Emotional Vocalizations Alter Behaviors and Neurochemical Release into the Amygdala

Reviewer #1 (Public Review):

The manuscript addresses a fundamental question about how different types of communication signals differentially affect brain state and neurochemistry. In addition, their manuscript highlights the various processes that modulate brain responses to communication signals, including prior experience, sex, and hormonal status. Overall, the manuscript is well-written and the research is appropriately contextualized.

That being said, it remains important for the authors to think more about their analytical approaches. In particular, the effect of normalization and the explicit outlining and interpretations of statistical models. As mentioned in the original review, the normalization of neurochemical data seems unnecessary given the repeated-measures design of their analysis and by normalizing all data to the baseline data and including this baseline data in the repeated measures analysis, one artificially creates a baseline period with minimal variation that dramatically differs in variance from other periods (akin to heteroscedasticity). If the authors want to analyze how a stimulus changes neurochemical concentrations, they could analyze the raw data but depict normalized data in their figures (similar to other papers). Or they could analyze group differences in the normalized data of the two stimulus periods (i.e., excluding the baseline period used for normalization).

It would also be useful for the authors to provide further discussion of the potential contributions of different types of experiences (mating vs. restraint) to the change in behavior and neurochemical responses to the vocalization playbacks and to try to disentangle sensory and motor contributions to neurochemical changes.

Reviewer #3 (Public Review):

The work by Ghasemahmad et al. has the potential to significantly advance our understanding of how neuromodulators provide internal-state signals to the basolateral amygdala (BLA) while an animal listens to social vocalizations.

Ghasemahmad et al. made changes to the manuscript that have significantly improved the work. In particular, the transparency in showing the underlying levels of Ach, DA, and 5HIAA is excellent. My previous concerns have been adequately addressed.

Author Response

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

eLife assessment

This important study advances our understanding of the ways in which different types of communication signals differentially affect mouse behaviors and amygdala cholinergic/dopaminergic neuromodulation. Researchers interested in the complex interaction between prior experience, sex, behavior, hormonal status, and neuromodulation should benefit from this study. Nevertheless, the data analysis is incomplete at this stage, requiring additional analysis and description, justification, and - potentially - power to support the conclusions fully. With the analytical part strengthened, this paper will be of interest to neuroscientists and ethologists.

GENERAL COMMENTS ON REVIEWS AND REVISIONS

Experimental design

Here we address questions from several reviewers regarding our periods of neuromodulator and behavioral analysis. First, we recognize that the text would benefit from an overview of the experimental structure different from the narrative we provide in the first paragraphs of the Results. We now include this near the beginning for the Materials and Methods (page 17). We further articulate that the 10-minute time periods were dictated by the sampling duration required to perform accurate neurochemical analyses (and to reserve half of the sample in the event of a catastrophic failure of batch-processing samples). Since neurochemical release may display multiple temporal components (e.g., ACh: Aitta-aho et al., 2018) during playback stimulation, and since these could differ across neurochemicals of interest, we decided to collect, analyze, and report in two stimulus periods as well as one Pre-Stim control. We now clarify this in additional text in the Material and Methods (p. 24, lines 20-22; p. 26, lines 17-19). We decided not to include analyses of the post-stimulus period because this is subject to wider individual and neuromodulator-specific effects and because it weakens statistical power in addressing the core question—the change in neuromodulator release DURING vocal playback.

We also sought to clarify the meaning of the periods “Stim 1” and “Stim 2”; they are two data collection periods, using the same examplar sequences in the same order. We have added statements in the Material and Methods (p. 18, lines 4-7; Fig. caption, p. 39, lines 11-13) to clarify these periods.

For behavioral analyses, observation periods were much shorter than 10 mins, but the main purpose of behavioral analyses in this report is to relate to the neurochemical data. As a result, we matched the temporal features of the behavioral and neurochemical analyses (p. 22, lines 17-22). We plan a separate report, focused exclusively on a broader set of behavioral responses to playback, that may examine behaviors at a more granular level.

Data and statistical analyses

Reviewers 1 and 3 expressed concerns about our normalization of neurochemical data, suggesting that it diminishes statistical power or is not transparent. We note that normalization is a very common form of data transformation that does not diminish statistical power. It is particularly useful for data forms in which the absolute value of the measurement across experiments may be uninformative. Normalization is routine in microdialysis studies, because data can be affected by probe placement and factors affecting neurochemical recovery and processing. Recent examples include:

Li, Chaoqun, Tianping Sun, Yimu Zhang, Yan Gao, Zhou Sun, Wei Li, Heping Cheng, Yu Gu, and Nashat Abumaria. "A neural circuit for regulating a behavioral switch in response to prolonged uncontrollability in mice." Neuron (2023).

