Mouse Ultrasonic Vocalizations Research Articles

Brainstem control of vocalization and its coordination with respiration.

Phonation critically depends on precise controls of laryngeal muscles in coordination with ongoing respiration. However, the neural mechanisms governing these processes remain unclear. We identified excitatory vocalization-specific laryngeal premotor neurons located in the retroambiguus nucleus (RAmVOC) in adult mice as being both necessary and sufficient for driving vocal cord closure and eliciting mouse ultrasonic vocalizations (USVs). The duration of RAmVOC activation can determine the lengths of both USV syllables and concurrent expiration periods, with the impact of RAmVOC activation depending on respiration phases. RAmVOC neurons receive inhibition from the preBötzinger complex, and inspiration needs override RAmVOC-mediated vocal cord closure. Ablating inhibitory synapses in RAmVOC neurons compromised this inspiration gating of laryngeal adduction, resulting in discoordination of vocalization with respiration. Our study reveals the circuits for vocal production and vocal-respiratory coordination.

Midbrain neurons important for the production of mouse ultrasonic vocalizations are not required for distress calls

A fundamental feature of vocal communication is that animals produce vocalizations with different acoustic features in different behavioral contexts (contact calls, territorial calls, courtship calls, etc.). The midbrain periaqueductal gray (PAG) is a key region that regulates vocal production, and artificial activation of the PAG can elicit the production of multiple species-typical vocalization types.1,2,3,4,5,6,7,8,9 How PAG circuits are organized to regulate the production of different vocalization types remains unknown. On the one hand, studies have found that partial PAG lesions abolish the production of some vocalization types while leaving others intact,3,8,10,11 suggesting that different populations of PAG neurons might control the production of different vocalization types. On the other hand, electrophysiological recordings have revealed individual PAG neurons that increase their activity during the production of multiple vocalization types,12,13,14 suggesting that some PAG neurons may regulate the production of more than one vocalization type. To test whether a single population of midbrain neurons regulates the production of different vocalization types, we applied intersectional methods to selectively ablate a population of midbrain neurons important for the production of ultrasonic vocalizations (USVs) in mice. We find that, although ablation of these PAG-USV neurons blocks USV production in both males and females, these neurons are not required for the production of distress calls. Our findings suggest that distinct populations of midbrain neurons control the production of different vocalization types.

Composite receptive fields in the mouse auditory cortex.

A central question in sensory neuroscience is how neurons represent complex natural stimuli. This process involves multiple steps of feature extraction to obtain a condensed, categorical representation useful for classification and behaviour. It has previously been shown that central auditory neurons in the starling have composite receptive fields composed of multiple features. Whether this property is an idiosyncratic characteristic of songbirds, a group of highly specialized vocal learners or a generic property of sensory processing is unknown. To address this question, we have recorded responses from auditory cortical neurons in mice, and characterized their receptive fields using mouse ultrasonic vocalizations (USVs) as a natural and ethologically relevant stimulus and pitch-shifted starling songs as a natural but ethologically irrelevant control stimulus. We have found that these neurons display composite receptive fields with multiple excitatory and inhibitory subunits. Moreover, this was the case with either the conspecific or the heterospecific vocalizations. We then trained the sparse filtering algorithm on both classes of natural stimuli to obtain statistically optimal features, and compared the natural and artificial features using UMAP, a dimensionality-reduction algorithm previously used to analyse mouse USVs and birdsongs. We have found that the receptive-field features obtained with both types of the natural stimuli clustered together, as did the sparse-filtering features. However, the natural and artificial receptive-field features clustered mostly separately. Based on these results, our general conclusion is that composite receptive fields are not a unique characteristic of specialized vocal learners but are likely a generic property of central auditorysystems. KEY POINTS: Auditory cortical neurons in the mouse have composite receptive fields with several excitatory and inhibitory features. Receptive-field features capture temporal and spectral modulations of natural stimuli. Ethological relevance of the stimulus affects the estimation of receptive-field dimensionality.

