Rats emit aversive 44-kHz ultrasonic vocalizations during prolonged Pavlovian fear conditioning

Reviewer #1 (Public Review):

Olszyński and colleagues present data showing variability from canonical "aversive calls", typically described as long 22 kHz calls rodents emit in aversive situations. Similarly long but higher-frequency (44 kHz) calls are presented as a distinct call type, including analyses both of their acoustic properties and animals' responses to hearing playback of these calls. While this work adds an intriguing and important reminder, namely that animal behavior is often more variable and complex than perhaps we would like it to be, there is some caution warranted in the interpretation of these data.

The exclusive use of males is a major concern lacking adequate justification and should be disclosed in the title and abstract to ensure readers are aware of this limitation. With several reported sex differences in rat vocal behaviors this means caution should be exercised when generalizing from these findings. The occurrence of an estrus cycle in typical female rats is not justification for their exclusion. Note also that male rodents experience great variability in hormonal states as well, distinguishing between individuals and within individuals across time. The study of endocrinological influences on behavior can be separated from the study of said behavior itself, across all sexes. Similarly, concerns about needing to increase the number of animals when including all sexes are usually unwarranted (see Shansky [2019] and Phillips et al. [2023]).

Regarding the analysis where calls were sorted using DBSCAN based on peak frequency and duration, my comment on the originally reviewed version stands. It seems that the calls are sorted by an (unbiased) algorithm into categories based on their frequency and duration, and because 44kHz calls differ by definition on frequency and duration the fact that the algorithm sorts them as a distinct category is not evidence that they are "new calls [that] form a separate, distinct group". I appreciate that the authors have softened their language regarding the novelty and distinctness of these calls, but the manuscript contains several instances where claims of novelty and specificity (e.g. the subtitle on line 193) is emphasized beyond what the data justifies.

The behavioral response to call playback is intriguing, although again more in line with the hypothesis that these are not a distinct type of call but merely represent expected variation in vocalization parameters. Across the board animals respond rather similarly to hearing 22 kHz calls as they do to hearing 44 kHz calls, with occasional shifts of 44 kHz call responses to an intermediate between appetitive and aversive calls. This does raise interesting questions about how, ethologically, animals may interpret such variation and integrate this interpretation in their responses. However, the categorical approach employed here does not address these questions fully.

I appreciate the amendment in discussing the idea of arousal being the key determinant for the increased emission of 44kHz, and the addition of other factors. Some of the items in this list, such as annoyance/anger and disgust/boredom, don't really seem to fit the data. I'm not sure I find the idea that rats become annoyed or disgusted during fear conditioning to be a particularly compelling argument. As such the list appears to be a collection of emotion-related words, with unclear potential associations with the 44kHz calls.

Later in the Discussion the authors argue that the 44kHz aversive calls signal an increased intensity of a negative valence emotional state. It is not clear how the presented arguments actually support this. For example, what does the elongation of fear conditioning to 10 trials have to do with increased negative emotionality? Is there data supporting this relationship between duration and emotion, outside anthropomorphism? Each of the 6 arguments presented seems quite distant from being able to support this conclusion.

In sum, rather than describing the 44kHz long calls as a new call type, it may be more accurate to say that sometimes aversive calls can occur at frequencies above 22 kHz. Individual and situational variability in vocalization parameters seems to be expected, much more so than all members of a species strictly adhering to extremely non-variable behavioral outputs.

Author response:

The following is the authors’ response to the original reviews.

We would like to express our gratitude to the reviewers for their suggestions and critiques as we continually strive to enhance the quality of the manuscript. We improved it, by incorporating the reviewers’ suggestions, changing the content and numbering of figures (Figs 1, 3S1 were edited; 4 figures were moved to supplemental materials), and adding several analyses suggested by the reviewers along with accompanying figures (1S2, 1S3) and tables (1 and 2). These analyses include investigating the link between freezing behavior and 44-kHz calls as well as their sound mean power and duration. Also, we have introduced detailed information regarding the experiments performed as well as expanded the description and discussion of the results section. Finally, we added the information about 44-kHz calls reported by another group – which was inspired by our findings.

Below is the point-by-point response to the reviewers’ comments.

Reviewer #1 (Public Review):

Olszyński and colleagues present data showing variability from canonical "aversive calls", typically described as long 22 kHz calls rodents emit in aversive situations. Similarly long but higher-frequency (44 kHz) calls are presented as a distinct call type, including analyses both of their acoustic properties and animals' responses to hearing playback of these calls. While this work adds an intriguing and important reminder, namely that animal behavior is often more variable and complex than perhaps we would like it to be, there is some caution warranted in the interpretation of these data. The authors also do not provide adequate justification for the use of solely male rodents. With several reported sex differences in rat vocal behaviors this means caution should be exercised when generalizing from these findings.

We fully agree that our data should be interpreted with caution and we followed the Reviewer’s suggestions along these lines (see below). Also, we appreciate the suggestion to explore the prevalence of 44-kHz calls in female subjects, which would indeed represent an important and intriguing extension of our research. However, due to present financial constraints, we can only plan such experiments. To address the comment, we have added the sentence: “Here we are showing introductory evidence that 44-kHz vocalizations are a separate and behaviorally-relevant group of rat ultrasonic calls. These results require further confirmations and additional experiments, also in form of repetition, including research on female rat subjects.”

