Separation slang – Laboratory mice use low-frequency call repertoire during physical separation

Reviewer #1 (Public review):

Summary:

Adult laboratory mice produce ultrasonic vocalizations during free social interactions, as well as lower-frequency, voiced calls (squeaks) during aversive contexts. The question of whether mice possess a more complex repertoire of vocalizations has been of great interest to scientists studying rodent vocal behavior. In the current study, the authors analyze the rates and acoustic features of vocalizations produced by pairs of mice that are allowed to interact across a barrier, which prevents direct physical interaction. In this context, they find that same-sex (but not opposite-sex) pairs of mice produce vocalizations that are lower in frequency than the typical 70 kHz ultrasonic vocalizations produced during free interactions and that are also distinct from squeaks. These lower frequency vocalizations were observed in both male-male and female-female pairs, as well as in same-sex pairs from multiple mouse strains. The authors also report that call rates and acoustic features are not affected in male-male pairs that have been treated with the anxiolytic drug buspirone, suggesting that anxiety is not a major driver of vocalization in this behavioral context.

Strengths:

(1) The observation that same-sex pairs of mice produce lower frequency (<70 kHz) vocalizations in this behavioral context is novel.

(2) The consideration of multiple types of pairs (female-female, male-male, and female-male), as well as the inclusion of multiple strains of mice and barriers with different hole diameters, are all strengths of the study.

(3) The authors include detailed analyses of vocalization acoustic features, as well as detailed tracking of mouse positions relative to the barrier.

Weaknesses:

The categorization applied to vocalizations based on their mean frequencies is poorly supported and ignores the distinction in laryngeal production mechanism between voiced and ultrasonic vocalizations. Specifically, the authors are likely lumping together voiced and ultrasonic vocalizations into their "low frequency" (< 30 kHz) category, while they reserve the term "ultrasonic" exclusively for the subset of ultrasonic vocalizations with the highest mean frequencies (> 50 kHz). This categorization scheme also does not align well with past work on lower frequency rodent vocalizations, which complicates the comparison of the present findings to that past work.

In some analyses, the authors report that different groups of mice produce different relative proportions of vocalization types (as defined by mean frequency) but then compare acoustic features of vocalizations between groups after pooling all vocalizations together. The analyses of acoustic features conducted in this way may be confounded by the different proportions of vocalization types across groups.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors examine vocal communication during same-sex dyadic interactions in mice, comparing periods of physical separation (with limited sensory access) to direct social contact. They report that separation dramatically alters the vocal repertoire, shifting it away from canonical ultrasonic vocalizations (USVs) toward low-frequency vocalizations (LFVs) and broadband "noisy" calls. While LFVs and noisy calls have been described previously, largely in aversive contexts, this study provides a detailed, systematic characterization of these vocalizations during social interactions, thereby extending prior work.

The authors explore several experimental manipulations and analyses, including divider hole size, strain and sex differences, anxiolytic drug treatment, and correlations with spatial proximity, to infer potential functions of these call types. Although the dataset is rich, the results are largely descriptive, and many conclusions remain tentative. Several experimental variables are not fully controlled, and in some cases, the interpretation exceeds what the data can clearly support. Nonetheless, with improved experimental framing, additional analyses of existing data, and a clearer discussion of limitations, this work has the potential to make a valuable contribution by broadening the field's focus beyond USVs to understand a wider vocal repertoire relevant to social context.

Strengths:

Much work on mouse vocal communication focuses almost exclusively on USVs. This manuscript convincingly demonstrates that non-USV vocalizations (LFVs and noisy calls) are prominent and systematically modulated by social context, highlighting an underappreciated dimension of mouse communication. Furthermore, the authors employ several experimental manipulations, including sensory access, strain, sex, and pharmacological treatment, to assess changes in vocalization repertoire. This provides a valuable resource for the field and reveals robust context dependence of vocalization. The discussion is thoughtful and integrative, particularly in its consideration of potential communicative roles of LFVs and noisy calls and their relationship to sensory constraints and signal propagation, although these ideas will require further experimental validation.