Predictive Mouse Ultrasonic Vocalization Sequences: Uncovering Behavioral Significance, Auditory Cortex Neuronal Preferences, and Social-Experience-Driven Plasticity.

Mouse ultrasonic vocalizations (USVs) contain predictable sequential structures like bird songs and speech. Neural representation of USVs in the mouse primary auditory cortex (Au1) and its plasticity with experience has been largely studied with single-syllables or dyads, without using the predictability in USV sequences. Studies using playback of USV sequences have used randomly selected sequences from numerous possibilities. The current study uses mutual information to obtain context-specific natural sequences (NSeqs) of USV syllables capturing the observed predictability in male USVs in different contexts of social interaction with females. Behavioral and physiological significance of NSeqs over random sequences (RSeqs) lacking predictability were examined. Female mice, never having the social experience of being exposed to males, showed higher selectivity for NSeqs behaviorally and at cellular levels probed by expression of immediate early gene c-fos in Au1. The Au1 supragranular single units also showed higher selectivity to NSeqs over RSeqs. Social-experience-driven plasticity in encoding NSeqs and RSeqs in adult females was probed by examining neural selectivities to the same sequences before and after the above social experience. Single units showed enhanced selectivity for NSeqs over RSeqs after the social experience. Further, using two-photon Ca2+ imaging, we observed social experience-dependent changes in the selectivity of sequences of excitatory and somatostatin-positive inhibitory neurons but not parvalbumin-positive inhibitory neurons of Au1. Using optogenetics, somatostatin-positive neurons were identified as a possible mediator of the observed social-experience-driven plasticity. Our study uncovers the importance of predictive sequences and introduces mouse USVs as a promising model to study context-dependent speech like communications.SIGNIFICANCE STATEMENT Humans need to detect patterns in the sensory world. For instance, speech is meaningful sequences of acoustic tokens easily differentiated from random ordered tokens. The structure derives from the predictability of the tokens. Similarly, mouse vocalization sequences have predictability and undergo context-dependent modulation. Our work investigated whether mice differentiate such informative predictable sequences (NSeqs) of communicative significance from RSeqs at the behavioral, molecular, and neuronal levels. Following a social experience in which NSeqs occur as a crucial component, mouse auditory cortical neurons become more sensitive to differences between NSeqs and RSeqs, although preference for individual tokens is unchanged. Thus, speech-like communication and its dysfunction may be studied in circuit, cellular, and molecular levels in mice.

A review of ultrasonic vocalizations in mice and how they relate to human speech.

Mice communicate through audible vocalizations, which are within the human hearing range, and ultrasonic vocalizations (USVs), which are above the upper limit of human hearing. USVs are produced by rodents in social contexts including pup separation, territorial, and courting assays. Like birdsong, an established model for human speech, USVs in mice have been used as a model for understanding human communication. Their utility as a model of social communication is illustrated in neurodevelopmental conditions with a genetic basis, like autism spectrum disorders and Rett syndrome. As mice do not exhibit clear visual cues when they vocalize, the source of vocalization is often assumed. Therefore, there is potential to better discern the true vocal contribution of individual mice if the upper limit of human hearing were to be extended. Currently, there are efforts to increase the precision of sound-localizing technology, which will develop our understanding of communication in mice and other animal models.

Mouse and rat ultrasonic vocalizations in neuroscience and neuropharmacology: State of the art and future applications.

Mice and rats emit ultrasonic vocalizations (USVs), which may express their arousal and emotional states, to communicate with each other. There is continued scientific effort to better understand the functions of USVs as a central element of the rodent behavioral repertoire. However, studying USVs is not only important because of their ethological relevance, but also because they are widely applied as a behavioral readout in various fields of biomedical research. In mice and rats, a large number of experimental models of brain disorders exist and studying the emission of USVs in these models can provide valuable information about the health status of the animals and the effectiveness of possible interventions, both environmental and pharmacological. This review (i) provides an updated overview of the contexts in which ultrasonic calling behaviour of mice and rats has particularly high translational value, and (ii) gives some examples of novel approaches and tools used for the analysis of USVs in mice and rats, combining qualitative and quantitative methods. The relevance of age and sex differences as well as the importance of longitudinal evaluations of calling and non-calling behaviour is also discussed. Finally, the importance of assessing the communicative impact of USVs in the receiver, that is, through playback studies, is highlighted.