It is important to note that the data presented in the current manuscript originates primarily from previously conducted experiments. These earlier experiments employed male subjects only; it was due to established evidence indicating that the female estrus cycle significantly influences ultrasonic vocalization (Matochik et al., 1992). Adhering to controls for the estrus cycle would require a greater number of female subjects than males, which would not only increase animal suffering but also escalate the demands of human labor and financial costs.

Firstly, the authors argue that the shift to higher-frequency aversive calls is due to an increase in arousal (caused by the animals having received multiple aversive foot shocks towards the end of the protocols). However, it cannot be ruled out that this shift would be due to factors such as the passage of time and increase in fatigue of the animals as they make vocalizations (and other responses) for extended periods of time. In fact the gradual frequency increase reported for 22 kHz calls and the drop in 44 kHz calls the next day in testing is in line with this.

Answer: We would like to point out that the “increased-arousal” hypothesis, declared in the manuscript, is only a hypothesis – as reflected by the wording used. However, we changed the beginning of the sentence in question from “It could be argued” to “We would like to propose a hypothesis” to emphasize the speculative aspect of the proposed explanation behind the increase of 44-kHz ultrasonic emissions.

Also, we do agree that other factors could contribute to the increased emission of 44kHz calls. These factors could include: heightened fear, stress/anxiety, annoyance/anger, disgust/boredom, grief/sadness, despair/helplessness, and weariness/fatigue. We are listing these potential factors in the discussion. Also, we added: “It is not possible, at this stage, to determine which factors played a decisive role. Please note that the potential contribution of these factors is not mutually exclusive”. However, we propose a list of arguments supporting the idea that 44-kHz vocalizations communicate an increased negative emotional state. Among these arguments were the conclusions drawn from additional analyses – mostly inspired by the fatigue hypothesis proposed by the Reviewer #1. In particular, we investigated changes in the sound mean power and duration of 22-kHz and 44-kHz calls. Specifically, we showed that the mean power of 44-kHz vocalizations did not change, and was higher than that of 22-kHz vocalizations (Fig. 1S2EF).

Finally, the Reviewer #1 listed “the gradual frequency increase reported for 22 kHz calls and the drop in 44 kHz calls the next day” as arguments for the fatigue hypothesis. We do not agree that the “increase” should be interpreted as a sign of fatigue [Producing and maintaining higher frequency calls require greater effort from the vocalizer, on which we elaborated in the manuscript], also we are not sure what “drop in 44 kHz calls” the Reviewer is referring to [We assume it refers to less 44-kHz calls during testing vs. training; we suppose that the levels of arousal are lower in the test due to shorter session time and lack of shocks, which additionally contributes to fear extinction].

Secondly, regarding the analysis where calls were sorted using DBSCAN based on peak frequency and duration, it is not surprising that the calls cluster based on frequency and duration, i.e. the features that are used to define the 44 kHz calls in the first place. Thus presenting this clustering as evidence of them being truly distinct call types comes across as a circular argument.

Answer: The DBSCAN sorting results were to convey that when changing the clustering ε value, the degree of cluster separation, the 44-kHz vocalizations remained distinct from the 22-kHz and various short-call clusters that merged. In other words: 44-kHz calls remained separate from long 22-kHz, short 22-kHz and 50-kHz vocalizations, which all consolidated into one common cluster. As a result, in this mathematical analysis, 44-kHz vocalizations remained distinct without applying human biases. Additionally, frequency and duration are the two most common features used to define all types of calls (Barker et al., 2010; Silkstone & Brudzynski, 2019a, 2019b; Willey & Spear, 2013). In summary, we did not expect the analysis to isolate out the 44-kHz calls, and we were surprised by this result.

The sparsity of calls in the 30-40 kHz range (shown in the individual animal panels in Figure 2C) could in theory be explained by some bioacoustics properties of rat vocal cords, without necessarily the calls below and above that range being ethologically distinct.

Answer: We respectfully disagree with the argument regarding sparsity. It is important to note that, during prolonged fear conditioning experiments, we observed an increased incidence of 44-kHz calls (Fig. 1E-G) of up to >19% (Fig. 1S2AB) of the total ultrasonic vocalizations during specific inter-trial intervals. Also, it is possible that in observed experimental circumstances almost every fifth call could be attributed to the vocal apparatus as an artifact of its functioning (assuming we are interpreting the Reviewer’s argument correctly). While we do not believe this to be the case, we acknowledge the importance of considering such a hypothesis.

The behavioral response to call playback is intriguing, although again more in line with the hypothesis that these are not a distinct type of call but merely represent expected variation in vocalization parameters. Across the board animals respond rather similarly to hearing 22 kHz calls as they do to hearing 44 kHz calls, with occasional shifts of 44 kHz call responses to an intermediate between appetitive and aversive calls. This does raise interesting questions about how, ethologically, animals may interpret such variation and integrate this interpretation in their responses. However, the categorical approach employed here does not address these questions fully.

Answer: We are unsure of the Reviewer’s critique in this paragraph and will attempt to address it to the best of our understanding. Our finding of up to >19% of long seemingly aversive, 44-kHz calls, at a frequency in the define appetitive ultrasonic range (usually >32 kHz) is unexpected rather than “expected”. We would agree that aversive call variation is expected, but not in the appetitive frequency range.

