Response to Assini et al.

tightly correlated with emission of 22 kHz calls (Figure 1D). This is expected as call emission is mechanistically linked with breathing [5,7]. Accordingly, rats maintained long uninterrupted exhalations to accommodate continuous emission of prolonged 22 kHz calls (Figure 1A bottom; Figure S2A). Thus, the observed changes in sniff-cycle duration following face-toface interactions were mirroring the increases in 22 kHz calls (Figure 1E). Sniff duration also tracked the time course of 22 kHz emission following face-to-face interactions, with both measures peaking 1 second following interactions and returning to baseline after 5 seconds (Figure S2B). Overall, sniff cycles with 22 kHz calls were approximately five times longer than non-22 kHz sniffs during social interactions (Figure 1F). This clear separation by duration makes it possible to identify the emission of 22 kHz calls based on sniff cycle duration alone. To determine whether 22 kHz calls were likely present in the data reported in Wesson [1], we directly measured the duration of sniffs from the four example panels illustrating decreases in sniff rate following face-to-face interactions. As expected, sniffs reported for dominant rats were short and thus incompatible with 22 kHz call production (Figure S2C). For submissive rats, however, we estimate that 41% of the reported sniffs had 22 kHz calls based on their long duration (Figure 1G; and see Supplemental Experimental Procedures). It is therefore likely that the large reductions in sniff rate following face-to-face interactions in Wesson [1] were due to disruption of sniffing behavior by 22 kHz vocal production. What aspects of rat behavior communicate dominance during social interactions? Wesson [1] showed that sniffing patterns correlate with submissive status during face-to-face encounters. 22 kHz calls are known to signal dominance over long timescales. As expected, submissive rats could be reliably identified by their general emission of 22 kHz calls. Our finding that these calls are rapidly triggered after face-to-face encounters further implicates this signal in communication at faster timescales. During ultrasound emission, rats make prolonged exhalations against a constricted larynx [7], reflected in our intranasal recordings as periods of flat signal. Mechanistically, sniffing and vocal output carry redundant information. Rats are known to perceive and react to the loud (60–80 dB) 22 kHz alarm calls [8], making these salient signals unlikely to be ignored by conspecifics. Even so, sniffing and vocalization are only part of a larger ensemble of correlated behaviors comprising social interactions, including also whisking and postural changes [9, 10]. Disentangling the effects of each individual behavior would require targeted manipulations. Together with Wesson [1] and Wolfe et al. [10], our studies demonstrate that face-toface encounters are relevant discrete events in the establishment of social dominance.