22 and 50 kHz rat ultrasonic vocalization playback reveals sex differences in behavior and cFos in brain regions associated with affective processing.

Adult rats communicate using ultrasonic vocalization (USV) frequencies indicating negative (22 kHz) or positive (50 kHz) affective states. Playback of USVs can serve as an ethologically translational method to study affective processing in response to socially communicated states. However, few studies have examined behavioral and neural effects of USV playback in both male and female rats. Here, adult male and female Sprague-Dawley rats experienced a 20-min open field test (OFT) with either silence, 22 kHz, or 50 kHz recorded USV playback. Center exploration and locomotor activity were analyzed to characterize sex differences in playback effects. Results suggest that females display greater sensitivity to frequency-specific effects of USV playback in this paradigm compared to males. 50 kHz USV playback evoked an immediate increase in center exploration and locomotor activity in females, indicating the appetitive nature of 50 kHz USVs. Initially, 22 kHz playback inhibited center exploration in the OFT compared to 50 kHz. However, females exhibited a switch in behavioral strategy in response to 22 kHz following prolonged playback. Following OFT, neural activity was quantified via the immediate early gene cFos. Results from cFos quantification showed sex- and region-specific differences in neural recruitment in areas of the brain associated with affective processing, including the prefrontal cortex, amygdala, bed nucleus of the stria terminalis, and nucleus accumbens. Taken together, this work provides a normative baseline for understanding how sex influences behavioral and neural responses to USV playback, which can be leveraged to study anxiety, communication, and affect in an ethologically relevant assay.