Kindly note the findings by Saito et al. (2019), which claim that frequency band plays the main role in rat ultrasonic perception. It is possible that the higher peak frequency of 44kHz calls may be a strong factor in their perception by rats, which is, however, modified by the longer duration and the lack of modulation.

Also, from our experience, it is quite challenging to demonstrate different behavioral responses of naïve rats to pre-recorded 22-kHz (aversive) vs. 50-kHz (appetitive) vocalizations. Therefore, to demonstrate a difference in response to two distinct, potentially aversive, calls, i.e., 22-kHz vs. 44-kHz calls, to be even more difficult (as to our knowledge, a comparable experiment between short vs. long 22-kHz ultrasonic vocalizations, has not been done before).

Therefore, we do not take lightly the surprising and interesting finding that “animals respond rather similarly to hearing 22 kHz calls as they do to hearing 44 kHz calls, with occasional shifts of 44 kHz call responses to an intermediate between appetitive and aversive calls”. We would rather put this description in analogous words: “the rats responded similarly to hearing 44-kHz calls as they did to hearing aversive 22-kHz calls, especially regarding heartrate change, despite the 44-kHz calls occupying the frequency band of appetitive 50-kHz vocalizations” and “other responses to 44-kHz calls were intermediate, they fell between response levels to appetitive vs. aversive playback” – which we added to the Discussion.

Finally, we acknowledge that our findings do not present a finite and complete picture of the discussed aspects of behavioral responses to the presented ultrasonic stimuli (44-kHz vocalizations). Therefore, we have incorporated the Reviewer’s suggestion in the discussion. The added sentence reads: “Overall, these initial results raise further questions about how, ethologically, animals may interpret the variation in hearing 22-kHz vs. 44-kHz calls and integrate this interpretation in their responses.”

In sum, rather than describing the 44kHz long calls as a new call type, it may be more accurate to say that sometimes aversive calls can occur at frequencies above 22 kHz. Individual and situational variability in vocalization parameters seems to be expected, much more so than all members of a species strictly adhering to extremely non-variable behavioral outputs.

Answer: The surprising fact that there are presumably aversive calls that are beyond the commonly applied thresholds, i.e. >32 kHz, while sharing some characteristics with 22-kHz calls, is the main finding of the current publication. Whether they be finally assigned as a new type, subtype, i.e. a separate category or become a supergroup of aversive calls with 22-kHz vocalizations is of secondary importance to be discussed with other researchers of the field of study.

However, we would argue – by showing a comparison – that 22-kHz calls occur at durations of <300 ms and also >300 ms, and are, usually, referred to in literature as short and long 22-kHz vocalizations, respectively (not introduced with a description that “sometimes 22kHz calls can occur at durations below 300 ms”). These are then regarded and investigated as separate groups or classes usually referred to as two different “types” (e.g., Barker et al., 2010) or “subtypes” (e.g., Brudzynski, 2015). Analogously, 44-kHz vocalizations can also be regarded as a separate type or a subtype of 22-kHz calls. The problem with the latter is that 22-kHz vocalizations are traditionally and predominantly defined by 18–32 kHz frequency bandwidth (Araya et al., 2020; Barroso et al., 2019; Browning et al., 2011; Brudzynski et al., 1993; Hinchcliffe et al., 2022; Willey & Spear, 2013).

Reviewer #2 (Public Review):

Olszyński et al. claim that they identified a "new-type" ultrasonic vocalization around 44 kHz that occurs in response to prolonged fear conditioning (using foot-shocks of relatively high intensity, i.e. 1 mA) in rats. Typically, negative 22-kHz calls and positive 50-kHz calls are distinguished in rats, commonly by using a frequency threshold of 30 or 32 kHz. Olszyński et al. now observed so-called "44-kHz" calls in a substantial number of subjects exposed to 10 tone-shock pairings, yet call emission rate was low (according to Fig. 1G around 15%, according to the result text around 7.5%).

Answer: We are thankful for praising the strengths. Please note Figure 1G referred to 10-trial Wistar rats during delay fear conditioning session in which 44-kHz constituted 14.1% of ultrasonic vocalizations. The 7.5% number in results refers to the total of vocalizations analyzed across all animal groups used in fear conditioning experiments. These values have been updated in the current version of the manuscript. Also, please note – 44-kHz calls constituted up to 19.4% of calls, on average, in one of the ITI during fear conditioning session. However, the prevalence of aversive calls and of 44-kHz vocalizations in particular varied. It varied between individual rats; we added the text: “for n = 3 rats, 44-kHz vocalizations accounted for >95% of all calls during at least one ITI (e.g., 140 of total 142, 222 of 231, and 263 of 265 tallied 44-kHz calls), and in n = 9 rats, 44-kHz vocalizations constituted >50% of calls in more than one ITI.” See also further for the description of the array of experiments analyzed and the prevalence/percentage of 44-kHz calls encountered (Tab. 1, Fig. 1S3).

Weaknesses: I see a number of major weaknesses.