Gálvez-Márquez, Donovan K., Mildred Salgado-Ménez, Perla Moreno-Castilla, Luis Rodríguez-Durán, Martha L. Escobar, Fatuel Tecuapetla, and Federico Bermudez-Rattoni. "Spatial contextual recognition memory updating is modulated by dopamine release in the dorsal hippocampus from the locus coeruleus." Proceedings of the National Academy of Sciences 119, no. 49 (2022): e2208254119.

Holly, Elizabeth N., Christopher O. Boyson, Sandra Montagud-Romero, Dirson J. Stein, Kyle L. Gobrogge, Joseph F. DeBold, and Klaus A. Miczek. "Episodic social stress-escalated cocaine self-administration: role of phasic and tonic corticotropin releasing factor in the anterior and posterior ventral tegmental area." Journal of Neuroscience 36, no. 14 (2016): 4093-4105.

Bagley, Elena E., Jennifer Hacker, Vladimir I. Chefer, Christophe Mallet, Gavan P. McNally, Billy CH Chieng, Julie Perroud, Toni S. Shippenberg, and MacDonald J. Christie. "Drug-induced GABA transporter currents enhance GABA release to induce opioid withdrawal behaviors." Nature neuroscience 14, no. 12 (2011): 1548-1554.

However, since all reviewers requested raw values of neurochemicals, we provide these in supplementary tables 1-3. The manuscript references these table early in the Results (p. 6, lines 18-19) and in the Material and Methods (p. 27, lines 3-4)

All reviewers commented on correlation analyses that we presented, with different perspectives. Reviewer 2 questioned the validity of such analyses, performed across experimental groups, while Reviewer 1 pointed out that the analyses were redundant with the GLM. We agree with these criticisms, and note the challenges associated with correlations involving behaviors for which there is a “floor” in the number of observations. As a result, we have removed most correlation analyses from the manuscript. The text and figures have been modified accordingly. Due these changes, we have to decline requests of Reviewer 3 to include many more such analyses. While correlation analyses could still be performed between neurochemicals and behaviors for each group, the relatively small size of each experimental group, the large number of groups, and the even larger numbers of pairings between neurochemicals and behavior, the statistical power is very low. The only correlations we utilize in the manuscript concern the interpretation of our increased acetylcholine levels.

As part of this revision, we re-ran our statistical analyses on neuromodulators because of a calculation error in 3 animals (regarding baseline values). In a few instances, a significance level changed, but none of these changed a conclusion regarding neuromodulator changes under our experimental conditions.

Other revisions

INTRODUCTION: We modified the Introduction to provide both a more general framework and specific gaps in our understanding relating neuromodulators with vocal communication.

DISCUSSION: We have added material in the first two pages of the Discussion to provide more framework to our conclusions, to address the issues of the temporal aspects of neurochemical release and behavioral observations, and to identify limitations that should be addressed in future studies.

FIGURES: All figures are now in the main part of the manuscript. We modified most figures in response to reviewer comments. We removed neuromodulator – behavior correlations from several figures. We modified all box plots to ensure that all data points are visible. The visible data points match the numbers reported in figure captions. We brought 5-HIAA data into the main figures reporting on neuromodulator results.

Public Reviews:

Reviewer #1 (Public Review):

The manuscript addresses a fundamental question about how different types of communication signals differentially affect brain states and neurochemistry. In addition, the manuscript highlights the various processes that modulate brain responses to communication signals, including prior experience, sex, and hormonal status. Overall, the manuscript is well-written and the research is appropriately contextualized. The authors are thoughtful about their quantitative approaches and interpretations of the data.

That being said, the authors need to work on justifying some of their analytical approaches (e.g., normalization of neurochemical data, dividing the experimental period into two periods (as opposed to just analyzing the entire experimental period as a whole)) and should provide a greater discussion of how their data also demonstrate dissociations between neurochemical release in the basolateral amygdala and behavior (e.g., neurochemical differences during both of the experimental periods but behavioral differences only during the first half of the experimental period). The normalization of neurochemical data seems unnecessary given the repeated-measures design of their analysis and could be problematic; by normalizing all data to the baseline data (p. 24), one artificially creates a baseline period with minimal variation (all are "0"; Figures 2, 3 & 5) that could inflate statistical power.

Please see our general responses to structure of observation periods and normalization of neuromodulator data. Normalization is a common and appropriate procedure in microdialysis studies that does not alter statistical power.

We have included a section in the Discussion concerning the temporal relationship between behavioral responses and neurochemical changes in response to vocal playback (p. 12, lines 3-17). We note where the linkage is particularly strong (e.g., ACh release and flinching). This points to a need to examine these phenomena with finer temporal resolution, but also with the recognition that the brain circuits driving a behavioral response may extend beyond the BLA.