Weaknesses:

There are several concerns regarding experimental design and data interpretation that could be addressed to strengthen the manuscript.

(1) The terminology used for vocalization types is confusing and needs better clarification. The authors refer to Grimsley et al. (2016) multiple times, yet they use the same names for their vocalizations while applying different definitions. This makes it very difficult to compare the two papers. Since this study and Grimsley et al. use different mouse strains (FVB vs CBA), a direct comparison of absolute frequencies may also not be appropriate. Please explicitly clarify the definitions of the call types (e.g., frequency range, voiced vs. USV) and explain how they relate to those in the previous study earlier in the manuscript.

(2) In the initial experiment, mice always experience separation first (15 minutes), followed by unification (5 minutes), using novel same-sex dyads. Multiple factors besides physical contact could influence vocalization across this sequence, including habituation to the arena, reduced anxiety over time, or increasing familiarity with the partner despite physical separation. It is unclear whether the authors have tested the reverse order (unification first, followed by separation). If not, this limitation should be explicitly acknowledged. In addition, examining whether vocalizations or behaviors change over the course of the 15-minute separation period, for example, by comparing early vs late phases, could help disentangle effects of habituation from those of physical separation per se.

(3) The conclusion that separation-induced LFVs are unlikely to be anxiety-driven may overinterpret the buspirone experiment (Figure 8). Vehicle injections themselves produced large changes in call rate and call-type distribution, raising concerns about stress or arousal induced by the injection procedure. Comparisons between buspirone-treated animals and untreated animals are therefore problematic, as these groups differ in their experimental histories, including the number of exposures. The manuscript would benefit from independent measures confirming the anxiolytic efficacy of buspirone compared to vehicle injection in this paradigm, such as behavioral readouts of anxiety. In addition, the experimental design requires a clearer description. It is not always clear whether the same dyads were tested twice, or how social familiarity, contextual familiarity, and habituation to injections were handled. Male data comparing first and second exposures should also be included as supplementary figures to allow direct comparison with the excluded female dataset.

(4) The idea that noisy calls function to attract conspecific attention is intriguing. However, in Figure 5, all call types, including LFVs and USVs, are most likely to occur when mice are already in close proximity during separation, which seems inconsistent with a long-distance signaling role. Analyses of the temporal relationship between vocalizations and behavior would strengthen this claim. For example, it would be informative to test whether bouts of noisy calls precede approach behavior or a reduction in inter-animal distance. Examining whether calls occur before, during, or after orientation toward the partner could further clarify whether these vocalizations actively modulate social behavior.

(5) The effects of divider hole size on vocal repertoire are striking but difficult to interpret. Unexpectedly, small holes and no holes yield similar call distributions, whereas large holes produce a markedly different profile dominated by LFVs, which also differs from free interactions. If large holes allow greater tactile or close-range interaction, the reduction in USVs and MFV is counterintuitive. Incorporating behavioral metrics such as distance, orientation, or specific interaction types alongside call classification would greatly aid interpretation and help link vocal output to interaction quality rather than divider type alone.

(6) Throughout the study, vocalizations are pooled across both animals in the dyad. Because the arena is neutral rather than a home cage, either animal could be initiating vocalization. Assigning calls to individuals, where possible, using spatial or acoustic cues, would substantially strengthen functional interpretations. Even limited analyses, e.g., identifying which animal vocalizes first or whether calls precede approach by the partner, could provide important insight into the communicative role of different call types.

Author Response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

Adult laboratory mice produce ultrasonic vocalizations during free social interactions, as well as lower-frequency, voiced calls (squeaks) during aversive contexts. The question of whether mice possess a more complex repertoire of vocalizations has been of great interest to scientists studying rodent vocal behavior. In the current study, the authors analyze the rates and acoustic features of vocalizations produced by pairs of mice that are allowed to interact across a barrier, which prevents direct physical interaction. In this context, they find that same-sex (but not opposite-sex) pairs of mice produce vocalizations that are lower in frequency than the typical 70 kHz ultrasonic vocalizations produced during free interactions and that are also distinct from squeaks. These lower frequency vocalizations were observed in both male-male and female-female pairs, as well as in same-sex pairs from multiple mouse strains. The authors also report that call rates and acoustic features are not affected in male-male pairs that have been treated with the anxiolytic drug buspirone, suggesting that anxiety is not a major driver of vocalization in this behavioral context.