While the descriptive approach applied is useful, the findings have only focused importance and scope, given the low prevalence of "44 kHz" calls and limited attempts made to systematically manipulate factors that lead to their emission. In fact, the data presented appear to be derived from reanalyses of previously conducted studies in most cases and the main claims are only partially supported. While reading the manuscript, I got the impression that the data presented here are linked to two or three previously published studies (Olszyński et al., 2020, 2021, 2023). This is important to emphasize for two reasons:

(1) It is often difficult (if not impossible) to link the reported data to the different experiments conducted before (and the individual experimental conditions therein). While reanalyzing previously collected data can lead to important insight, it is important to describe in a clear and transparent manner what data were obtained in what experiment (and more specifically, in what exact experimental condition) to allow appropriate interpretation of the data. For example, it is said that in the "trace fear conditioning experiment" both single- and grouphoused rats were included, yet I was not able to tell what data were obtained in single- versus group-housed rats. This may sound like a side aspect, however, in my view this is not a side aspect given the fact that ultrasonic vocalizations are used for communication and communication is affected by the social housing conditions.

Answer: Preparing the current manuscript, we indeed used data collected during fear conditioning experiments which were described previously (Olszyński et al., 2021; Olszyński et al., 2022). Please note, however, that vocalization behavior during the fear conditioning itself was not the main subject of these publications. Our previous publications (Olszyński et al., 2020; Olszyński et al., 2021; Olszyński et al., 2022) present primarily ultrasonic-vocalization data from playback-part of experiments whereas here we analyze recordings obtained during fear conditioning experiments, thus we are analyzing new parts, i.e., not yet analyzed, of previously published studies. Also, we have performed additional experiments.

In the first version of the current manuscript, we did not attempt to demonstrate exactly which calls were recorded in which conditions as the focus was to demonstrate that 44-kHz calls were emitted in several different fear-conditioning experiments. Also, as the experiments were not performed simultaneously and are results from different experimental situations, we would prefer to not compare these results directly.

However, in the current version of the manuscript, we have introduced an additional reference system, based on Tab. 1, to more clearly indicate which rats have been employed in each analysis, e.g. the group of “Wistar rats that undergone 10 trials of fear conditioning” are described as “Tab. 1/Exp. 1-3/#2,4,8,13; n = 46”, i.e., these are the rats listed in rows 2, 4, 8, and 13 of Tab. 1.

We have also tried to unify the analyses, in terms of rats used, as much as possible. Finally, we have also introduced Fig. 1S3 to demonstrate the prevalence of 44-kHz calls in all experiments analyzed with the note that “the experiments were not performed in parallel”.

Regarding the Reviewer’s concerns about analyzing single- and pair-housed rats together. We have examined ultrasonic vocalizations emitted and freezing behavior in these two groups.

• Ultrasonic vocalizations; when comparing the number of vocalizations, their duration, peak frequency and latency to first occurrence, equally for all types of calls and divided into types (short 22-kHz, long 22-kHz, 44-kHz, 50-kHz), the only difference was observed in peak frequency in 50-kHz vocalizations (50.7 ± 2.8 kHz for paired vs. 61.8 ± 3.1 kHz for single rats; p = 0.0280, Mann-Whitney). Since 50-kHz calls are not the subject of the current publication, we did not investigate this difference further. Also, this difference was not observed during playback experiments (Olszyński et al., 2020, Tab. 1).

• Freezing. There were no differences between single- and pair-housed groups in freezing behavior, both in the time before first shock presentation and during fear conditioning training (Mann-Whitney).

In summary, since the two groups did not differ in relevant ultrasonic features and freezing, we decided to present the results obtained from these rats together. However, we agree with the Reviewer, and it is possible that social housing conditions may in fact affect the emission of 44-kHz vocalizations, which could be a subject of another project – involving, e.g., larger experimental groups observed under hypothesis-oriented and defined conditions.

(2) In at least two of the previously published manuscripts (Olszyński et al., 2021, 2023), emission of ultrasonic vocalizations was analyzed (Figure S1 in Olszyński et al., 2021, and Fig. 1 in Olszyński et al., 2023). This includes detailed spectrographic analyses covering the frequency range between 20 and 100 kHz, i.e. including the frequency range, where the "newtype" ultrasonic vocalization, now named "44 kHz" call, occurs, as reflected in the examples provided in Fig. 1 of Olszyński et al. (2023). In the materials and methods there, it was said: "USV were assigned to one of three categories: 50-kHz (mean peak frequency, MPF >32 kHz), short 22-kHz (MPF of 18-32 kHz, <0.3 s duration), long 22-kHz (MPF of 18-32 kHz, >0.3 s duration)". Does that mean that the "44 kHz" calls were previously included in the count for 50-kHz calls? Or were 44 kHz calls (intentionally?) left out? What does that mean for the interpretation of the previously published data? What does that mean for the current data set? In my view, there is a lack of transparency here.

Answer: As mentioned above, we indeed used data collected during fear conditioning experiments which were described previously (Olszyński et al., 2021; Olszyński et al., 2022). However, in these publications, ultrasonic vocalizations emitted during playback experiments were the main subject, while the ultrasonic calls emitted during fear conditioning (performed before the playback) were only analyzed in a preliminary way. As a result, the 44-kHz vocalizations analyzed in the current manuscript were not included in the previous analyses. In particular, in Olszyński et al. (2021), we counted the overall number of ultrasonic vocalizations before fear conditioning session to determine the basal ultrasonic emissions (Fig. S1). Then, our next article (Olszyński et al., 2022), we analyzed again the number of all ultrasonic vocalizations before fear conditioning (Fig. S1) and restricted the analysis of vocalizations during fear conditioning to 22-kHz calls (Tab. S1 and S2).