Contribution of the ventral pouch in the production of mouse ultrasonic vocalizations.

Mouse ultrasonic vocalizations (USVs) are of communicative significance and can serve as one of the major tools for behavioral phenotyping in mouse models of neurological disorders with social communication deficits. Understanding and identifying the mechanisms and role of laryngeal structures in generating USVs is crucial to understanding neural control of its production, which is likely dysfunctional in communication disorders. Although mouse USV production is accepted to be a whistle-based phenomenon, the class of whistle is debatable. Contradictory accounts exist on the role of a specific rodent intralaryngeal structure-the ventral pouch (VP), an air-sac-like cavity, and its cartilaginous edge. Also, inconsistencies in the spectral content of fictive USVs and real USVs in models without the VP points us to re-examine the role of the VP. We use an idealized structure, based on previous studies, to simulate a two-dimensional model of the mouse vocalization apparatus with the VP and without the VP. Our simulations were performed using comsol Multiphysics to examine characteristics of vocalizations beyond the peak frequency (f_{p}), like pitch jumps, harmonics, and frequency modulations, important in context-specific USVs. We successfully reproduced some of the crucial aspects of mouse USVs mentioned above, as observed through the spectrograms of simulated fictive USVs. Conclusions about the lack of a role of the mouse VP were previously made in studies primarily examining f_{p}. We investigated the impact of the intralaryngeal cavity and the alar edge on the simulated USV features beyond f_{p}. For the same combinations of parameters, removing the ventral pouch resulted in an alteration of the call characteristics, dramatically removing the variety of calls observed otherwise. Our results thus provide evidence supporting the hole-edge mechanism and the possible role of the VP in mouse USV production.

Ultrasonic Vocalizations in Adult C57BL/6J Mice: The Role of Sex Differences and Repeated Testing.

Ultrasonic vocalizations (USVs) are a major tool for assessing social communication in laboratory mice during their entire lifespan. At adulthood, male mice preferentially emit USVs toward a female conspecific, while females mostly produce ultrasonic calls when facing an adult intruder of the same sex. Recent studies have developed several sophisticated tools to analyze adult mouse USVs, especially in males, because of the increasing relevance of adult communication for behavioral phenotyping of mouse models of autism spectrum disorder (ASD). Little attention has been instead devoted to adult female USVs and impact of sex differences on the quantitative and qualitative characteristics of mouse USVs. Most of the studies have also focused on a single testing session, often without concomitant assessment of other social behaviors (e.g., sniffing), so little is still known about the link between USVs and other aspects of social interaction and their stability/variations across multiple encounters. Here, we evaluated the USVs emitted by adult male and female mice during 3 repeated encounters with an unfamiliar female, with equal or different pre-testing isolation periods between sexes. We demonstrated clear sex differences in several USVs' characteristics and other social behaviors, and these were mostly stable across the encounters and independent of pre-testing isolation. The estrous cycle of the tested females exerted quantitative effects on their vocal and non-vocal behaviors, although it did not affect the qualitative composition of ultrasonic calls. Our findings obtained in B6 mice, i.e., the strain most widely used for engineering of transgenic mouse lines, contribute to provide new guidelines for assessing ultrasonic communication in male and female adult mice.

Automatic segmentation and classification of mice ultrasonic vocalizations.

