Simultaneous Measurement of Audible and Ultrasonic Vocalizations

Components of vocalizations in the audible (20 Hz–16 kHz) and ultrasonic (25 ± 4 kHz) ranges were simultaneously measured after hindlimb withdrawal assays using an automatic computerized vocalization system consisting of a full-spectrum USB ultrasound microphone (max sampling rate: 384 kHz) and UltraVox XT four-channel recording and analysis system (Noldus Information Technology, Leesburg, VA, USA). Rats were briefly anesthetized with isoflurane (2–3%; precision vaporizer, Harvard Apparatus) and placed in the customized recording chamber for stable recordings of vocalizations evoked by natural stimulation. After the rat recovered from anesthesia and habituated to the recording chamber for 30 min, hindlimb withdrawal thresholds were evaluated (see Section 2.5.2) and the calibrated forceps with a force transducer were used for vocalization assays. Vocalizations were evoked by a brief (10 s), continuous noxious stimulus applied to the left knee joint (arthritis pain model; stimulus: 1500 g/30 mm²) or to the left hindpaw (neuropathic pain model; stimulus: 500 g/6 mm²) as described in our previous studies [20 (link),28 (link),29 (link),37 (link),38 (link),42 (link)]. Vocalizations were automatically detected for 1 min and total durations of audible and ultrasonic components of vocalizations following the onset of mechanical stimulus were analyzed using UltraVox 3.2 software (Noldus Information Technology). For vocalization analyses, audible calls were labeled using frequency ranges of 20 Hz–16 kHz and ultrasonic components of calls were labeled using frequency ranges of 21–29 kHz. The following call descriptions were also specified: minimum amplitude, 50 units; minimum duration, 1 ms; maximum duration, 2000 ms; minimum gap between calls, 1 ms. Calls that fit these criteria were detected for each recording. At the conclusion of each experiment, call statistics for each recording were exported as a text file. The duration (in ms) for each individual call was summed for each 1 min recording period to give the total duration of audible and ultrasonic components of vocalizations for each rat.