Also, we re-reviewed all the data used in our previous playback publications. Overall, 44-kHz calls were extremely rare in playback parts of the experiments. There were no 44-kHz calls in the playback data used in Olszyński et al. (2022) and Olszyński et al. (2020). In Olszyński et al. (2021), one rat produced eight 44-kHz calls. These 44-kHz calls constituted 0.03% of all vocalizations analyzed in the experiment (8/24888) and were included in the total number of calls analyzed (but not in the 50-kHz group), they were not described in further detail in that publication.

Moreover, whether the newly identified call type is indeed novel is questionable, as also mentioned by the authors in their discussion section. While they wrote in the introduction that "high-pitch (>32 kHz), long and monotonous ultrasonic vocalizations have not yet been described", they wrote in the discussion that "long (or not that long (Biały et al., 2019)), frequency-stable high-pitch vocalizations have been reported before (e.g. Sales, 1979; Shimoju et al., 2020), notably as caused by intense cholinergic stimulation (Brudzynski and Bihari, 1990) or higher shock-dose fear conditioning (Wöhr et al., 2005)" (and I wish to add that to my knowledge this list provided by the authors is incomplete). Therefore, I believe, the strong claims made in abstract ("we are the first to describe a new-type..."), introduction ("have not yet been described"), and results ("new calls") are not justified.

Answer: We would argue that 44-kHz vocalizations were indeed reported but not described. As far as we are concerned, an in-depth analysis of the properties and experimental circumstance of emission of long, high-frequency calls has not yet been performed. These researchers have observed, at least to a degree, similar calls to the ones we observed – as we mentioned in the discussion section. However, since these reported 44-kHz vocalizations were not fully described, we can only guess that they may be similar to ours. We speculate that perhaps like us, these researchers unknowingly recorded 44-kHz calls in their experiments and may also be able to describe them more extensively when re-analyzing their data as we have done here.

Possibly, it was difficult to find reports on vocalizations, similar to the 44-kHz calls that we observed, because of the canonical and accepted definitions of ultrasonic vocalization types. Biały et al. (2019) allocated them as a part of 22-kHz group, perhaps because their calls were often of a step variation having both low and high components. Shimoju et al. (2020) grouped them along with 50-kHz vocalizations because they appeared during stroking rats held vertically; this procedure was compared to tickling which usually elicits appetitive calls.

The Reviewer #2 states there are other publications to complete the list. We are aware of other articles authored by the same team as Shimoju et al. (2020) with different first authors. However, they are reporting similar findings to the cited article. Otherwise, we would gladly cite a more complete list of publications showing atypical, long, monotonous highfrequency vocalizations, similar to those observed in our experiments. Therefore, we would argue that ultrasonic vocalizations which were long, flat, high in frequency, and repeatedly occurring in a defined behavioral situation, have not been reported before. However, concerning the strong claims of novelty of our finding, we toned them down where we found this was warranted.

In general, the manuscript is not well written/ not well organized, the description of the methods is insufficient, and it is often difficult (if not impossible) to link the reported data to the experiments/ experimental conditions described in the materials and methods section.

Answer: The description of the methods has been adjusted and expanded. We added the requested link to each particular experiment as a formula “Tab. 1/Exp. nos./# nos.” which shows, each time, which experiments and experimental groups were analyzed. The list of the experiments and groups is found in the Tab. 1.

For example, I miss a clear presentation of basic information: 1) How many rats emitted "44 kHz" calls (in total, per experiment, and importantly, also per experimental condition, i.e. single- versus group-housed)?

Answer: We now clearly show which experiments were performed and how many animals were tested in each condition (Tab. 1), while the prevalence of 44-kHz calls amongst experimental conditions and animal groups is shown in Fig. 1S3. Also, we included information regarding the number of animals and treatment of each group of rats when reporting results. For example, we are stating that:

(1a) “53 of all 84 conditioned Wistar rats (Tab. 1/Exp. 1-3/#2,4,6-8,13, Figs 1B, 1E, 1S1BC) displayed” 44-kHz vocalizations – as a general assessment; these numbers are different from those in the first version of the Ms, when we are mentioning Wistar rats conditioned 6 or 10 times only.

(1b) “From this group of rats (n = 46), n = 41 (89.1%) emitted long 22-kHz calls, and 32 of them (69.6%) emitted 44-kHz calls” – this time referring only to 10-times conditioned Wistar rats as the biggest group that could be analyzed together (Figs 1F, 1G, 1S2A).

(1c) “for n = 3 rats, 44-kHz vocalizations accounted for >95% of all calls during at least one ITI (e.g., 140 of total 142, 222 of 231, and 263 of 265 tallied 44-kHz calls), and in n = 9 rats, 44kHz vocalizations constituted >50% of calls in more than one ITI.”

(2) Out of the ones emitting "44 kHz" calls, what was the prevalence of "44 kHz" calls (relative to 22- and 50-kHz calls, e.g. shown as percentage)?

Answer: The prevalence of 44-kHz vocalizations in all investigated experiments and groups is shown in Fig. 1S3CD. Also, more information regarding the percentage of 44-kHz calls was demonstrated in Fig. 1S2AB where we calculated the distribution of 44-kHz calls to 22-kHz calls in Wistar rats, in 10-trial fear conditioning, across the length of the session.