Strengths:

(1) The observation that same-sex pairs of mice produce lower frequency (<70 kHz) vocalizations in this behavioral context is novel.

(2) The consideration of multiple types of pairs (female-female, male-male, and female-male), as well as the inclusion of multiple strains of mice and barriers with different hole diameters, are all strengths of the study.

(3) The authors include detailed analyses of vocalization acoustic features, as well as detailed tracking of mouse positions relative to the barrier.

Weaknesses:

The categorization applied to vocalizations based on their mean frequencies is poorly supported and ignores the distinction in laryngeal production mechanism between voiced and ultrasonic vocalizations. Specifically, the authors are likely lumping together voiced and ultrasonic vocalizations into their "low frequency" (< 30 kHz) category, while they reserve the term "ultrasonic" exclusively for the subset of ultrasonic vocalizations with the highest mean frequencies (> 50 kHz). This categorization scheme also does not align well with past work on lower frequency rodent vocalizations, which complicates the comparison of the present findings to that past work.

We thank the reviewer for their assessment. Firstly, we did not use mean frequencies, but peak frequencies of each single call.

The distinction between ‘voiced’ and ‘whistled’ vocalizations based on their spectral-temporal features is hardly possible. While evidence in form of audio recordings made from both deer mouse and grasshopper mouse in helium-enriched air suggests vocalizations with lower fundamental frequency being ‘voiced’ (Pasch et al., 2017; Riede et al., 2022), a computational model considering the laryngeal anatomy of Mus musculus estimates fundamental frequencies of vocalizations at subglottal phonation threshold pressures usual for USVs to be in the range of 1 – 5 kHz and approaching 10 kHz for higher subglottal pressures usually found in the production of ‘voiced’ vocalizations (Pasch et al., 2017). Furthermore, a recent study in the singing mouse (Scotinomys teguina) found minimal fundamental frequencies of single song notes, produced by a whistle mechanism, to be about 4 kHz (Zheng et al., 2025). Thus, the presence of low fundamental (peak) frequencies in mouse vocalizations alone appears to be insufficient for deducing the production mechanism of these vocalizations.

We did not observe differences in acoustic features clearly separating our ‘LFV’ calls into two groups suggestive of different production mechanisms. Thus, we cannot rule out that our ‘LFV’ class contains vocalizations produced by different mechanisms. However, we did not observe any squeaks in our experiments and can therefore rule out that this prominent type of ‘voiced’ call is lumped together with other calls in the ‘LFV’ calls.

While the questions regarding production mechanism, the neurocircuitry involved, and the context-dependent choice of which mechanism to use is intriguing/enticing, the distinction between ‘voiced’ and ‘whistled’ vocalizations lies beyond the scope of our manuscript. Instead, the neurocircuitry involved in mouse vocalization production, particularly USVs and squeaks has been revealed by other laboratories. Optogenetical activation of RAm Nts neurons elicited emission of both audible vocalizations (fundamental frequencies of 10 kHz and below) and USVs in awake mice in a stimulus-dependent manner (Veerakumar et al., 2023). Furthermore, optogenetical activation of RAm-vocalization neurons led to immediate measurable adduction of vocal folds and emission of canonical USVs (Park et al., 2024). While different populations of PAG neurons are responsible for the production both squeaks and USVs (Ziobro et al., 2024), the two input streams seem to converge on RAm vocalization neurons, as silencing the output of these neurons abolished both squeak and USV emission completely (Park et al., 2024). Thus, while near complete closing of the vocal folds is necessary for the production of canonical USVs (Mahrt et al., 2016; Park et al., 2024), it is not clear which degree of vocal fold opening would result in what fundamental frequencies.

We will add a paragraph on this issue to the discussion in the next version of the manuscript.