This paper addresses the development of a system for classifying mouse ultrasonic vocalizations (USVs) present in audio recordings. The automatic labeling process for USVs is usually divided into two main steps: USV segmentation followed by the matching classification. Three main contributions can be highlighted: (i) a new segmentation algorithm, (ii) a new set of features, and (iii) the discrimination of a higher number of classes when compared to similar studies. The developed segmentation algorithm is based on spectral entropy analysis. This novel segmentation approach can detect USVs with 94% and 74% recall and precision, respectively. When compared to other methods/software, our segmentation algorithm achieves a higher recall. Regarding the classification phase, besides the traditional features from time, frequency, and time-frequency domains, a new set of contour-based features were extracted and used as inputs of shallow machine learning classification models. The contour-based features were obtained from the time-frequency ridge representation of USVs. The classification methods can differentiate among ten different syllable types with 81.1% accuracy and 80.5% weighted F1-score. The algorithms were developed and evaluated based on a large dataset, acquired on diverse social interaction conditions between the animals, to stimulate a varied vocal repertoire.

Blast trauma affects production and perception of mouse ultrasonic vocalizations.

Blast trauma from explosions affects hearing and communication in a significant proportion of soldiers. Many veterans report difficulty communicating, especially in noisy and reverberant environments, which contributes to complex mental health problems including anxiety and depression. However, the relationship between communication and perceptual problems after a blast has received little scientific attention. In the current studies, the effects of blast trauma on the production and perception of ultrasonic vocalizations (USVs) by CBA/CaJ mice, a common animal model for hearing and communication disorders, was explored. Overall, mice change the total number of vocalizations, the proportion produced of each syllable category, and the peak frequency, bandwidth, and duration of their vocalizations after blast exposure. Further, the perception of USVs is affected after blast trauma, with an immediate worsening of detection for most USV categories in the first 1-5 days after blasts, which later recovers. This study is the first to examine changes in the production and perception of communication signals after blast traumas in mice and is an important step towards developing treatments for blast-induced hearing and communication disorders.

HybridMouse: A Hybrid Convolutional-Recurrent Neural Network-Based Model for Identification of Mouse Ultrasonic Vocalizations.

Mice use ultrasonic vocalizations (USVs) to convey a variety of socially relevant information. These vocalizations are affected by the sex, age, strain, and emotional state of the emitter and can thus be used to characterize it. Current tools used to detect and analyze murine USVs rely on user input and image processing algorithms to identify USVs, therefore requiring ideal recording environments. More recent tools which utilize convolutional neural networks models to identify vocalization segments perform well above the latter but do not exploit the sequential structure of audio vocalizations. On the other hand, human voice recognition models were made explicitly for audio processing; they incorporate the advantages of CNN models in recurrent models that allow them to capture the sequential nature of the audio. Here we describe the HybridMouse software: an audio analysis tool that combines convolutional (CNN) and recurrent (RNN) neural networks for automatically identifying, labeling, and extracting recorded USVs. Following training on manually labeled audio files recorded in various experimental conditions, HybridMouse outperformed the most commonly used benchmark model utilizing deep-learning tools in accuracy and precision. Moreover, it does not require user input and produces reliable detection and analysis of USVs recorded under harsh experimental conditions. We suggest that HybrideMouse will enhance the analysis of murine USVs and facilitate their use in scientific research.

LMT USV Toolbox, a Novel Methodological Approach to Place Mouse Ultrasonic Vocalizations in Their Behavioral Contexts-A Study in Female and Male C57BL/6J Mice and in Shank3 Mutant Females.

Ultrasonic vocalizations (USVs) are used as a phenotypic marker in mouse models of neuropsychiatric disorders. Nevertheless, current methodologies still require time-consuming manual input or sound recordings clean of any background noise. We developed a method to overcome these two restraints to boost knowledge on mouse USVs. The methods are freely available and the USV analysis runs online at https://usv.pasteur.cloud. As little is currently known about usage and structure of ultrasonic vocalizations during social interactions over the long-term and in unconstrained context, we investigated mouse spontaneous communication by coupling the analysis of USVs with automatic labeling of behaviors. We continuously recorded during 3 days undisturbed interactions of same-sex pairs of C57BL/6J sexually naive males and females at 5 weeks and 3 and 7 months of age. In same-sex interactions, we observed robust differences between males and females in the amount of USVs produced, in the acoustic structure and in the contexts of emission. The context-specific acoustic variations emerged with increasing age. The emission of USVs also reflected a high level of excitement during social interactions. We finally highlighted the importance of studying long-term spontaneous communication by investigating female mice lacking Shank3, a synaptic protein associated with autism. While the previous short-time constrained investigations could not detect USV emission abnormalities, our analysis revealed robust differences in the usage and structure of the USVs emitted by mutant mice compared to wild-type female pairs.