Additionally, the values are listed in the sentence regarding all Wistar rats which underwent 10 trials of fear conditioning: “these vocalizations were less frequent following the first trial (1.2 ± 0.4% of all calls), and increased in subsequent trials, particularly after the 5th (8.8 ± 2.8%), through the 9th (19.4 ± 5.5%, the highest value), and the 10th (15.5 ± 4.9%) trials, where 44-kHz calls gradually replaced 22-kHz vocalizations in some rats (Fig. 1F, 1S2B, Video 1; comp Fig. 1D vs. 1E).”

(3) How did this ratio differ between experiments and experimental conditions?

Answer: The prevalence of 44-kHz vocalizations in all experimental conditions is shown in Fig. 1S3. However, the direct comparison of results obtained in different conditions was not the goal of the present work. Also, we would argue, that such direct comparisons of results of different experiments would not be allowed. These experiments were done with different groups of animals, at different times, with different timetables of experimental manipulations.

However, we are comfortable to state that:

• There were more 44-kHz vocalizations during fear conditioning training than testing in all fear-conditioned Wistar rats;

• We observed more 44-kHz vocalizations in Wistar rats compared to SHR.

(4) Was there a link to freezing? Freezing was apparently analyzed before (Olszyński et al., 2021, 2023) and it would be important to see whether there is a correlation between "44-kHz" calls and freezing. Moreover, it would be important to know what behavior the rats are displaying while such "44-kHz" calls are emitted? (Note: Even not all 22-kHz calls are synced to freezing.) All this could help to substantiate the currently highly speculative claims made in the discussion section ("frequency increases with an increase in arousal" and "it could be argued that our prolonged fear conditioning increased the arousal of the rats with no change in the valence of the aversive stimuli"). Such more detailed analyses are also important to rule out the possibility that the "new-type" ultrasonic vocalization, the so-called "44 kHz" call, is simply associated with movement/ thorax compression.

Answer: We analyzed freezing behavior and its association with ultrasonic emissions. The emission of 44-kHz vocalizations was associated with freezing. The results are now described and presented in the manuscript, i.e., Tab. 2, its legend and the description in Results: “Freezing during the bins of 22-kHz calls only (p < 0.0001, for both groups) and during 44-kHz calls only bins (p = 0.0003) was higher than during the first 5 min baseline freezing levels of the session. Also, the freezing associated with emissions of 44-kHz calls only was higher than during bins with no ultrasonic vocalizations (p = 0.0353), and it was also 9.9 percentage points higher than during time bins with only long 22-kHz vocalizations, but the difference was not significant (p = 0.1907; all Wilcoxon)” and “To further investigate this potential difference, we measured freezing during the emission of randomly selected single 44-kHz and 22-kHz vocalizations. The minimal freezing behavior detection window was reduced to compensate for the higher resolution of the measurements (3, 5, 10, or 15 video frames were used). There was no difference in freezing during the emission of 44-kHz vs. 22-kHz vocalizations for ≥150ms-long calls (3 frames, p = 0.2054) and for ≥500-ms-long calls (5 frames, p = 0.2404; 10 frames, p = 0.4498; 15 frames, p = 0.7776; all Wilcoxon, Tab. 2B).”

Please note, that the general observation that "frequency increases with an increase in arousal" is not our claim but a general rule derived from large body of observations and proposed by the others (Briefer et al., 2012); we changed the wording of this statement to: “frequency usually increases with an increase in arousal (Briefer et al., 2012)”.

The figures currently included are purely descriptive in most cases - and many of them are just examples of individual rats (e.g. majority of Fig. 1, all of Fig. 2 to my understanding, with the exception of the time course, which in case of D is only a subset of rats ("only rats that emitted 44-kHz calls in at least seven ITI are plotted" - is there any rationale for this criterion?)), or, in fact, just representative spectrograms of calls (all of Fig. 3, with the exception of G, all of Fig. 4).

Answer: Please note, the former figures 2, 4, 6, and 8 have been now moved to supplementary figures 1S1, 2S1, 3S1, and 4S1 – to better organize the presentation of data. Figures 1, 3, 5, 7 are now 1, 2, 3, 4 respectively. In regards to presenting data from individual rats, this was to show the general patterns of ultrasonic-calls distributions observed. Showing the full data set as seen in Fig. 5A (now Fig. 3A) would obscure the readability of the graph without using mathematical clustering techniques such as DBSCAN.

Concerning the Reviewer’s #2 question regarding the criterion of “minimum seven ITI”, we selected the highest vocalizers by taking animals above the 75th percentile of the number of ITI with 44-kHz calls. However, in the current version of the manuscript, we decided to omit this part of the analysis and the accompanying part of the figure, since it did not provide any additional informative value (apart from employing questionable criterion).

Moreover, the differences between Fig. 5 and Fig. 6 are not clear to me. It seems Fig. 5B is included three times - what is the benefit of including the same figure three times?

Answer: We hope that designating Fig. 6 as supplementary to Fig. 5 (now Figs 3S1 and 3, respectively) will make interpreting them more streamlined. Fig. 6A (now Fig. 3S1A) is a more detailed look on information presented in Fig. 5B (now Fig. 3B) with spectrogram images of ultrasonic vocalizations from different areas of the plot. Also, Fig. 3B (former Fig. 5B) was removed from Fig. 3S1B (former Fig. 6B).

