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73 protocols using cm16 cmpa

1

Automated Ultrasonic Vocalization Analysis

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Vocalization recordings were done using a condenser microphone (CM16/CMPA, Avisoft Bioacoustics, Germany) connected to an ultrasound‐recording interface (UltraSoundGate 116Hb, Avisoft Bioacoustics). Calls counting was done using DeepSqueak (Coffey et al., 2019 (link)) on MATLAB 2018b. A short rat call network was used for the detection of USVs in the recordings with the detection settings set as “high recall” to ensure the detection of as many calls as possible. The other detection parameters were set as followed: total analysis length of 0, analysis chunk length of 6, frame overlap of 0.001 s, frequency low cut‐off of 30 kHz, frequency high cut‐off of 120 kHz, and score threshold of 0. After detection was finalized, the files were revised by an experimenter blind to testing conditions to exclude false‐positive findings. Vocalizations were manually classified as previously described (Romano et al., 2013 (link)). Calls were classified into a syllable repertoire of 11 different syllables: chevron, complex, composite, downward, flat, frequency step, short, two‐component, upward, unstructured, and calls that did not fit any of the syllables classifications were called unknown. Due to the low number of unknown calls, this type of USV was not included in the repertoire analysis.
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2

Self-Grooming Behavior Captured by Ultrasonic Audio

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Audio recording of blue laser induced self-grooming was performed in a sound-attenuated chamber. Within this chamber, a condenser ultrasound microphone (CM16/CMPA, Avisoft Bioacoustics) was fixed above a clean cage. Mice were habituated to the sound attenuated chamber and underwent 4 laser stimulation trials using parameters described above (20 Hz 10 ms pulses for 10 s) with a 5 min interval between stimulations. Acoustic data were acquired at 192 kHz to capture potential audible and ultrasonic sounds related to self-grooming. Data were acquired, visualized, and quantified using Raven Pro v1.4 software (the Cornell Lab of Ornithology).
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3

Ultrasonic Vocalization Analysis in Mice

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Each male was tested in its home cage. The vocalizations were recorded, after the introduction of a receptive female, for 4 min with an Ultrasound gate microphone CM16/CMPA (Avisoft Bioacoustics), which was connected to an Ultrasound recording interface plugged into a computer equipped with the recording software Avisoft-SASLab Pro 5.2.09, and then analyzed using SASLab Pro (Avisoft Bioacoustics). Spectrograms were generated for each detected call (frequency resolution: fast Fourrier transform length: 512; frame size: 100%; overlap: 50%). The parameters used for the automatic quantification of the USVs were: cutoff frequency of 30kHz and element separation based on an automatic single threshold with a hold time of 15 ms. Syllables were identified and grouped into three main categories (Figure S1A), as previously described (Ey et al. 2013 (link)). The total number and duration of USVs, the number and duration of each syllable, and the percentage of each category (number of syllables of each category/total number) were analyzed.
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4

Ultrasonic Vocalizations in Rats

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Ultrasonic vocalizations produced by the rats were recorded using four microphones (condenser ultrasound CM16/CMPA, frequency range 10–200 kHz, Avisoft Bioacoustics, Berlin, Germany) placed under the elevated platforms (Figure 1A). Data were acquired using UltraSoundGate 416H at a sampling rate of 250 kHz and 16-bit resolution using Avisoft-RECORDER USGH software (Avisoft Bioacoustics, Berlin, Germany).
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5

Vocal behavior analysis in mice

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Vocal behavior was recorded using an ultrasonic condenser microphone (CM16/CMPA, Avisoft Bioacoustics, Berlin, Germany) and was analyzed using Avisoft Bioacoustics SAS lab software. Markers indicating the start and end of each emitted vocalization and a label indicating the vocalization type (based on category descriptions by Grimsley et al., 2016 (link)) were inserted manually. Data were then extracted automatically from Avisoft for syllable duration, peak frequency, and the number of instances of vocalization type. For the first 10 min of playback in each trial, we assessed both the number of vocalizations and the proportion of USVs emitted by experimental subjects while listening to the playback. USV proportion, calculated by dividing the number of USVs emitted by the total number of vocalizations emitted, provided an additional measure of vocal behavior beyond USV calling rate (e.g., Scattoni et al., 2008 (link)) or the total number of vocalizations emitted (e.g., Grimsley et al., 2016 (link)), including each of the non-USV social vocalizations emitted by CBA/CaJ mice.
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6

Mouse Vocalization Analysis for Fear Conditioning

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Vocalization of the mouse during the fear conditioning was recorded using an ultrasonic microphone (CM16/CMPA, Avisoft) amplified (UltraSoundGate 116H, Avisoft) and digitized at 250 kHz (by a software, Avisoft-RECORDER USGH, Avisoft). For the visualization of the vocalization, we generated a spectrogram using a multitaper method58 (link). In this analysis, multiple time tapers were designed using a set of 6 series of discrete prolate spheroidal sequences with the time half bandwidth parameter set to 359 . The length of these tapers was set to 512 data samples (~2 ms at 250 kHz). The stored waveform was multiplied by each taper and transformed into the frequency domain. These multiplied waveforms were averaged in the frequency domain. This procedure could produce a stable spectrotemporal representation of vocal sound with background noise attenuated.
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7

