Recorder usgh software

Experiments were conducted in a modular operant conditioning cage with a grid floor, pellet receptacle, and pellet dispenser (Behavioral Test Packages for Rat, Med Associates Inc., Fairfax, VT, USA) controlled by Med-PC^® V software (version 5.08, Med Associates Inc.). Additionally, there was a USV condenser microphone CM16/CMPA (UltraSoundGate, Avisoft Bioacoustics, Glienicke/Nordbahn, Germany) placed in the glass ceiling of the cage and an ultrasonic speaker (Vifa, Avisoft Bioacoustics) placed in the corner of the cage, next to the pellet receptacle, connected to an UltraSoundGate Player 116 (Avisoft Bioacoustics). Both USV-playback and recording were performed using Avisoft Recorder USGH software (version 1.0.0.1, Avisoft Bioacoustics). The locomotor activity was recorded with a FLIR^® camera (Teledyne FLIR LCC, Wilsonville, OR, USA) mounted behind or above the cage and controlled via Spinnaker^® SDK software (version 1.15.0.63, Teledyne FLIR LCC, Wilsonville, OR, USA); home-cage activity was recorded with a Basler camera (acA1300-60gc, Basler AG, Ahrensburg, Germany) controlled via EthoVision XT software (version 10, Noldus, Wageningen, Netherlands). Non-flavored sucrose pellets weighing 45 mg (TestDiet^®, St. Louis, MO, USA) were used as rewards in conditioning. The cage was cleaned and thoroughly wiped using 10% EtOH between animals.

One day after the conditioning, the rats were transferred into individual experimental cages, identical to home cages (plastic; 37 × 25 × 16 cm), and transported to the experimental room, where under white light, in the absence of the experimenter and other rats in the room, acoustic stimuli were presented through an ultrasonic speaker (Vifa, Avisoft Bioacoustics, Berlin, Germany), placed just above the shorter side of the cage, connected to an UltraSoundGate Player 116 (Avisoft Bioacoustics). USV emitted by the rat were recorded by a CM16/CMPA condenser microphone (UltraSoundGate, Avisoft Bioacoustics) placed 33 cm above the center of the cage floor, 20 cm away from the speaker. In this configuration, calls from the speaker were still visible in the recording (monitoring of playback), but they were distinctively weaker than USV emitted from the cage. Both playback and recording were performed using Avisoft Recorder USGH software (Avisoft Bioacoustics). The locomotor activity of the animal was recorded with a camera (acA1300-60gc, Basler AG, Ahrensburg, Germany) mounted above the cage and EthoVision XT software (version 10, Noldus, Wageningen, The Netherlands). Signals from radiotelemetric transmitters were collected by receivers located under the cage floor and then recorded by Ponemah software (version 6.32, Data Sciences International, St. Paul, MN, USA).

The experimental setup consisted of a box (‘tickle box’) lined with black foam rubber, with a plexiglass separation wall in the middle. The bottom of the separation wall was a mesh wire stripe, allowing the animals to see, hear and smell each other. The experimental environment was kept dim (∼20 lx). Video material was recorded under infrared illumination with two or three cameras: a top view and a side view camera with 30 fps (Imaging Source, Germany), and a side view with 240 fps (H5PRO – modified GoPro Hero 5, Back-bone, Ottawa, Canada). Ultrasonic vocalizations were recorded with four microphones (condenser ultrasound CM16/CMPA, frequency range 10–200 kHz, Avisoft Bioacoustics, Berlin, Germany) at a sampling rate of 250 kHz and with a 16-bit resolution using Avisoft-RECORDER USGH software (Avisoft Bioacoustics, Berlin, Germany). The microphone positioning enabled assigning calls to demonstrator and observer rat, respectively. The audio recording, video recording and electrophysiological recording were synchronized using TTL pulses. The setup is illustrated in Figure 1A.