The Introduction could benefit from a priori predictions about the differential release of specific neuromodulators based on previous literature.

We added some material to the Introduction to provide additional rationale for the study. However, we did not attempt to develop predictions for the range of neuromodulators that we sought to test. The literature can lead to opposite predictions for a given neuromodulator. For example, acetylcholine could be associated with both positive and negative valence. Instead, we note in the Introduction the association of both DA and ACh with vocalizations.

The manuscript would also benefit from a description of space use and locomotion in response to different valence vocalizations.

We have provided additional descriptions of space use and video tracking data in Material and Methods (p. 23, lines 1-6). We now report a few correlations based on these data in the Results to demonstrate that increased ACh in Restraint males and Mating estrus females was not related to the amount of locomotion (p. 9, lines 8-14).

Nevertheless, the current manuscript seems to provide some compelling support for how positive and negative valence vocalizations differentially affect behavior and the release of acetylcholine and dopamine in the basolateral amygdala. The research is relevant to broad fields of neuroscience and has implications for the neural circuits underlying social behavior.

Reviewer #2 (Public Review):

Ghasemahmad et al. report findings on the influence of salient vocalization playback, sex, and previous experience, on mice behaviors, and on cholinergic and dopaminergic neuromodulation within the basolateral amygdala (BLA). Specifically, the authors played back mice vocalizations recorded during two behaviors of opposite valence (mating and restraint) and measured the behaviors and release of acetylcholine (ACh), dopamine (DA), and serotonin in the BLA triggered in response to those sounds.

Strength: The authors identified that mating and restraint sounds have a differential impact on cholinergic and dopaminergic release. In male mice, these two distinct vocalizations exert an opposite effect on the release of ACh and DA. Mating sounds elicited a decrease of Ach release and an increase of DA release. Conversely, restraint sounds induced an increase in ACh release and a trend to decrease in DA. These neurotransmission changes were different in estrus females for whom the mating vocalization resulted in an increase of both DA and ACh release.

Weaknesses: The behavioral analysis and results remain elusive, and although addressing interesting questions, the study contains major flaws, and the interpretations are overstating the findings.

Although Reviewer 2 raises several valid issues that we have addressed in our response and revision, we believe that none represent “major flaws” in the study that challenge the validity of our central conclusions. In brief, we will:

--provide enhanced description of behaviors (pp. 22-23 and Table 1)

--clarify / modify box-plot representations of data (p 28. Lines 3-9)

--point to our methods that describe corrections for multiple comparisons (p. 27; lines 15-16)

--revise figures to clarify sample size (Figs. 3-6)

Reviewer #3 (Public Review):

Ghasemahmad et al. examined behavioral and neurochemical responses of male and female mice to vocalizations associated with mating and restraint. The authors made two significant and exciting discoveries. They revealed that the affective content of vocalizations modulated both behavioral responses and the release of acetylcholine (ACh) and dopamine (DA) but not serotonin (5-HIAA) in the basolateral amygdala (BLA) of male and female mice. Moreover, the results show sex-based differences in behavioral responses to vocalizations associated with mating. The authors conclude that behavior and neurochemical responses in male and female mice are experience-dependent and are altered by vocalizations associated with restraint and mating. The findings suggest that ACh and DA release may shape behavioral responses to context-dependent vocalizations. The study has the potential to significantly advance our understanding of how neuromodulators provide internal-state signals to the BLA while an animal listens to social vocalizations; however, multiple concerns must be addressed to substantiate their conclusions.

Major concerns:

1. The authors normalized all neurochemical data to the background level obtained from a single pre-stimulus sample immediately preceding playback. The percentage change from the background level was calculated based on a formula, and the underlying concentrations were not reported. The authors should report the sample and background concentrations to make the results and analyses more transparent. The authors stated that NE and 5-HT had low recovery from the mouse brain and hence could not be tracked in the experiment. The authors could be more specific here by relating the concentrations to ACh, DA, and 5-HIAA included in the analyses.

Please see our general statement regarding normalization of neurochemical data. We have added supplemental tables that shows concentrations of dopamine, acetylcholine, 5-HIAA. We do not report serotonin or noradrenalin since these were below the detection threshold.

1. For the EXP group, the authors stated that each animal underwent 90-min sessions on two consecutive days that provided mating and restraint experiences. Did the authors record mating or copulation during these experiments? If yes, what was the frequency of copulation? What other behaviors were recorded during these experiences? Did the experiment encompass other courtship behaviors along with mating experiences? Was the female mouse in estrus during the experience sessions?