Isolation-Induced Ultrasonic Vocalizations in Seizure Pups

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To assess changes in USV behavior following seizures, we examined USVs on PD12 using the isolation-induced USV paradigm previously described [15 (link), 16 (link)]. All vocalizations were recorded in the afternoon (between 1 and 5 pm), approximately 24 hours after the last seizure for the HSL paradigm and 48 hours later for the LSL paradigm. Briefly, all pups were weighed and transferred to a holding cage with fresh bedding, warmed by an electronic heating pad to ambient nesting temperature (~35° C). Ultrasonic vocalizations were recorded using a condenser microphone (CM16/CMPA, Avisoft Bioacoustics, Germany) connected to an ultrasound-recording interface (UltraSoundGate 116Hb, Avisoft Bioacoustics), which allowed assessment of all USVs on a continuous spectrum from 0–125 kilohertz (kHz). Each pup was individually placed into another housing pan, within an acrylic sound-attenuating chamber (40 cm × 40 cm × 30 cm) where USVs were recorded for 2 minutes. Following recording, the pups were placed back into the holding cage with their littermates. This procedure was repeated until each pup in the litter was tested. An experimenter remained in the room during all recordings. At the conclusion of testing, pups were returned to the home cage.
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8

Eliciting Adult Mouse Ultrasonic Vocalizations

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The methods for adult USV collection have been previously described (Wöhr et al., 2011 ). Briefly, one week prior to testing, male mice were paired with females for a 5-minute duration. This was done to standardize the social experience in males, a necessary prerequisite for eliciting adult vocalizations (Scattoni et al., 2008 ). Twenty-four hours prior to testing, estrus was induced in females by placing male bedding into the female cage that had been previously introduced to the test males. On the day of testing, fresh urine was collected from females that were in estrus and 20 uL of the urine was then pipetted into the center of a testing chamber. The chamber was sound attenuating to ensure an accurate detection of the test mouse’s USVs. Males were placed in the chamber and all vocalizations were recorded for a 5-minute duration using a condenser microphone (CM16/CMPA, Avisoft Bioacoustics, Germany) coupled with a recording interface (UltraSoundGate 116Hb, Avisoft Bioacoustics). After testing, the males were returned to their home cages.
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9

Ultrasonic Vocalizations in Mouse Sexual Interactions

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A male mouse that had been caged alone at least for 7 days was brought in its home cage into the test room under dim light. The home cage was placed inside a sound-attenuating Styrofoam box for USV recording. A microphone (CM16/CMPA, Avisoft Bioacoustics e.K., Glienicke, Germany) was fitted onto a transparent lid with a hole in the center. The microphone was connected to a USV recording device (UltraSoundGate 116H, Avisoft Bioacoustics e.K.). After a 30-min habituation period, an estrus or diestrus female mouse was introduced into the cage, and the USV calls during sexual interactions were recorded for 5 min. The sampling rate was set at 250 kHz and the bit depth was formatted to 16 bits for recording by RECORDER Hardlock software program (Avisoft Bioacoustics e.K). The recorded data were processed with SASLab Pro software program (Avisoft Bioacoustics e.K.). Signals with frequencies less than 35 kHz and noise with vertical patterns were removed. Each call in the recorded data was automatically determined (Hold time: 10 ms, Overlap: 75%, Hamming window). Dominant frequency, bandwidth, and duration were determined automatically with SASLab.
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10

Assessing Ultrasonic Vocalizations in Seizure Pups

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We examined ultrasonic vocalizations (USVs) on PD12 using an isolation-induced paradigm to assess changes in vocalization production following seizure and treatment administration on PD10. Prior to the recording phase, all pups were transferred to a new housing cage with fresh bedding that was warmed with a heating pad to an ambient temperature of ∼35°C. During recording, one at a time pups were placed into a separate housing pan placed within an acrylic sound-attenuating chamber. Vocalizations were recorded for 2 min for each pup. The recording apparatus consisted of a condenser microphone (CM16/CMPA, Avisoft Bioacoustics, Germany) which was connected to an ultrasound-recording interface (UltraSoundGate 116Hb, Avisoft Bioacoustics) and recorded all USVs on a continuous spectrum from 0 to 125kHz. Following recording, pups were placed back into the warmed housing pan with littermates. This procedure was repeated in sequence until all pups were recorded, and upon completion all pups were returned to their original home cage. The total number of recording sessions allowed for pups to not be separated from their home cage and mothers for longer than 30 minutes.
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