The Difference between Male and Female Rats in Terms of Freezing and Aversive Ultrasonic Vocalization in an Active Avoidance Test

Round 1

Reviewer 1 Report

Manuscript of Alexandrov et al. deals with behavioral performance and ultrasonic 22kHz vocalizations (USV) in Wistar rats in a two-way shuttle box active avoidance paradigm. Authors describe different strategies in males and females, with freezing being typical for males. USVs were found in both sexes.

Studying USVs in aversive, shock-controlled situations dates back to the 80s, so one would expect quite an innovative approach to push forward our knowledge in this field. From this point of view, I find (unfortunately) very little that this manuscript can offer. Actually, the main finding is that (diestrus) females outperform males in active avoidance, which itself is a good starting point (and was observed in numerous studies), but the following analysis of USVs is rather disappointing. 

Rats naturally display great variability in terms of USVs - some individuals are very vocal, and some are almost mute. Thus it would be interesting to see if an individual's USV frequency is just related to the aversive situation in the active avoidance or is also present in less stressful experience such as an open field. In addition, the authors should discuss in more detail why they did not observe a well-known relation between impaired performance in two-way avoidance and increased level of vocalization. Measuring a level of stress (corticosterone, heart rate) would be helpful for better understanding why some males vocalized during freezing while others did not. Were the WAG/Rij rats carefully examined for potential confounding effects of epileptic seizures during the experiment?

Minor point:

Table 1, row 92: ...the number of WAG/Rij males who successfully performed the test was LOWER, not higher.

Author Response

Dear Reviewer,

Thank you for your feedback and careful revision of our paper. We highly appreciate the time and effort that you dedicated to evaluating our paper. Your critiques focused on the novelty of our study, the overall clarity of the document, and theoretical issues. We have made substantial revisions to the manuscript based on your feedback. Please find our point-to-point answers below.

Manuscript of Alexandrov et al. deals with behavioral performance and ultrasonic 22kHz vocalizations (USV) in Wistar rats in a two-way shuttle box active avoidance paradigm. Authors describe different strategies in males and females, with freezing being typical for males. USVs were found in both sexes.

Studying USVs in aversive, shock-controlled situations dates back to the 80s, so one would expect quite an innovative approach to push forward our knowledge in this field.

Response. We sincerely appreciate your critical comments. Thank you for bringing these shortcomings to light. Yes, we are using old techniques, such as active avoidance and analysis of 22-25 kHz ultrasonic vocalization. Avoidance has been a longstanding technique since the late 60's (Riccio and colleagues, 1968). We found the study of V. Cuomo and co-authors who examined ultrasonic calling during stressful situations more than twenty years ago (in 1992). In the revised Introduction we cited Cuomo et al. 1992: “it is surprising how few scientists have documented USVs during learning sessions in rats and used aversive ultrasonic calls as a sign of a negative emotional state”. We comprehensively revised the Introduction and included new findings.

From this point of view, I find (unfortunately) very little that this manuscript can offer. Actually, the main finding is that (diestrus) females outperform males in active avoidance, which itself is a good starting point (and was observed in numerous studies), but the following analysis of USVs is rather disappointing. 

Response. In the 'Conclusions' section, we have detailed our findings, provided a concise overview of our novel discoveries, and highlighted strong points. Rats are often employed in cognitive studies due to their high capability for learning, but only a small number of studies have determined emotional states through ultrasonic vocalizations. This has also been mentioned in Introduction.

Rats naturally display great variability in terms of USVs - some individuals are very vocal, and some are almost mute. Thus it would be interesting to see if an individual's USV frequency is just related to the aversive situation in the active avoidance or is also present in less stressful experience such as an open field.

Response. This remark focuses on the variability of the ultrasonic vocalizations of rats in various stressful conditions. Adult rats are known to emit two different kinds of ultrasonic vocalizations: the appetitive type (50 kHz) and the aversive type (22-25 kHz) as summarized by Stefan M. Brudzynski (e.g., doi:10.3390/BRAINSCI11050605). We totally agree with the reviewer: ultrasonic vocalizations emitted by adult rats are heterogeneous, but within the category 50 kHz, as noted by Jennifer M. Wright et al (doi:10.1007/s00213-010-1859-y). The aversive type of USVs (22-25 kHz) is very stable (in frequency) and very prolonged (from 100 ms up to several seconds). It is a unique behavioral indicator of emotional state in rats, though they are not commonly used. This is mentioned now in the Introduction.