In some analyses, the authors report that different groups of mice produce different relative proportions of vocalization types (as defined by mean frequency) but then compare acoustic features of vocalizations between groups after pooling all vocalizations together. The analyses of acoustic features conducted in this way may be confounded by the different proportions of vocalization types across groups.

We displayed the relative distribution of the different call classes demonstrating that 80% of the call repertoire during the separation consisted of noisy calls and ‘LFV’. Thus, the per individual averaged acoustic features e.g. peak frequency would be predominantly shaped by the features of these two call classes. However, we agree with the reviewer’s criticism and will provide a more detailed display and analysis of the acoustic features of each call class.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors examine vocal communication during same-sex dyadic interactions in mice, comparing periods of physical separation (with limited sensory access) to direct social contact. They report that separation dramatically alters the vocal repertoire, shifting it away from canonical ultrasonic vocalizations (USVs) toward low-frequency vocalizations (LFVs) and broadband "noisy" calls. While LFVs and noisy calls have been described previously, largely in aversive contexts, this study provides a detailed, systematic characterization of these vocalizations during social interactions, thereby extending prior work.

The authors explore several experimental manipulations and analyses, including divider hole size, strain and sex differences, anxiolytic drug treatment, and correlations with spatial proximity, to infer potential functions of these call types. Although the dataset is rich, the results are largely descriptive, and many conclusions remain tentative. Several experimental variables are not fully controlled, and in some cases, the interpretation exceeds what the data can clearly support. Nonetheless, with improved experimental framing, additional analyses of existing data, and a clearer discussion of limitations, this work has the potential to make a valuable contribution by broadening the field's focus beyond USVs to understand a wider vocal repertoire relevant to social context.

Strengths:

Much work on mouse vocal communication focuses almost exclusively on USVs. This manuscript convincingly demonstrates that non-USV vocalizations (LFVs and noisy calls) are prominent and systematically modulated by social context, highlighting an underappreciated dimension of mouse communication. Furthermore, the authors employ several experimental manipulations, including sensory access, strain, sex, and pharmacological treatment, to assess changes in vocalization repertoire. This provides a valuable resource for the field and reveals robust context dependence of vocalization. The discussion is thoughtful and integrative, particularly in its consideration of potential communicative roles of LFVs and noisy calls and their relationship to sensory constraints and signal propagation, although these ideas will require further experimental validation.

Weaknesses:

There are several concerns regarding experimental design and data interpretation that could be addressed to strengthen the manuscript.

(1) The terminology used for vocalization types is confusing and needs better clarification. The authors refer to Grimsley et al. (2016) multiple times, yet they use the same names for their vocalizations while applying different definitions. This makes it very difficult to compare the two papers. Since this study and Grimsley et al. use different mouse strains (FVB vs CBA), a direct comparison of absolute frequencies may also not be appropriate. Please explicitly clarify the definitions of the call types (e.g., frequency range, voiced vs. USV) and explain how they relate to those in the previous study earlier in the manuscript.

The existence and use of various distinct classification systems for mouse vocalizations is well known and the need to agree on a common classification system is consensus in the field. Thus, it was not our intention to complicate mouse call classification even more.

Grimsley at al. (2016) reserve the ‘low frequency’ band solely for squeaks (or “low frequency harmonics”). Hence, it appears straight forward to name mouse calls with “mean dominant frequencies” falling between squeaks and USVs, “mid-frequency tonal vocalizations (MFVs)” (Grimsley et al., 2016). We did not observe the emission of squeaks in our experiments, but instead we observed tonal vocalizations in a peak frequency spectrum encompassing both squeaks and Grimsley and colleagues’ ‘MFVs’, representing the lowest peak frequencies we observed (< 32 kHz). Furthermore, we observed vocalizations in the range of 32 – 50 kHz (which were not low frequency components of canonical USVs) and of > 50 kHz (corresponding to canonical USVs). Leaning on the terminology of Grimsley and colleagues (2016), we thought it to be straightforward to name these call classes according to their location on the frequency spectrum: low frequency vocalizations (LFVs; up to 32 kHz), encompassing squeaks, but also Grimsley and colleagues’ MFVs, middle frequency vocalizations (MFVs; 32 – 50 kHz), and finally canonical USVs (> 50 kHz). Admittedly, choosing ‘MFVs’ for mouse calls with different acoustic features than those described by Grimsley and colleagues (2016) has caused unnecessary confusion. We therefore consider adapting our classification scheme for the next version of the manuscript.

