Ultrasoundgate cm16 cmpa microphone

USV, mostly 50-kHz-calls, are easily induced by manipulating the animal in a way that mimics the rough-and-tumble play in juvenile rats or, literally, by tickling them [111 (link)]. Tickling sessions can be used to induce playfulness and social joyfulness and the number of emitted USV may be used as a reflection and measure of positive affective states of rats [112 (link)]. Our rats were tickled for five consecutive days at PND 45–49, as described previously [113 (link),114 (link),115 (link)]. After the first three days of learning/habituation, at days 4 and 5, i.e., PND 48 and 49, the rats were taken for further analysis. Each day the rats were transported into the 58 × 37 × 20 cm tickling cage for a 30 s wait period. Then the tickling session was initiated with gentle poking of the animal’s sides, rubbing its scruff, then flipping the rat on its back and tickling it with rapid finger movements around the belly. The tickling lasted for 15 s and was followed by a 15 s period when the animal was allowed to follow experimenter’s hand. The tickling-follow scheme was repeated four times and the whole tickling session lasted for 120 s. USV were recorded with an UltraSoundGate CM16/CMPA microphone placed 30 cm above the cage, collected using Avisoft Recorder software, and analyzed using the SASLab Pro software (all from Avisoft Bioacustics, Glienicke/Nordbahn, Germany).

Ultrasonic vocalizations (USVs) were quantified as a potential measure of social communication after TBI or sham-operation at p21. Recording of USV's was performed at ∼p80, one week following the resident-intruder task (Cohort 1) or housing isolation (Cohort 2). Mice were placed into an open-topped plexiglass enclosure, which was either circular (for female bedding stimulus; 8.5 cm diameter) or rectangular (for addition of a male or female stimulus mouse; 7×15 cm; see figure S2). This enclosure was placed either on the bench top (Cohort 1) or in a sound-attenuating chamber (Cohort 2; Med Associates Inc, St Albans, VT) and the test mouse was allowed a 30 min habituation period. Vocalizations were then recorded from an Avisoft UltraSoundGate CM16/CMPA microphone (1–180,000 Hz; Berlin, Germany) suspended a fixed distance above the enclosure. Recordings were collected using a National Instruments data acquisition board and processed in MATLAB v7.10.1 (MathWorks, Natick, MA). After a 30 sec ‘baseline’ recording period (no stimulus), the stimulus was added to the enclosure and vocalizations were recorded for the following 120 sec. As the number of calls during baseline measurements was negligible (0–3 calls per test) and did not differ between treatment groups, analyses presented are for the 120 sec when the stimulus was present. USV's were recorded during three different paradigms: (1) addition of a novel male mouse; (2) addition of a novel female mouse; and (3) addition of female bedding, a 3–4 cm diameter clump of soiled bedding collected fresh from a cage of group-housed, stimulus female mice [50] (link). ‘Cohort 1’ consisted of mice which had previously undergone the resident-intruder test and were later used for scent marking and histology experiments (n = 10/group; figure 1a). ‘Cohort 2’ was a second group of mice who had no other behavioral assessments prior to USV recordings (n = 7–8/group; figure 1b). Mice that produced fewer than 5 calls during the recording period following addition of the stimulus were excluded as ‘non-responders.’
Sound files were filtered and de-noised using custom built software in MATLAB v7.10.1. Individual USV calls were detected using an automated detection algorithm as previously described [36] (link). Call detection was then checked by visual inspection of the spectrogram and calculated sound pressure envelope. False detections (scratches and other noises mistaken for calls) were removed and missed calls were added to the call tally. All analyses were performed independently by two investigators blinded to injury condition. When two mice were present during a test (i.e. addition of a male or female stimulus mouse), the total calls emitted were analyzed together. Calls emitted during encounters with a female stimulus were presumed to originate from the male test subject; although females have the capacity to vocalize in the ultrasonic range, previous studies have demonstrated that they rarely do so when paired with a male [51] , [52] (link), [53] (link), [54] (link). Parameters that were analyzed included total number of calls, number of calls with median frequency above a set threshold (75 kHz), call duration, median call frequency, latency to the first call, and call burst characteristics. The median frequency of a call was calculated using the technique outlined in [36] (link). Histograms of the square root of the number of calls within mean median frequency bins of 2.886 kHz were plotted to determine the frequency threshold of 75 kHz. Call bursts were verified by a trained observer by quantifying the inter-call intervals (ICI), the time between the end of one call and the start of the next. ICI's were log-transformed to examine the temporal distribution of calls [36] (link), and a burst interval threshold was identified. Once bursts were identified by implementing the burst interval threshold, the mean burst duration, number of calls contained within bursts, and fraction of calls in bursts was calculated.