In addition, the authors should discuss in more detail why they did not observe a well-known relation between impaired performance in two-way avoidance and increased level of vocalization. Measuring a level of stress (corticosterone, heart rate) would be helpful for better understanding why some males vocalized during freezing while others did not.

Response. Thank you for this suggestion. Now we discuss the correlation between unsuccessful performance in two-way avoidance and the level of aversive ultrasonic vocalization (Sections 3.2 and 3.3).
We completely agree that measuring hormones associated with stress (corticosterone), the physical manifestation of stress (heart rate) and the activity of the hypothalamic–pituitary–adrenal axis would be beneficial for better understanding why some males vocalized during freezing. This idea is added to future directions (Section 3.2).

Were the WAG/Rij rats carefully examined for potential confounding effects of epileptic seizures during the experiment?

Response. The experiment did not involve the assessment of WAG/Rij rats for epilepsy. There was no rationale to examine epileptic activity, because absence seizures do not occur when a rat is in a stressful situation or actively engaged in an activity (doi:10.1016/0920-1211(87)90005-2; doi:10.1023/a:1026179013847; doi: 10.3389/fphar.2020.00395). This seizure type occurs when a rat is in a resting condition, in a passive state and during drowsiness. This information is added to Sections 3.3 and 4.1.

Minor point:

Table 1, row 92: ...the number of WAG/Rij males who successfully performed the test was LOWER, not higher.

Response. Thank you for notifying this error. It is corrected.

Reviewer 2 Report

The present article entitled ‘The rat male's behavioral strategy of the active avoidance test: freezing and aversive ultrasonic vocalization’ by Alexandrov and colleagues investigates the emotional response and behavioral strategies of rats during active avoidance learning using ultrasonic vocalizations (USVs) as indicators. Active avoidance is a behavioral paradigm used to study conditioned learning, where rats learn to associate a negative outcome with a preceding event. In this study, the authors focus on male and female rats from two strains: Wistar Albino Rats from Rijswijk (WAG/Rij rats) and a non-epileptic control strain (NEW). The rats were trained to shuttle between compartments in a two-alternative choice paradigm when presented with a warning signal (tone or flash) to avoid an electric shock. The authors found that male rats exhibited higher levels of aversive USVs and freezing behavior compared to female rats. Furthermore, a higher percentage of male rats failed to learn the avoidance task successfully. The results suggest that male rats may have a more cautious behavioral strategy, possibly driven by their negative emotional reaction, as indicated by increased aversive USVs. In contrast, female rats showed a higher success rate in learning the avoidance task and had a lower incidence of freezing behavior.

In general, I think the idea of this article is really interesting and the authors’ fascinating observations on this timely topic may be of interest to the readers of Physiologia. However, some comments, as well as some crucial evidence that should be included to support the author’s argumentation, needed to be addressed to improve the quality of the manuscript, its adequacy, and its readability prior to the publication in the present form. My overall judgment is to publish this paper after the authors have carefully considered my suggestions below, in particular reshaping parts of the ‘Introduction’ and ‘Methods’ sections by adding more evidence.

Please consider the following comments:

• I suggest changing the title. In my opinion, in the present form it is wordy/misleading for the readers and not enough informative, and it could benefit from clearer language and structure. The use of phrases like "rat male's behavioral strategy" and "active avoidance test" may not be immediately understandable to a broad audience. 

• Abstract: In my opinion, Authors should consider rephrasing this section. According to the Journal’s guidelines, the Abstract should contain most of the following kinds of information in brief form. Please, consider giving a more synthetic overview of the paper's key points: I would suggest rephrasing the results and conclusion to make them clear for readers to understand.

• A graphical abstract that will visually summarize the main findings of the manuscript is highly recommended.

• Introduction: This section provides a good overview of active avoidance as a behavioral paradigm but could benefit from more clarity and organization. Still, in my opinion, it would be beneficial to provide more context and background information on the importance of studying active avoidance and its relevance to animal and human behavior, as well as to delve deeper into the specific neural circuits and mechanisms of conditioning involved in active avoidance. Exploring these aspects would provide a more comprehensive understanding of the underlying processes and shed light on the intricate relationship between behavior, emotion, and neural activity (DOI: 10.3390/biomedicines11030945). By elucidating the neural circuits, such as the amygdala, hippocampus, and prefrontal cortex, that are known to be involved in fear conditioning and active avoidance, the Authors can establish a stronger link between behavior and the underlying neural mechanisms (https://doi.org/10.1016/j.neubiorev.2023.105163; DOI: 10.1111/psyp.14020). 