Investigating the acoustic directionality of mouse ultrasonic vocalizations

Across the animal kingdom, ultrasonic vocalizations (USVs) are used as a means of communication, navigation, and predation, and in some species (e.g., echolocating bats and whales), the directionality of the signal facilitates these processes. The utility and characteristics of mouse USVs, however, are less clear. Using an 8-channel microphone array, we recorded, localized, and assigned USVs to individual mice that were in isolation (n = 28) or socially interacting (male-female dyads, n = 13; 4-mouse, mixed-sex groups, n = 11). Males emitted a total of 3675 (100%), 22 268 (84.48%), and 73 488 (82.05%) of the assigned vocalizations when in isolation, interacting with a female, or socializing in a group. By examining the relationship between the USV sound intensity on the eight microphones and the head directions of the mice, we observed that male mice consistently produced directional USVs across each of the three behavioral contexts. Specifically, USV intensity was strongest in the direction that the mouse was facing and weakest in the opposite direction. Overall, these findings directly demonstrate that male mouse USVs are directional and may provide insight into the communicative function of mouse vocalizations.

A humanized version of Foxp2 affects ultrasonic vocalization in adult female and male mice.

The transcription factor FoxP2 is involved in setting up the neuronal circuitry for vocal learning in mammals and birds and is thought to have played a special role in the evolution of human speech and language. It has been shown that an allele with a humanized version of the murine Foxp2 gene changes the ultrasonic vocalization of mouse pups compared to pups of the wild-type inbred strain. Here we tested if this humanized allele would also affect the ultrasonic vocalization of adult female and male mice. In a previous study, in which only male vocalization was considered and the mice were recorded under a restricted spatial and temporal regime, no difference in adult vocalization between genotypes was found. Here, we use a different test paradigm in which both female and male vocalizations are recorded in extended social contact. We found differences in temporal, spectral and syntactical parameters between the genotypes in both sexes, and between sexes. Mice carrying the humanized Foxp2 allele were using higher frequencies and more complex syllable types than mice of the corresponding wildtype inbred strain. Our results support the notion that the humanized Foxp2 allele has a differential effect on mouse ultrasonic vocalization. As mice carrying the humanized version of the Foxp2 gene show effects opposite to those of mice carrying disrupted or mutated alleles of this gene, we conclude that this mouse line represents an important model for the study of human speech and language evolution.

Loss of cGMP-dependent protein kinase II alters ultrasonic vocalizations in mice, a model for speech impairment in human microdeletion 4q21 syndrome

Chromosome 4q21 microdeletion leads to a human syndrome that exhibits restricted growth, facial dysmorphisms, mental retardation, and absent or delayed speech. One of the key genes in the affected region of the chromosome is PRKG2, which encodes cGMP-dependent protein kinase II (cGKII). Mice lacking cGKII exhibit restricted growth and deficits in learning and memory, as seen in the human syndrome. However, vocalization impairments in these mice have not been determined. The molecular pathway underlying vocalization impairment in humans is not fully understood. Here, we employed cGKII knockout (KO) mice as a model for the human microdeletion syndrome to test whether vocalizations are affected by loss of the PRKG2 gene. Mice emit ultrasonic vocalizations (USVs) to communicate in social situations, stress, and isolation. We thus recorded ultrasonic vocalizations as a model for human speech. We isolated postnatal day 5–7 pups from the nest to record and analyze USVs and found significant differences in vocalizations of KO mice relative to wild-type and heterozygous mutant mice. KO mice produced fewer calls that were shorter duration and higher frequency. Because neuronal activation in the arcuate nucleus in the hypothalamus is important for the production of animal USVs following isolation from the nest, we assessed neuronal activity in the arcuate nucleus of KO pups following isolation. We found significant reduction of neuronal activation in cGKII KO pups after isolation. Taken together, our studies indicate that cGKII is important for neuronal activation in the arcuate nucleus, which significantly contributes to the production of USVs in neonatal mice. We further suggest cGKII KO mice can be a valuable animal model to investigate pathophysiology of human microdeletion 4q21 syndrome