Regarding the comparison of call classes between different mouse strains, strain differences of spectral-temporal features of call classes have been described for canonical USVs (e.g. Scattoni et al., 2008). However, the acoustic features as well as call repertoire are still quite comparable. Furthermore, we have additionally tested both CBA/J and C57BL/6J mice in our study confirming the presence of both noisy calls, ‘LFVs’, ‘MFVs’, and ‘USVs’ in the vocal repertoire of these two strains.

We will provide a more detailed display and analysis of the acoustic features of the call classes with the next version of the manuscript.

(2) In the initial experiment, mice always experience separation first (15 minutes), followed by unification (5 minutes), using novel same-sex dyads. Multiple factors besides physical contact could influence vocalization across this sequence, including habituation to the arena, reduced anxiety over time, or increasing familiarity with the partner despite physical separation. It is unclear whether the authors have tested the reverse order (unification first, followed by separation). If not, this limitation should be explicitly acknowledged. In addition, examining whether vocalizations or behaviors change over the course of the 15-minute separation period, for example, by comparing early vs late phases, could help disentangle effects of habituation from those of physical separation per se.

We had not tested mice in the reverse order, beginning with 5 minutes of unification followed by 15 minutes of separation. Therefore, we acknowledge this limitation of our study and will address it explicitly in the next version of our manuscript. We appreciate the reviewer’s note regarding the inclusion of vocalizations over time and aim to provide this analysis in the next version of the manuscript.

(3) The conclusion that separation-induced LFVs are unlikely to be anxiety-driven may overinterpret the buspirone experiment (Figure 8). Vehicle injections themselves produced large changes in call rate and call-type distribution, raising concerns about stress or arousal induced by the injection procedure. Comparisons between buspirone-treated animals and untreated animals are therefore problematic, as these groups differ in their experimental histories, including the number of exposures. The manuscript would benefit from independent measures confirming the anxiolytic efficacy of buspirone compared to vehicle injection in this paradigm, such as behavioral readouts of anxiety. In addition, the experimental design requires a clearer description. It is not always clear whether the same dyads were tested twice, or how social familiarity, contextual familiarity, and habituation to injections were handled. Male data comparing first and second exposures should also be included as supplementary figures to allow direct comparison with the excluded female dataset.

We agree with the reviewer’s point that the injection procedure itself appeared to have an impact on vocalization behavior. In fact, we had included the ‘untreated’ cohort in Fig. 8 despite their different experimental history to appreciate the potential impact of injection onto vocal behavior.

Furthermore, we appreciate the reviewer’s point of confirming the anxiolytic effect of buspirone treatment with further behavioral readouts and aim to provide such analysis in the next version of the manuscript.

Regarding the reviewer’s query for clearer experimental design description, the same dyads were tested twice. All mice lived in groups in their home cage, however, they had not met the individual they would face during the experiment before the first experiment. We will improve the description of the experimental design addressing the reviewer’s points in the next version of the manuscript.

(4) The idea that noisy calls function to attract conspecific attention is intriguing. However, in Figure 5, all call types, including LFVs and USVs, are most likely to occur when mice are already in close proximity during separation, which seems inconsistent with a long-distance signaling role. Analyses of the temporal relationship between vocalizations and behavior would strengthen this claim. For example, it would be informative to test whether bouts of noisy calls precede approach behavior or a reduction in inter-animal distance. Examining whether calls occur before, during, or after orientation toward the partner could further clarify whether these vocalizations actively modulate social behavior.

We appreciate the reviewer’s remarks regarding the apparent inconsistencies between noisy calls as conspecific attraction calls and their occurrence in close mouse-to-mouse proximity. We must concede that the size of our testing arena limited the maximum distances mice could achieve. Thus, we aim to provide a more extensive analysis including approach behavior and changes of inter-animal distances for resubmission of the manuscript as suggested by the reviewer.