A systematic comparison of experimental conditions is limited to Fig. 7 and Fig. 8, the figures depicting the playback results (which led to the conclusion that "the responses to 44-kHz aversive calls presented from the speaker were either similar to 22-kHz vocalizations or in between responses to 22-kHz and 50-kHz playbacks", although it remains unclear to me why differences were seen b e f o r e the experimental manipulation, i.e. the different playback types in Fig. 8B).

Answer: There were indeed instances of such before-differences. Such differences were observed in our previous studies (Olszyński et al., 2020, Tabs S9-12; Olszyński et al., 2021, Tabs S7; Olszyński et al., 2022, Tabs S4, S9, S13, S17, S18) and were most likely due to analyzing multiple comparisons. However, we think that the carry-over effect, mentioned by the Reviewer #2 (see below), also played a role.

Related to that, I miss a clear presentation of relevant methodological aspects: 1) Why were some rats single-housed but not the others?

Answer: As stated before, data were collected from our previous experiments and the observation of 44-kHz vocalizations in fear conditioning was an emergent discovery as we decided to analyze ultrasonic recordings from fear conditioning procedures. Single-housed animals were part of our experiment comparing fear conditioning and social situation on the perception of ultrasonic playback as described in Olszyński et al. (2020). Aside from this experiment, all other rats were housed in pairs.

(2) Is the experimental design of the playback study not confounded? It is said that "one group (n = 13) heard 50-kHz appetitive vocalization playback while the other (n = 16) 22-kHz and 44kHz aversive calls". How can one compare "44 kHz" calls to 22- and 50-kHz calls when "44 kHz" calls are presented together with 22-kHz calls but not 50-kHz calls? What about carry-over effects? Hearing one type of call most likely affects the response to the other type of call. It appears likely that rats are a bit more anxious after hearing aversive 22-kHz calls, for example. Therefore, it would not be very surprising to see that the response to "44 kHz" calls is more similar to 22-kHz calls than 50-kHz calls.

Of note, in case of the other playback experiment it is just said that rats "received appetitive and aversive ultrasonic vocalization playback" but it remains unclear whether "44 kHz" calls are seen as appetitive or aversive. Later it says that "rats were presented with two 10-s-long playback sets of either 22-kHz or 44-kHz calls, followed by one 50-kHz modulated call 10-s set and another two playback sets of either 44-kHz or 22-kHz calls not previously heard" (and wonder what data set was included in the figures and how - pooled?). Again, I am worried about carry-over effects here. This does not seem to be an experimental design that allows to compare the response to the three main call types in an unbiased manner.

Answer: We apologize for being confounding and brief in our original description of the playback experiments. We wanted to avoid confusion associated with including several additional playback signals (please note some are not related to the current comparisons and include different 50-kHz ultrasonic subtypes and two different subtypes of short 22-kHz calls). We lengthened the description of these playback experiments in the current version.

In general, including more than one type of ultrasonic calls as playback has a risk of a carry-over effect as well as a habituation effect (the responses become weak). However, it greatly reduces the number of required animals. Finally, regarding the first experiment, we chose 3 playbacks to compare the rats’ reactions, as this was the most conservative choice we thought of.

We would like to highlight that we wanted to compare specifically the rats’ responses to 22-kHz vs. 44-kHz playback (as well as the effects of playback of different subtypes 50-kHz calls, which is not the subject of the current work). Therefore, we would argue, that the design of both experiments is actually unbiased regarding this key comparison (responses to 22-kHz vs. 44-kHz playback). In both experiments, 22-kHz and 44-kHz playbacks were included in the same sequences of stimuli and counterbalanced regarding their order (i.e., taking into account possible carry-over effects), and presented to the same rats. We regarded the group of rats that heard 50-kHz recordings as a baseline/control, since we know from previous playback studies what reactions to expect from rats exposed to these vocalizations (and 22-kHz playback), while in the second experiment, we reduced the 50-kHz playback to one set in order to minimize possible habituation to multiple playbacks.

We agree that the design of both experiments does not allow for full comparison of the effects of aversive playbacks to 50-kHz playback. Also, we agree that some carry-over effects could play a role. It was mentioned in the discussion: ”Please factor in potential carryover effects (resulting from hearing playbacks of the same valence in a row) in the differences between responses to 50-kHz vs. 22/44-kHz playbacks, especially, those observed before the signal (Fig. 4AB).” However, we would still argue that the observed lack of difference in heartrate response (Fig. 4A) and the differences regarding the number of 50-kHz calls emitted (e.g., Fig. 4S1F) are void of the constraints raised by the Reviewer #2.

We acknowledge that our studies do not give a complete picture of 44-kHz ultrasonic perception in relation to other ultrasonic bands and, given the possibility, we would like to perform more in-depth and focused experiments to study this aspect of 44-kHz calls in the future.

Finally, regarding the second experiment, the description of the rats now includes that they “received 22-kHz, 44-kHz, and 50-kHz ultrasonic vocalization playback”, while the description of the experiment itself includes: “Responses to the pairs of playback sets were averaged”.

Of note, what exactly is meant by "control rats" in the context of fear conditioning is also not clear to me. One can think of many different controls in a fear conditioning experiment.

More concrete information is needed.

