Ultrasoundgate 416h

Female mice in estrus were identified using vaginal lavage, followed by cytological staining (Giemsa solution, Polysciences) and visual assessment. Male mice were habituated to the experimental arena (40 × 20 cm) for 3 min. Next, an unfamiliar female mouse in estrus was added to the arena, and the mice were allowed to interact freely for 3 min. Ultrasonic vocalizations (USVs) between 40 and 250 Hz produced by the male mice were recoded using a preamplifier (UltraSoundGate 416 H, Avisoft Bioacoustics) connected to a microphone (UltraSoundGate CM16, Avisoft Bioacoustics). The total number of USVs and their duration was analyzed using SASLabPro (Avisoft Bioacoustics).

The acoustic measurements began in March 2018. Equipment measuring the acoustic activity of the soil fauna was installed across the three treatments (irrigation/irrigation stop/control, Fig 1A). Three acoustic sensors were distributed randomly in the control (K1–K3), two in the irrigation, and one in the irrigation stop treatment (B1–B3) at distinct distances from the recording computer (Fig 1C), and two additional sensors were mounted to distinguish airborne and soil sounds (K4, B4).
The recording equipment consisted of an array of specially designed contact microphones (see S1 Fig), covering a soil depth of approx. 10 cm and a volume of approx. 1000 cm³. The signal from the sensors was amplified by +40 dB (a factor of 100) using modified hydrophone preamplifiers by Avisoft, Berlin. Signals from both plots were digitized by two 4-channel audio interfaces (Avisoft UltraSoundGate 416h) and recorded on a minicomputer at 10-min intervals (20-s recordings) using Avisoft’s Recorder software (Fig 1C). The loss of signal strength due to long audio cables (25 m) was compensated by +18 dB in the internal amplifiers of the audio interfaces, and the captured signals were recorded with a high-pass filter of 0.05 KHz (512 taps, filtering low-frequency noise out) and a sampling rate of 50 KHz. The whole system was powered by a fuel cell.

As performed in our laboratory, locomotor activity and 50-kHz USVs were recorded simultaneously in customized sound-attenuating chambers (Kim et al., 2021 (link); Lee et al., 2022 (link)). The chamber consisted of two boxes to minimize exterior noise (inside box: 54 × 38 × 35 cm, outside box: 68 × 50 × 51 cm). A condenser ultrasonic microphone (Ultramic250K; Dodotronic, Castel Gandolfo, Italy) and a digital camera were positioned at the center of the ceiling of the chamber. The 50-kHz USVs were recorded using the ultrasonic microphone with an UltraSoundGate 416H data acquisition device (Avisoft Bioacoustics, Glienicke, Germany). Ultrasonic vocal signals were band-filtered between 38 and 96 kHz for the 50-kHz USVs and analyzed using Avisoft-SASLab Pro (version 4.2; Avisoft Bioacoustics, Glienicke, Germany). Locomotor activity was measured with a video-tracking system (Ethovision XT; Noldus Information Technology BV, Wageninge, Netherlands). After recording baseline activity for 30 min, the rats were given an intraperitoneal injection of cocaine (15 mg/kg) and/or MS and monitored for up to 60 min after cocaine injection. The data were expressed as the distance traveled for locomotor behaviors and the numbers of 50-kHz USVs during each 10 min period.

Vocalizations were recorded using an UltraSoundGate condenser microphone CM16/CMPA (Avisoft Bioacoustics; Berlin, GER) connected via an UltraSoundGate 416H audio device (Avisoft Bioacoustics) to a personal computer. Acoustic data were recorded as a WAV file with a sampling rate of 250 kHz in 16‐bit format by Avisoft RECORDER USGH (version 4.2.30). Specific recording conditions for each experiment are described below. Avisoft SASLab Pro (version 5.2.15) with a 256 fast Fourier transform was used to generate spectrograms. An investigator blinded to the experimental condition identified calls manually. Calls were categorized as either ultrasonic (>20 kHz) or audible (<20 kHz) mouse squeaks. A separate investigator blinded to the experimental condition listened to the audio files to confirm mouse squeaks. The false‐positive rate for audible mouse squeaks called from the spectrogram was 2.5%.

We measured pup ultrasonic vocalizations in a social isolation task at P7, P9, and P11. The ultrasonic vocalizations of each animal were monitored for 2 min in a sound-attenuating chamber (Med Associates Inc.). For Atoh1^Cre/+;Vglut2^fl/fl mice and littermate controls, vocalizations were recorded using a CM16 microphone (Avisoft Bioacoustics). Sound was amplified and digitized using UltraSoundGate 416H at a 250 kHz sampling rate, and bit depth of 16 while Avisoft RECORDER software was used to collect the recordings. For Ntsr1^Cre;Vglut2^fl/fl mice and littermate controls, we measured vocalizations using a Noldus microphone and UltraVox XT software. Due to minimal congruency between the number of vocalizations recorded between these two recording systems^{79 (link)}, we only compared mutants to control littermates whose vocalizations were measured using the same system.

Ultrasonic vocalisations were obtained from five male C57BL/6 mice in response to urine samples from conspecific females in oestrus, across 11 recording sessions. The urine stimulus consisted of the tip of one sterile cotton bud soaked in a mixture of freshly collected urine from at least two animals. Sounds were recorded at a sampling frequency of 250 kHz (Avisoft Bioacoustics, Germany) using a condenser ultrasonic microphone (CM16/CMPA), recording interface (UltrasoundGate 416H), and software (Avisoft Recorder version 5.2.09, all from Avisoft Bioacoustics).
The stimulus set used in the playback experiments (‘intact/original songs’) consisted of a subset of seven songs produced by three C57BL/6 male mice (median age 22 wk) over six recording sessions. The sound files were high-pass filtered above 40 kHz and were denoised using the frequency-domain noise reduction algorithm in Avisoft SASlab Pro (version 5.2.09, Avisoft Bioacoustics). Syllable detection and segmentation was verified manually. Sound files were up-sampled to 260,420 Hz with anti-aliasing for playback.