Low-dimensional learned feature spaces quantify individual and group differences in vocal repertoires.

Increases in the scale and complexity of behavioral data pose an increasing challenge for data analysis. A common strategy involves replacing entire behaviors with small numbers of handpicked, domain-specific features, but this approach suffers from several crucial limitations. For example, handpicked features may miss important dimensions of variability, and correlations among them complicate statistical testing. Here, by contrast, we apply the variational autoencoder (VAE), an unsupervised learning method, to learn features directly from data and quantify the vocal behavior of two model species: the laboratory mouse and the zebra finch. The VAE converges on a parsimonious representation that outperforms handpicked features on a variety of common analysis tasks, enables the measurement of moment-by-moment vocal variability on the timescale of tens of milliseconds in the zebra finch, provides strong evidence that mouse ultrasonic vocalizations do not cluster as is commonly believed, and captures the similarity of tutor and pupil birdsong with qualitatively higher fidelity than previous approaches. In all, we demonstrate the utility of modern unsupervised learning approaches to the quantification of complex and high-dimensional vocal behavior.

Lactobacillus reuteri effects on maternal separation stress in newborn mice.

Probiotic Lactobacillus reuteri DSM 17938 (LR 17938) is beneficial to infants with colic. To understand its mechanism of action, we assessed ultrasonic vocalizations (USV) and brain pain/stress genes in newborn mice exposed to maternal separation stress. Pups were exposed to unpredictable maternal separation (MSU or SEP) or MSU combined with unpredictable maternal stress (MSU + MSUS or S + S), from postnatal days 5 to 14. USV calls and pain/stress/neuroinflammation-related genes in the brain were analyzed. We defined 10 different neonatal call patterns, none of which increased after MSU. Stress reduced overall USV calls. Orally feeding LR 17938 also did not change USV calls after MSU. However, LR 17938 markedly increased vocalizations in mice allowed to stay with their dams. Even though LR 17938 did not change MSU-related calls, LR 17938 modulated brain genes related to stress and pain. Up-regulatedgenes following LR 17938 treatment were opioid peptides, kappa-opioid receptor 1 genes, and CD200, important in anti-inflammatory signaling. LR 17938 down-regulated CCR2 transcripts, a chemokine receptor, in the stressed neonatal brain. USV calls in newborn mice are interpreted as "physiological calls" instead of "cries." Feeding LR 17938 after MSU did not change USV calls but modulated cerebral genes favoring pain and stress reduction and anti-inflammatory signaling. We defined mouse ultrasonic vocalization (USV) call patterns in this study, which will be important in guiding future studies in other mouse strains. Newborn mice with maternal separation stress have reduced USVs, compared to newborn mice without stress, indicating USV calls may represent "physiological calling" instead of "crying." Oral feeding of probiotic Lactobacillus reuteri DSM 17938 raised the number of calls when newborn mice continued to suckle on their dams, but not when mice were under stress. The probiotic bacteria had a dampening effect on monocyte activation and on epinephrine and glutamate-related stress gene expression in the mouse brain.

Ultrasonic vocalizations in mice: relevance for ethologic and neurodevelopmental disorders studies.