• Methods:I suggest to provide a clear rationale for the selection of the control group in the experimental design. Indeed, this section should explicitly state how the control group was chosen and why it is an appropriate comparison for the active avoidance group. 

• Results: In my opinion, this section is well organized, but it illustrates findings in an excessively broad way, without really providing full statistical details, to ensure in-depth understanding and replicability of the findings. I suggest rewriting this section more accurately, for example including the actual p-values for each observed comparison. Also, the Authors should consider presenting the results in a more organized and visually appealing manner, such as using tables or graphs to summarize the data.

• In my opinion, I think the ‘Conclusions’ paragraph would benefit from some thoughtful as well as in-depth considerations by the authors, because as it stands, it is very descriptive but not enough theoretical as a discussion should be. Authors should make an effort, trying to explain the theoretical implication as well as the translational application of their research.

• In according to the previous comment, I would ask the authors to include a proper and defined ‘Limitations and future directions’ section before the end of the manuscript, in which authors can describe in detail and report all the technical issues brought to the surface.

• References: Authors should consider revising the bibliography, as there are several incorrect citations. Indeed, according to the Journal’s guidelines, they should provide the abbreviated journal name in italics, the year of publication in bold, the volume number in italics for all the references. 

I hope that, after these careful revisions, this paper can meet the Journal’s high standards for publication. 

I am available for a new round of revision of this article. 

I declare no conflict of interest regarding this manuscript. 

 

Best regards,

Reviewer

 

Minor editing of English language required.

Author Response

Dear Reviewer,

We highly appreciate your efforts and time in evaluating our paper. Thank you for your suggestions on ways to improve clarity, and to add more relevant references. We revised our review in accordance with your critical remarks. We have revised the manuscript significantly in light of your feedback, emphasizing the originality of our study and the theoretical considerations. Please find our point-to-point answers below.

Please consider the following comments:

• I suggest changing the title. In my opinion, in the present form it is wordy/misleading for the readers and not enough informative, and it could benefit from clearer language and structure. The use of phrases like "rat male's behavioral strategy" and "active avoidance test" may not be immediately understandable to a broad audience. 

Response. Thank you for bringing this issue to our attention. We agree that the term "strategy" is not applicable here as we are dealing with behavioral responses (or reactions). We reclassified freezing as a behavioral reaction, rather than a strategy, in response to the experimental context. We replaced the behavioral "strategy" with "response/reaction."

• Abstract: In my opinion, Authors should consider rephrasing this section. According to the Journal’s guidelines, the Abstract should contain most of the following kinds of information in brief form. Please, consider giving a more synthetic overview of the paper's key points: I would suggest rephrasing the results and conclusion to make them clear for readers to understand.

Response. The abstract has been revised to give a more concise summary of the main points.

• A graphical abstract that will visually summarize the main findings of the manuscript is highly recommended.

Response. This point has been addressed; the graphical abstract has been included in the submission.

• Introduction: This section provides a good overview of active avoidance as a behavioral paradigm but could benefit from more clarity and organization. Still, in my opinion, it would be beneficial to provide more context and background information on the importance of studying active avoidance and its relevance to animal and human behavior, as well as to delve deeper into the specific neural circuits and mechanisms of conditioning involved in active avoidance. Exploring these aspects would provide a more comprehensive understanding of the underlying processes and shed light on the intricate relationship between behavior, emotion, and neural activity (DOI: 10.3390/biomedicines11030945). By elucidating the neural circuits, such as the amygdala, hippocampus, and prefrontal cortex, that are known to be involved in fear conditioning and active avoidance, the Authors can establish a stronger link between behavior and the underlying neural mechanisms (https://doi.org/10.1016/j.neubiorev.2023.105163; DOI: 10.1111/psyp.14020). 

Response. The introduction has been revised to shed light on the intricate relationship between behavior, emotion, and neural activity. We highlighted adaptive and maladaptive kinds of avoidance, the central role of the amygdalo-cortical circuitry, and a questionable contribution of the hippocampus.

• Methods:I suggest to provide a clear rationale for the selection of the control group in the experimental design. Indeed, this section should explicitly state how the control group was chosen and why it is an appropriate comparison for the active avoidance group. 

