Rodent ultrasonic vocal interaction resolved with millimeter precision using hybrid beamforming.

Ultrasonic vocalizations (USVs) fulfill an important role in communication and navigation in many species. Because of their social and affective significance, rodent USVs are increasingly used as a behavioral measure in neurodevelopmental and neurolinguistic research. Reliably attributing USVs to their emitter during close interactions has emerged as a difficult, key challenge. If addressed, all subsequent analyses gain substantial confidence. We present a hybrid ultrasonic tracking system, Hybrid Vocalization Localizer (HyVL), that synergistically integrates a high-resolution acoustic camera with high-quality ultrasonic microphones. HyVL is the first to achieve millimeter precision (~3.4-4.8 mm, 91% assigned) in localizing USVs, ~3× better than other systems, approaching the physical limits (mouse snout ~10 mm). We analyze mouse courtship interactions and demonstrate that males and females vocalize in starkly different relative spatial positions, and that the fraction of female vocalizations has likely been overestimated previously due to imprecise localization. Further, we find that when two male mice interact with one female, one of the males takes a dominant role in the interaction both in terms of the vocalization rate and the location relative to the female. HyVL substantially improves the precision with which social communication between rodents can be studied. It is also affordable, open-source, easy to set up, can be integrated with existing setups, and reduces the required number of experiments and animals.

Most animals ­ from insects to mammals ­ use vocal sounds to communicate with each other. But not all of these sounds are audible to humans. Frogs, mice and even some primates can produce noises that are ultrasonic, meaning their frequency is so high they cannot be detected by the human ear. These 'ultrasonic vocalizations' are used to relay a variety of signals, including distress, courtship and defense. To understand the role ultrasonic vocalizations play in social interactions, it is important to work out which animal is responsible for emitting the sound. Current methods have a high error rate and often assign vocalizations to the wrong participant, especially if the animals are in close contact with each other. To solve this issue, Sterling et al. developed the hybrid vocalization localizer (HyVL), a system which detects ultrasonic sounds using two different types of microphones. The tool is then able to accurately locate where an ultrasonic vocalization is emitted from within a precision of millimeters. Sterling et al. used their new system to study courtship interactions between two to three mice. The experiments revealed that female courtship vocalizations were substantially rarer than previously reported when two mice were interacting. When three mice were together (one female, two males), Sterling et al. found that one of the male mice typically dominated the conversation. This result was also reflected by the male mouse locating themselves anogenitally to the female, as males tend to vocalize more when in this position. In neuroscience, researchers often measure ultrasonic vocalizations to monitor social interactions between rats and mice. HyVL could provide neuroscientists with a more affordable and easier to use platform for conducting these kinds of experiments, which are important for studying behavior and how the brain develops.

High-precision spatial analysis of mouse courtship vocalization behavior reveals sex and strain differences.

Mice display a wide repertoire of vocalizations that varies with sex, strain, and context. Especially during social interaction, including sexually motivated dyadic interaction, mice emit sequences of ultrasonic vocalizations (USVs) of high complexity. As animals of both sexes vocalize, a reliable attribution of USVs to their emitter is essential. The state-of-the-art in sound localization for USVs in 2D allows spatial localization at a resolution of multiple centimeters. However, animals interact at closer ranges, e.g. snout-to-snout. Hence, improved algorithms are required to reliably assign USVs. We present a novel algorithm, SLIM (Sound Localization via Intersecting Manifolds), that achieves a 2-3-fold improvement in accuracy (13.1-14.3 mm) using only 4 microphones and extends to many microphones and localization in 3D. This accuracy allows reliable assignment of 84.3% of all USVs in our dataset. We apply SLIM to courtship interactions between adult C57Bl/6J wildtype mice and those carrying a heterozygous Foxp2 variant (R552H). The improved spatial accuracy reveals that vocalization behavior is dependent on the spatial relation between the interacting mice. Female mice vocalized more in close snout-to-snout interaction while male mice vocalized more when the male snout was in close proximity to the female's ano-genital region. Further, we find that the acoustic properties of the ultrasonic vocalizations (duration, Wiener Entropy, and sound level) are dependent on the spatial relation between the interacting mice as well as on the genotype. In conclusion, the improved attribution of vocalizations to their emitters provides a foundation for better understanding social vocal behaviors.