In the mating experience, mounting or attempted mounting was required for the animal to be included in subsequent testing. Since the session lasted 90 minutes, more general courtship behavior was likely. However, we did not record detailed behaviors or track estrous stage for the mating experience. See p. 21, line 20-22.

1. For the mating playback, the authors stated that the mating stimulus blocks contained five exemplars of vocal sequences emitted during mating interactions. The authors should clarify whether the vocal sequences were emitted while animals were mating/copulating or when the male and female mice were inside the test box. If the latter was the case, it might be better to call the playback "courtship playback" instead of "mating playback".

We have modified the Results (p. 5, lines 18-20) and Materials and Methods (p. 21, lines 8-15) to clarify our meaning. We continue to use the term “mating” because this refers to a specific set of behaviors associated with mounting and copulation, rather than the more general term “courtship”. We also indicate that we based these behaviors on previous work (e.g., Gaub et al., 2016).

1. Since most differences that the authors reported in Figure 3 were observed in Stim 1 and not in Stim 2, it might be better to perform a temporal analysis - looking at behaviors and neurochemicals over time instead of dividing them into two 10-minute bins. The temporal analysis will provide a more accurate representation of changes in behavior and neurochemicals over time.

Please see our general response to the structuring of experimental periods. The 10-min periods are the minimum for the neurochemical analyses, and we adopted the same periods for behavioral analyses to match the two types of observations. Our repeated measures analysis is a form of temporal analysis, since it compares values in three observation periods.

1. In Figures 2 and 3, the authors show the correlation between Flinching behavior and ACh concentration. The authors should report correlations between concentrations of all neurochemicals (not just ACh) and all behaviors recorded (not just Flinching), even if they are insignificant. The analyses performed for the stim 1 data should also be performed on the stim 2 data. Reporting these findings would benefit the field.

Please see general comments regarding correlation analyses. We removed almost all such analyses and references to them from the manuscript based on concerns of the other reviewers.

1. The mice used in the study were between p90 - p180. The mice were old, and the range of ages was considerable. Are the findings correlated with age? The authors should also discuss how age might affect the experiment's results.

Our p90-p180 mice are not “old”. CBA/CaJ mice display normal hearing for at least 1 year (Ohlemiller, Dahl, and Gagnon, JARO 11: 605-623, 2010) and adult sexual and social behavior throughout our observation period. They are sexually mature adults, appropriate for this study. We decline to perform correlation analyses with age, both because this was not a question for this study and because the very large number of correlations, for each experimental group (as requested by reviewer #2), render this approach statistically problematic.

1. The authors reported neurochemical levels estimated as the animals listened to the sounds played back. What about the sustained effects of changes in neurochemicals? Are there any potential long-term effects of social vocalizations on behavior and neurochemical levels? The authors might consider discussing long-term effects.

We have not included discussion of long term effects of neuromodulatory release, both because our data analysis doesn’t address it (see response to Comment #10) and because we desired to keep the Discussion focused on topics more closely related to the results.

1. Histology from a single recording was shown in supplementary figure 1. It would benefit the readers if additional histology was shown for all the animals, not just the colored schematics summarizing the recording probe locations. Further explanation of the track location is also needed to help the readers. Make it clear for the readers which dextran-fluorescein labeling image is associated with which track in the schematic.

Based on the recent publications cited in our overall response to reviewer comments about statistical methods, our reporting of histological location of microdialysis exceeds the standard. We believe that the inclusion of all histology is unnecessary and not particularly helpful. Raw photomicrographs do not always illustrate boundaries, so interpretation is required. However, we added a second photomicrograph example and we identified which tracks correspond to these photomicrographs (see Figure 2; now in main body of manuscript).

1. The authors did not control for the sounds being played back with a speaker. This control may be necessary since the effects are more pronounced in Stim 1 than in Stim 2. Playing white noise rather than restraint or courtship vocalizations would be an excellent control. However, the authors could perform a permutation analysis and computationally break the relationship between what sound is playing and the neurochemical data. This control would allow the authors to show that the actual neurochemical levels are above or below chance.

We considered a potential “control” stimulus in our experimental design. We concluded, based on our previous work (e.g., Grimsley et al., 2013; Gadziola et al., 2016), that white noise is not or not necessarily a neutral stimulus and therefore the results would not clarify the responses to the two vocal stimuli. Instead, we opted to use experience as a type of control. This control shows very clearly that temporal patterns and across-group differences in neurochemical response to playback disappear in the absence of experience with the associated behavior.

1. The authors indicated that each animal's post-vocalization session was also recorded. No data in the manuscript related to the post-vocalization playback period was included. This omission was a missed opportunity to show that the neurochemical levels returned to baseline, and the results were not dependent on the normalization process described in major concern #1. The data should be included in the manuscript and analyzed. It would add further support for the model described in Figure 6.