Answer: This information was included in our previous publications. However, it was now provided in the method section of the current version of the manuscript. In general, control rats were subjected to the same procedures but did not receive electric shocks.

Literature included in the answers

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Recommendations For The Authors:

Reviewer #1 (Recommendations For The Authors):

Additional considerations:

The discussion of the "perfect fifth" and the proposition that this observation could be evidence of an evolutionary mechanism underlying it is rather far-fetched, especially for being presented in the Results section (with no supporting non-anecdotal evidence).

Answer: We agree with the Reviewer #1. The text was modified, the word “evolutionary” was deleted. Instead, we expended on the possible reason for prevalence of the perfect fifth in the current version of the manuscript; we added that the prevalence of the perfect fifth: “could be explained by the observation that all physical objects capable of producing tonal sounds generate harmonic vibrations, the most prominent being the octave, perfect fifth, and major third (Christensen, 1993, discussed in Bowling and Purves, 2015).”

It is not clear why Sprague-Dawleys were used as "receivers" in the playback experiment, when presumably the calls were recorded from Wistars and SHRs. While this does not critically impact the conclusions, within the species rats should be able to respond appropriately to calls made by rats of different genetic backgrounds, it adds an unnecessary source of variance.

Answer: Sprague-Dawley rats were used to test another normotensive strain of rats. Regarding the Reviewer’s main point – we beg to differ as we think that it is worth testing playback stimuli in different strains. Diverging the stimuli between different rat strains would add unnecessary variance and it seemed logical to use the same recordings to test effects in different strains. Please note that finally, in spite of this additional variance, the results of both playback experiments are, in general, similar – which may point to a universal effect of 44-kHz playback across rat strains.

It is pertinent to note that for the trace fear conditioning experiment, the rats had previously been exposed to a vocalization playback experiment. While such a pre-exposure is unlikely to be a very strong stressor, the possibility for it to influence the vocal behaviors of these rats in later experiments cannot be ruled out. It is also not clear what the control rats in this experiment experienced (home cage only?), nor what they were used for in analyses.

Answer: In the current version of the manuscript, we have described in greater detail all the experiments performed and analyzed. We would like to emphasize that both delay and trace fear conditioning experiments with radiotelemetric transmitters were not performed specifically to elicit any particular response during fear conditioning, rather that our observation of 44-kHz vocalizations emerged as a result of re-examining the audio recordings. As a result, this work summarizes our observations of 44-kHz calls from several different experiments. It is relevant to note, that 44-kHz vocalizations were observed “in rats which were exposed to vocalization playback experiment”, in rats before the playback experiments as well as in naïve rats, without transmitters implemented, trained in fear conditioning (Tab. 1/Exp. 1-3).

Our main message is that 44-kHz vocalizations were present in several experiments, with different conditions and subjects, while we are not attempting to compare in detail the results across the different experiments. In other words, we agree that pre-exposure to playback (and even more likely – transmitters implantation) could influence, but are not necessary, for 44-kHz ultrasonic emissions by the rats. To demonstrate this, we added a prolonged fear conditioning group with naïve Wistar rats (Exp. 3) to verify the emission of 44kHz calls in the absence of those experimental factors.

We modified the methods section to clarify the circumstances under which these discoveries were made, such as including the information regarding the control rats in trace fear conditioning. In particular we mention that: “Control rats were subjected to the exact same procedures but did not receive the electric shock at the end of trace periods”.

For Figure 1A-E, only example call distributions from individual rats are shown. It would perhaps be more informative to see the full data set displayed in this manner, with color/shape codes distinguishing individuals if desired.

Answer: Please note the Fig. 1S1 shows more examples of ultrasonic call distribution. Showing all the data would make it more difficult to read and interpret. The problem is partly amended in Fig. 3A.

It is not clear what is presented in Figure 2D vs. E, i.e. panel D is shown only for "selected rats" but the legend does not clarify how and why these rats were selected. It is also not clear why the legend reports p-values for both Friedman and Wilcoxon tests; the latter is appropriate for paired data which seems to be the case when the question is whether the call peak frequency alters across time, but the Friedman assumes non-paired input data.

Answer: The question refers to the current Fig. 1S2C panel (former Fig. 2E panel) and the former Fig. 2D panel. The latter was not included in the current version of the manuscript, since both reviewers opposed the presentation of “selected rats” only (see above). The full description of the Fig. 1S2C panel is now in the results section together with p-values for Friedman and Wilcoxon test. We used the latter to investigate the difference between the first and the last ITI (selected paired data), while the Friedman to investigate the presence of change within the chain of ten ITI – since it is a suitable test for a difference between two or more paired samples.

Reviewer #2 (Recommendations For The Authors):

The weaknesses listed in the public review need to be addressed.

Answer: We have done our best to address the weaknesses.

Notes: 1) Page and line numbers would have been useful.

Answer: We are including a separate manuscript version with page and line numbers.

.(2) English language needs to be improved.

Answer: The text has been checked by two native English speakers (one with a scientific background). Both only identified minor changes to improve the text which we applied.

(3) I am a bit unsure whether the comment about the Star Wars movie (1997) and the Game of Thrones series (2011) is supposed to be a joke.

Answer: These are indeed two genuine examples of the perfect fifth in human music that we hope are easily recognizable and familiar to readers. Parts of the same examples of the perfect fifth can also heard in the rat voice files provided.