Mice use ultrasonic vocalizations (USVs) to communicate each other and to convey their emotional state. USVs have been greatly characterized in specific life phases and contexts, such as mother isolation-induced USVs for pups or female-induced USVs for male mice during courtship. USVs can be acquired by means of specific tools and later analyzed on the base of both quantitative and qualitative parameters. Indeed, different ultrasonic call categories exist and have already been defined. The understanding of different calls meaning is still missing, and it will represent an essential step forward in the field of USVs. They have long been studied in the ethological context, but recently they emerged as a precious instrument to study pathologies characterized by deficits in communication, in particular neurodevelopmental disorders (NDDs), such as autism spectrum disorders. This review covers the topics of USVs characteristics in mice, contexts for USVs emission and factors that modulate their expression. A particular focus will be devoted to mouse USVs in the context of NDDs. Indeed, several NDDs murine models exist and an intense study of USVs is currently in progress, with the aim of both performing an early diagnosis and to find a pharmacological/behavioral intervention to improve patients’ quality of life.

Phenotyping in Mice Using Continuous Home Cage Monitoring and Ultrasonic Vocalization Recordings.

Over the last century, the study of mouse behavior has uncovered insights into brain molecular mechanisms while revealing potential causes of many neurological disorders. To this end, researchers have widely exploited the use of mutant strains, including those generated in mutagenesis screens and those produced using increasingly sophisticated genome engineering technologies. It is now relatively easy to access mouse models carrying alleles that faithfully recapitulate changes found in human patients or bearing variants of genes that provide data on those genes' functions. Concurrent with these developments has been an appreciation of the limitations of some current testing platforms, especially those monitoring complex behaviors. Out-of-cage observational testing is useful in describing overt persistent phenotypes but risks missing sporadic or intermittent events. Furthermore, measuring the progression of a phenotype, potentially over many months, can be difficult while relying on assays that may be susceptible to changes in the testing environment. In recent years, there has also been increasing awareness that measurement of behaviors in isolation can be limiting, given that mice attempt to hide behavioral cues of vulnerability. To overcome these limitations, laboratory animal science is capitalizing on progress in data capture and processing expertise. Moreover, as additional recording modes become commonplace, ultrasonic vocalization recording is an appealing focus, as mice use vocalizations in various social contexts. Using video and audio technologies, we record the voluntary, unprovoked behaviors and vocalizations of mice in social groups. Adoption of these approaches is undoubtedly set to increase, as they capture the round-the-clock behavior of mouse strains. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Continuous recording of home cage activity using the Home Cage Analyzer (HCA) system Support Protocol: Subcutaneous insertion of a radio frequency identification microchip in the inguinal area Basic Protocol 2: Continuous recording of mouse ultrasonic vocalizations in the home cage.

Classifying sex and strain from mouse ultrasonic vocalizations using deep learning.

Vocalizations are widely used for communication between animals. Mice use a large repertoire of ultrasonic vocalizations (USVs) in different social contexts. During social interaction recognizing the partner's sex is important, however, previous research remained inconclusive whether individual USVs contain this information. Using deep neural networks (DNNs) to classify the sex of the emitting mouse from the spectrogram we obtain unprecedented performance (77%, vs. SVM: 56%, Regression: 51%). Performance was even higher (85%) if the DNN could also use each mouse's individual properties during training, which may, however, be of limited practical value. Splitting estimation into two DNNs and using 24 extracted features per USV, spectrogram-to-features and features-to-sex (60%) failed to reach single-step performance. Extending the features by each USVs spectral line, frequency and time marginal in a semi-convolutional DNN resulted in a performance mid-way (64%). Analyzing the network structure suggests an increase in sparsity of activation and correlation with sex, specifically in the fully-connected layers. A detailed analysis of the USV structure, reveals a subset of male vocalizations characterized by a few acoustic features, while the majority of sex differences appear to rely on a complex combination of many features. The same network architecture was also able to achieve above-chance classification for cortexless mice, which were considered indistinguishable before. In summary, spectrotemporal differences between male and female USVs allow at least their partial classification, which enables sexual recognition between mice and automated attribution of USVs during analysis of social interactions.