We decided not to include analyses of the post-stimulus period because this period is subject to wider individual and neuromodulator-specific effects and because it weakens statistical power in addressing the core question—the change in neuromodulator release DURING vocal playback. We agree that the general question is of interest to the field, but we don’t think our study is best designed to answer that question.

1. The authors could use a predictive model, such as a binary classifier trained on the CSF sampling data, to predict the type of vocalizations played back. The predictive model could support the conclusions and provide additional support for the model in Figure 6.

We recognize that a binary classifier could provide an interesting approach to support conclusions. However, we do not believe that the sample size per group is sufficient to both create and test the classifier.

Reviewer #1 (Recommendations For The Authors):

Major comments:

• Introduction: It would be useful to set up an experimental framework before delving into the results. What are the predictions about specific neuromodulators based on previous literature?

Because this narrative is laid out in the first two paragraphs of the Results, which immediately follow the Introduction, we believe that additional text in the Introduction on the experimental framework is redundant. As stated above, detailing predictions for a range of neuromodulators would make for a long and not particularly illuminating Introduction. We instead have related our findings to more general understanding of DA and ACh in the Discussion.

• There really isn't a major difference in stimuli during the "Stim 1" and "Stim 2" phases, and it's not clear why the authors divided the experimental period into two phases. Therefore, the authors need to justify their experimental approach. For example, the authors could first anecdotally mention that behavioral responses to playbacks seem to be larger in the first half of the playbacks than during the second half, therefore they individually analyzed each half of the experimental period. Or adopt a different approach to justify their design. Overall, the analytical approach is reasonable but it is currently not justified.

See general comment for analysis periods. As noted, we clarified these issues in several locations with Materials and Methods (pp. 24, lines 20-22; p. 26, lines 17-19). We also sought to clarify the meaning of the periods “Stim 1” and “Stim 2”; they are two data collection periods, using the same examplar sequences in the same order. We have added statements in the Material and Methods (p. 18, lines 4-7; Fig. caption, p. 39, lines 11-13).

• The normalization of neurochemical data seems problematic and unnecessary. By normalizing all data to the baseline data (p. 24), one artificially creates a baseline period with minimal variation (all are "0"; Figures 2, 3 & 5) and this has implications for statistical power. Because the analysis is a within-subjects analysis, this normalization is not necessary for the analysis itself. It can be useful to normalize data for visualization purposes, but raw data should be analyzed. Indeed, behavioral data are qualitatively similar to the neurochemical data, and those data are not normalized to baseline values.

Please see our general comment on this issue. We believe normalization does not affect statistical power and is both the standard way and an appropriate way to analyze microdialysis results. We include concentrations of ACh, DA, and 5-HIAA in supplementary tables?

• The authors should include a discussion (in the Discussion section) of how behavior and neurochemical release are associated during the first half of the experimental session but not in the second half (e.g., differences in Ach and DA release between mating and restraint groups during stim 1 and 2, but behavioral differences only during stim 1).

We have included a section in the Discussion concerning the temporal relationship between behavioral responses and neurochemical changes in response to vocal playback. We note that the linkage is particularly strong in some cases (e.g., ACh release and flinching). This points to a need to examine these phenomena with finer temporal resolution, but also with the recognition that the brain circuits driving a behavioral response may extend beyond the BLA.

Minor comments:

• Keywords: add "serotonin" (even though there are no significant differences on 5-HIAA, people interested in serotonin would find this interesting).

Added to keywords list.

• Do the authors collect data on the vocalizations of mice in response to these playbacks?

We monitored vocalizations during playback, noting that vocalizations–especially “Noisy” vocalization–were common. However, we did not record vocalizations and are therefore unable quantify our observations.

• First line of page 7: readers do not know about "stim 1" and "stim 2". Therefore, the authors need to describe their approach to analyzing behavior and neurochemical release.

We first introduce these terms earlier, citing Figure 1D,E. We have added some additional wording for further clarification. page 7, lines 4-5.

• Make sure citations are uniformly formatted (e.g., Inconsistencies in: "As male and female mice emit different vocalizations during mating (Finton et al., 2017; J. M. S. Grimsley et al., 2013; Neunuebel et al., 2015; Sales (née Sewell), 1972)").

We have reviewed and corrected citations throughout the manuscript.

• Last paragraph of page 7: "attending behavior" has not been defined yet.

Table 1 contains our description of the behaviors analyzed in this study. We have now inserted a reference to Table 1 earlier in the Results (p. 6, line 12).