Response. We incorrectly used the term "control" in the submitted manuscript. We mentioned that “the control” non-epileptic NEW substrain was used together with WAG/Rij rats. Meaning that we use a minor rat substrain, which was selected as a control for WAG/Rij rats (Non-Epileptic WAG/Rij = NEW). We did not have an actual control in the current study, because we focused on the rat's behavioral performance of avoidance task and on aversive ultrasonic calls. Therefore, our study was exploratory in nature. We removed the misleading word “control” rats from the text and emphasized in Section 3.1 that our study was exploratory.

• Results: In my opinion, this section is well organized, but it illustrates findings in an excessively broad way, without really providing full statistical details, to ensure in-depth understanding and replicability of the findings. I suggest rewriting this section more accurately, for example including the actual p-values for each observed comparison. Also, the Authors should consider presenting the results in a more organized and visually appealing manner, such as using tables or graphs to summarize the data.

Response. We verified the Results section to ensure that we met the reviewer's critical feedback. The p-values for each observed comparison are stated in the text (with the exception of Figure legends, in which two or more p's were marked with an asterisk when they were less than 0.05). We use one table (Table 1) summarizing individual data and 3 graphs with experimental results. An error was discovered in Table 1, and the subsequent statistics were recalculated after the necessary corrections were made. Figure 2 shows the distribution of different test performance. Figure 3 has been revised to more accurately depict individual data of aUSVs. Figure 4 shows the results of a statistical analysis of USV density. We have no other clues as to how to arrange our Results in a visually appealing manner.

• In my opinion, I think the ‘Conclusions’ paragraph would benefit from some thoughtful as well as in-depth considerations by the authors, because as it stands, it is very descriptive but not enough theoretical as a discussion should be. Authors should make an effort, trying to explain the theoretical implication as well as the translational application of their research.

Response. We are truly grateful for this remark. We added more in-depth considerations in ‘Conclusions’. Rats have learning abilities and are widely used in cognitive studies, therefore, ultrasonic vocalizations. In this revision, we emphasize the theoretical implications and the translational value of our results.

• In according to the previous comment, I would ask the authors to include a proper and defined ‘Limitations and future directions’ section before the end of the manuscript, in which authors can describe in detail and report all the technical issues brought to the surface.

Response. We greatly appreciate this comment. Already in Introduction we added that active avoidance is an old technique. However, the study of active avoidance in rats (as models of neuropsychiatric human diseases) can provide important insights into emotional reactions, fear acquisition, fear reduction, stress reactions, and signs of depression.

• References: Authors should consider revising the bibliography, as there are several incorrect citations. Indeed, according to the Journal’s guidelines, they should provide the abbreviated journal name in italics, the year of publication in bold, the volume number in italics for all the references. 

Response.  The bibliography has been examined for incorrect citations. The bibliography has been expanded to include additional references. All references were examined to ensure that they corresponded to the "Multidisciplinary Digital Publishing Institute" citation style.

I hope that, after these careful revisions, this paper can meet the Journal’s high standards for publication. 

Response. We are extremely grateful for your suggestions and critical remarks. Your input is vital in ensuring that our paper is of the highest quality. We greatly appreciate your suggestions and critical remarks, as they help us to improve our paper.

Round 2

Reviewer 2 Report

Alexandrov and colleagues in the present article entitled ‘The rat male's behavioral strategy of the active avoidance test: freezing and aversive ultrasonic vocalization’ investigated the behavioral strategies and emotional responses of rats during an active avoidance test.

I only have one last suggestion to do: to further enhance the understanding of these results, it would be beneficial to explore several aspects in more depth. In this regard, I still suggest to deepen information on the relationship between behavior, emotion, and neural activity, in order to enhance the knowledge on neural substrates underlying fear conditioning and active avoidance (https://doi.org/10.1016/j.neubiorev.2023.105163; DOI: 10.17219/acem/165944; https://doi.org/10.3390/ijms24065926). 

Overall, this is a timely and needed work, and I only had one last comment to do, to fully support the authors’ claims. My overall judgment is this article but only after the authors have carefully considered my last suggestion.

I am always available for other reviews of such interesting and important articles.

Happy to help,

Reviewer

Author Response

I only have one last suggestion to do: to further enhance the understanding of these results, it would be beneficial to explore several aspects in more depth. In this regard, I still suggest to deepen information on the relationship between behavior, emotion, and neural activity, in order to enhance the knowledge on neural substrates underlying fear conditioning and active avoidance (https://doi.org/10.1016/j.neubiorev.2023.105163; DOI: 10.17219/acem/165944; https://doi.org/10.3390/ijms24065926).

Response. Thank you for bringing this issue to our attention. We added Section 3.4 to the Discussion, featuring theoretical considerations based on the proposed references.