4. Filter the sound files using as high-pass filter with a cutoff of 40 kHz.
   5. Reduce noise using the noise-reduction tool in post-processing software. Select and capture a segment of the file that contains no USVs as the noise-reduction profile, and run noise reduction with level 40.
2. Calibration of Playback Sounds
   1. Ensure that the space between ultrasound emitters and microphone is 10 cm.
   2. To calibrate sound pressure levels from the ultrasound emitters, perform ultrasonic monitoring using the configuration described in Step 2.1. Measure the sound pressure level by the microphone in decibels (dB).
   3. Using the attenuator and amplifiers, adjust the volume of the USVs generated by the ultrasound emitters to the same sound pressure level as the male USVs recorded in Step 3.
   4. When using the two ultrasound emitters and a sound file composed of USVs and background noise, confirm that the two ultrasound emitters exhibit the same sound pressure level using a calibration sound (e.g., a 75-kHz pure tone) before performing the behavioral test.
   5. When reproducing a sound file composed of two streams of USVs, adjust the two generated sound pressure levels to the same level, before testing.

6. Two-choice Test

1. Place the ultrasound emitters behind the meshes of the test box.
2. Close the gates to rooms A and B with an acrylic board, and habituate the female subject to the neutral zone for 30 min. Cover the box with acrylic board to avoid that the mouse can escape.
3. Start video recording using a CCD camera mounted above the box. The camera (not shown in Figure 1) covers the area of all three compartments.
4. Remove the gate and cover-board and allow the female to explore freely the test box, including the sound zones.
5. Once the female has investigated both meshes, and has returned to the neutral zone, start playing a 20 sec sound file repeatedly for 5 or 10 min.
6. Conduct behavioral monitoring for 5 or 10 min.
7. To reduce undesired olfactory cues deposited from the previous subject, clean the test cages between the tests with 70% ethanol.
8. Switch the place of ultrasound emitters A and B randomly to avoid inherent side bias effect towards rooms A or B.

7. Statistical Analyses

1. Using behavioral event-scoring software, analyze the following parameters: total number of entries in each room; total duration of stay in each middle zone; total duration of stay in each sound zone; and total duration of search of each mesh in front of ultrasound emitter. Figure 1 illustrates what is meant by “middle zone” and “sound zone.”
   Note: In cases where the mouse stays on the dividing line between the two zones (Figure 1, dashed line) decide the location by the direction of the head. When the head faces the mesh side, it is scored as a stay in the sound zone.
2. For each behavioral parameter, compare responses for rooms A and B using a Wilcoxon signed-rank test, or paired t-tests with a significance level of 0.05.