• Figure 2E and 3G: I find these correlations to be redundant with the GLMs. This is because the significant relationship is likely to be driven by group differences in behavior and in neurochemical release.

Please see general comments regarding correlation analyses. We removed such analyses and references to them from the manuscript.

• Page 2, 2nd paragraph, 2nd sentence: this paragraph seems to be rooted in comparing and contrasting experienced and inexperienced mice, so there should be explicit comparisons in each sentence. For example, the 2nd sentence should read: "Whereas EXP estrus females demonstrated increased flinching behaviors in response to mating vocalizations, INEXP ....". This paragraph overall could use some refining.

We believe this refers to page 9. We have revised the paragraph to clarify our findings (Beginning p. 9, line 23).

• Page 9: "Further, there were no significant differences across groups during Stim 1 or Stim 2 periods. These results contrast sharply with those from all EXP groups, in which both ACh and DA release changed significantly during playback (Figs. 2C, 2D, 3E, 3F)." While I understand their perspective, this is misleading because changes were only observed during the Stim 1 period.

We have slightly revised the wording in this paragraph, because the restraint males did not show significant ACh decreases. However, we do not believe our statements mislead readers just because some changes are observed in only one of the stimulation periods (p 10, lines 13-16).

• Last paragraph of page 14: it would be useful to mention the increase in flinching in experienced females in response to mating vocalizations.

We have added a sentence in this paragraph relating flinching in estrus females to increased ACh (p. 15, lines 18-20).

• Was there a full analysis of locomotion in response to playbacks? I see that locomotion was correlated with neurochemical release but was it different in response to different stimuli? Were there changes to the part of the arena that mice occupied in response to restraint vs. mating vocalizations? Given their methods section, it would be useful for the authors to mention the results of the analyses of these aspects of movement.

We have provided additional descriptions of space use and video tracking data in Material and Methods (p. 23, lines 1-6). We now report additional results associated with these analyses (p. 8, lines 13-15; p. 9, lines 8-14).

• I believe that each experimental mouse only heard one of the stimuli (given the analytical approach). Because it is plausible to measure neurochemical release in response to both types of stimuli, I encourage the authors to be more explicit about this aspect of the experimental design (e.g., mention in Results section).

Sentence modified to read: “Each mouse received playback of either the mating or restraint stimuli, but not both: same-day presentation of both stimuli would require excessively long playback sessions, the condition of the same probe would likely change on subsequent days, and quality of a second implanted probe on a subsequent day was uncertain.” (p. 7, lines 5-9).

• Figure 1A and 1B: add labels to the panels so readers don't have to read the legend to know what spectrogram is associated with what context.

We added these labels to Figure 1.

• Table 1: in the definition of "still and alert", should this mention "abrupt attending" instead of "abrupt freezing"? The latter isn't described.

Yes, we intended “abrupt attending”, and now indicated that in Table 1

Reviewer #2 (Recommendations For The Authors):

Major comments:

• The authors report they performed manual behavioral analysis, and provide a table defining the different behaviors. However, it remains unclear how some of these behaviors were detected (such as still-and-alert events). A thorough description of the criteria used to define these events needs to be provided.

We have modified some descriptions of manually analyzed behaviors in Table 1, and have added additional description of how we developed this set of behaviors for analysis in the study (pp. 22-23).

• The box plots do not appear to represent the "minimum, first quartile, median, third quartile, and maximum values." as specified on page 24 (Methods). Indeed, the individual data points sometimes do not reach the max or min of the bar plot, and sometimes are way beyond them.

We used the “inclusive median” function in Excel to generate final boxplots. These boxplots will sometimes result in a data point being placed outside of the whiskers. SPSS considers these to be “outliers”, but our GLM analysis includes these values. We describe this in Data Analysis section of Materials and Methods (p. 28, lines 3-9)

• Some of the data are replicated in different Figures: Figure 2A and Figure 3C. While this is acceptable, the authors did not correct for multiple comparisons (dividing the p value by the number of comparisons).

Our analysis included corrections for multiple comparisons, as we have indicated on p. 27, lines 15-16.

• Overall, the sample sizes are too small (for example in Figure 3, non-estrus females are at n=3), and are different in experiments where they should be equal (Figure 2B: mating stim 1 is at n=5 and mating stim 2 is at n=3).

We apologize that sample sizes were not properly displayed in figures. Please note that sample sizes are identified in the figure captions. For neuromodulator data, all sample sizes are at least 7. For behavioral data, the minimum sample size is 5. We have revised Figures 3-6 to ensure that all data points are visible.

• It remains unclear why the impact of mating vocalizations has been tested only in males.