(5) The effects of divider hole size on vocal repertoire are striking but difficult to interpret. Unexpectedly, small holes and no holes yield similar call distributions, whereas large holes produce a markedly different profile dominated by LFVs, which also differs from free interactions. If large holes allow greater tactile or close-range interaction, the reduction in USVs and MFV is counterintuitive. Incorporating behavioral metrics such as distance, orientation, or specific interaction types alongside call classification would greatly aid interpretation and help link vocal output to interaction quality rather than divider type alone.

We agree with the reviewer that the interpretation of the divider-hole-size-experiment are difficult and following this reviewer’s input, aim to provide additional behavioral analysis for the effect of divider hole size with the next version of the manuscript.

(6) Throughout the study, vocalizations are pooled across both animals in the dyad. Because the arena is neutral rather than a home cage, either animal could be initiating vocalization. Assigning calls to individuals, where possible, using spatial or acoustic cues, would substantially strengthen functional interpretations. Even limited analyses, e.g., identifying which animal vocalizes first or whether calls precede approach by the partner, could provide important insight into the communicative role of different call types.

We agree with the points raised by the reviewer regarding the importance of assigning recorded calls to the respective individual for deciphering the communicative role of different call types. Unfortunately, our system was only equipped with one condenser microphone therefore we are not able to assign calls to individual mice.

Literature:

Grimsley, J. M. S., Sheth, S., Vallabh, N., Grimsley, C. A., Bhattal, J., Latsko, M., Jasnow, A., & Wenstrup, J. J. (2016). Contextual Modulation of Vocal Behavior in Mouse: Newly Identified 12 kHz „Mid-Frequency“ Vocalization Emitted during Restraint. Frontiers in Behavioral Neuroscience, 10, 38. https://doi.org/10.3389/fnbeh.2016.00038

Mahrt, E., Agarwal, A., Perkel, D., Portfors, C., & Elemans, C. P. H. (2016). Mice produce ultrasonic vocalizations by intra-laryngeal planar impinging jets. Current Biology: CB, 26(19), R880–R881. https://doi.org/10.1016/j.cub.2016.08.032

Park, J., Choi, S., Takatoh, J., Zhao, S., Harrahill, A., Han, B.-X., & Wang, F. (2024). Brainstem control of vocalization and its coordination with respiration. Science (New York, N.Y.), 383(6687), eadi8081. https://doi.org/10.1126/science.adi8081

Pasch, B., Tokuda, I. T., & Riede, T. (2017). Grasshopper mice employ distinct vocal production mechanisms in different social contexts. Proceedings. Biological Sciences, 284(1859), 20171158. https://doi.org/10.1098/rspb.2017.1158

Riede, T., Kobrina, A., Bone, L., Darwaiz, T., & Pasch, B. (2022). Mechanisms of sound production in deer mice (Peromyscus spp.). The Journal of Experimental Biology, 225(9), jeb243695. https://doi.org/10.1242/jeb.243695

Scattoni, M. L., Gandhy, S. U., Ricceri, L., & Crawley, J. N. (2008). Unusual repertoire of vocalizations in the BTBR T+tf/J mouse model of autism. PloS One, 3(8), e3067. https://doi.org/10.1371/journal.pone.0003067

Veerakumar, A., Head, J. P., & Krasnow, M. A. (2023). A brainstem circuit for phonation and volume control in mice. Nature Neuroscience, 26(12), 2122–2130. https://doi.org/10.1038/s41593-023-01478-2

Zheng, X. M., Harpole, C. E., Davis, M. B., & Banerjee, A. (2025). Vocal repertoire expansion in singing mice by co-opting a conserved midbrain circuit node. Current Biology: CB, 35(23), 5762-5778.e6. https://doi.org/10.1016/j.cub.2025.10.036

Ziobro, P., Woo, Y., He, Z., & Tschida, K. (2024). Midbrain neurons important for the production of mouse ultrasonic vocalizations are not required for distress calls. Current Biology: CB, 34(5), 1107-1113.e3. https://doi.org/10.1016/j.cub.2024.01.016