We assume the reviewer meant that only males were tested in restraint. We now indicate that our preliminary evidence indicated no difference in behavioral responses to restraint vocalization between males and females, so we opted to perform the neurochemical analysis for restraint only in males (page 22 lines 4-5). If there were no limitations to time and cost, we would have preferred to test responses to restraint in females as well. We note that such inclusion would have added up to 4 experimental groups (estrus and non-estrus groups in both EXP and INEXP groups).

• The correlation between the number of flinching and ACh release changes (Figure 2E) visually appears to be opposite between mating and restraint playbacks. The authors should perform independent correlations for these 2 playbacks.

Please see general comments regarding correlation analyses. We removed such analyses and references to them from the manuscript.

• The authors state that their findings "indicate that behavioral responses to salient vocalizations result from interactions between sex of the listener or context of vocal stimuli with the previous behavioral experience associated with these vocalizations.". However, in male mice, they do not report any difference in previous experience on flinching for both restraint and mating sounds, as well as no difference in rearing for the restrain sounds (Figure 4A-B). Thus, the discussion of these results should be completely revisited.

We revised the paragraph in question (p. 9, line 22 through p. 10, line 9). For instance, we note that significant differences between EXP male-mating and male-restraint flinching do not exist between the INEXP groups. We believe that the last sentence correctly summarizes findings described in this paragraph.

• For serotonin experiments in Figure S2 there are strong outliers (150% increase in 5HIAA release). Did the authors correlate these levels with the behavior of the animals?

Outliers are identified by the Excel function that generated the boxplots, but we have no reason to consider these as outliers and exclude them. As noted above, we have clarified that these “outliers” are the result of the Excel function in the Materials and Methods (p. 28, lines 3-9) and we have revised the plotting of data points

Minor comments:

• Mating vocalization playback is mainly emitted by males, thus, instead of a positive valence signal, this could also be interpreted as a competitive signal to other males.

There is support in the literature for viewing our mating stimulus as having positive valence. Gaub et al., 2016 describe the emission of stepped calls, lower frequency harmonics, and increased sound level as indicators of “positive emotion”. We have shown (Grimsley et al, 2013) that the female LFH vocalization can be highly attractive to male mice, under the right conditions, indicating something like “sex is happening”. The inclusion of both the male and female vocalizations in our stimuli was a key piece of our experimental design, based on our understanding of the contributions of both vocalizations to the meaning of the overall acoustic experience.

• Figure 1 should include panel titles.

No change. This information is available in the Figure caption.

• n=31 should be indicated in the EXP group.

We’re not sure where the reviewer is referring to this value.

• The color legend of Figure 1E is absent, making the Figure not understandable.

We added text in the Figure 1 caption to indicate that each color represents a different exemplar. We don’t think a legend provides additional useful information.

• The point of making two blocks (stim 1 and stim2) should be stated more clearly.

Please see general statement regarding experimental blocks. We have modified our description of these in an Experimental overview section in the Material and Methods.

• Including raw data of micro-dialysis in the supplementary figures would allow assessment of the variability and quality of the measurements.

We have added concentrations of neurochemicals in supplemental tables 1-3.

• Baseline (prestimulus) number of flinch and rearing should systematically be indicated (missing in Figure 4).

The focus in this figure is on the differences that occur in Stim 1 values. There are no differences between EXP and INEXP animals of any group during the Pre-Stim period. We now state that in the Figure 4 caption.

• Discussion: "increase in AMPA/NMDA currents". We believe the authors are referring to the ratio of AMPA to NMDA currents. This sentence should be reformulated.

These are modified to refer to “… the AMPA/NMDA current ratio…” in two locations in the Discussion (p. 14, lines 8-9; p. 15, line 4)

• Overall the discussion is very speculative and should rely more on the data.

We believe that the Discussion provides appropriate speculation that is based on our experimental data and previous literature. We have added a paragraph to identify limitations of our findings and recommendations of future experiments to resolve some issues (p. 12, lines 3-17)

Reviewer #3 (Recommendations For The Authors):

Minor concerns:

1. The authors stated that USVs are most likely to be emitted by males, and LFH are likely to be emitted by females. However, Oliveira-Stahl et al. 2023, Matsumoto et al. 2022, Warren et al. 2018, Heckman et al. 2017, Neunuebel et al., 2015 showed that females also emit USVs. The authors should mention that USVs are emitted by both males and females and discuss how the sex of the vocalizing animal (both males and females) can influence neuromodulator release.

The reviewer slightly mis-stated the wording of our text, changing the meaning significantly. Our wording is “These sequences included ultrasonic vocalizations (USVs) with harmonics, steps, and complex structure, mostly emitted by males, and low frequency harmonic calls (LFHs) emitted by females (Fig. 1A,C)…” This phrasing is correct and carefully chosen. The Discussion in Oliveira-Stahl et al 2023 (p. 10-11) supports our statement: “The exact fraction of USVs emitted by females as concluded in all previous studies on dyadic courtship has varied, ranging from 18%, 17.5%, and 16% to 10.5% in the present study…”.

1. The authors should explain why ECF from BLA was collected unilaterally from the left hemisphere.

p. 23, lines 9-11: We inserted a sentence to explain why we targeted the BLA unilaterally. “Since both left and right amygdala are responsive to vocal stimuli in human and experimental animal studies (Wenstrup et al., 2020), we implanted microdialysis probes into the left amygdala to maintain consistency with other studies in our laboratory..” Beyond that, the choice was arbitrary.

1. The authors said each animal recovered in its home cage for four days before the playback experiment. A 4-day period may not be sufficient for every animal to recover from surgery, so the authors should describe how a mouse's recovery was assessed.

p. 23, lines 20-23: We provide more description about the recovery and how it was assessed. Except for a few animals that were not included in the experiments, all animals recovered within 4 days.

1. The authors stated that each animal was exposed to 90-min sessions with mating and restraint behaviors in a counterbalanced design. This description for Figure 1D should also include the duration of the mating and restraint experience.

The Results that immediately precede citation to this figure include this information.

1. The authors stated, "Data are reported only from mice with more than 75% of the microdialysis probe implanted within the BLA". What are the implications of having 25% of the probe outside the BLA? The authors should shed more light on this by discussing this issue as it relates to the findings and commenting on where the other 25% of the probe was located.

We inserted a sentence to explain the rationale for this inclusion criterion. “We verified placement of microdialysis probes to minimize variability that could arise because regions surrounding BLA receive neurochemical inputs from different sources (e.g., cholinergic inputs to putamen and central amygdala).” (p. 25, lines 21-23).

All brain regions that surround BLA, dorsal, medial, ventral, or lateral, could have been sampled by the “other” 25%. Some of these, e.g., the central amygdala or caudate-putamen, have different sources of cholinergic input that may not have the same release pattern. We do not think it is worthy of further speculation in the Discussion. Due to the high cost of the neurochemical analysis, we often did not process the neurochemistry data if histology indicated that a probe missed the BLA target.

1. The authors confirmed that the estrus stage did not change during the experiment day by evaluating and comparing estrus prior to and after data collection. This strategy was a fantastic experimental approach, but the authors should have discussed the results. How did the results the authors included change when the females were in estrus before but not after data collection? What percentage of females started in estrus but ended in metestrus? Assuming that some females changed estrus state, were these animals excluded from the analyses?

All animals were in the same estrus state at the beginning and end of the playback session.

7). Authors cite Neunuebel et al., 2015 for the sentence "As male and female mice emit different vocalizations during mating". However, Neunuebel et al., 2015 showed vocalizations emitted during chasing--not mating. If mating is a general term for courtship, then this reference is appropriate, but see major concern #3.

In the Results (p. 8, line 5), we changed the phrasing to “courtship and mating” to include the Neunubel et al study.

As we indicate in our response to Public Comment #3, we have modified the Results (p. 5, lines 18-20) and Materials and Methods (p. 21, lines 8-15) to clarify our meaning. We continue to use the term “mating” because this refers to a specific set of behaviors associated with mounting and copulation, rather than the more general term “courtship”. We also indicate that we based these behaviors on previous work (e.g., Gaub et al., 2016).

1. Authors interpret Figure 3F as DA release showed a "consistent" increase during mating playback across all three experimental groups. However, the increase in the estrus female group is inconsistent, as seen in the graph. This verbiage should be reworded to describe the data more accurately.

p. 8, line 23 “consistent” was deleted.

1. In all the box plots, multiple data points overlay each other. A more transparent way of showing the data would be adding some jitter to the x value to make each data point visible. The mean (X's) in Figure 3D (pre-stim mating and mating estrus) are difficult to see, as are all the data points in mating non-estrus. Adding all the symbols to the figure legend or a key in the figure instead of the method section would aid the reader and make the plots easier to interpret

We have revised the boxplots to ensure that all data points are visible.

1. Some verbiage used in the discussion should be toned down. For example, "intense" experiences and "emotionally charged" vocalizations should be removed.

We have not changed these terms, which we believe are appropriate to describe these experiences and vocalizations.

1. The authors include "Emotional Vocalizations" in the title. It would be beneficial if the authors included more detail and references in the introduction to help set up the emotional content of vocalizations. It may benefit a broader readership as typically targeted by eLife.

We now cite Darwin and some more recent publications that articulate the general understanding that social vocalizations carry emotional content.