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 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.

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

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

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.

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.

2. 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?

3. 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".

4. 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.

5. 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.

6. The mice used in the study were between p90 - p180. Although CBA/CaJ mice display normal hearing, sexual behaviors, and social behaviors for at least 1 year (Ohlemiller, Dahl, and Gagnon, JARO 11: 605-623, 2010), the age of the mice covers a range of 90 days. It would strengthen the authors' argument 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 if there were no correlations between the magnitude of the neural responses and age.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

Author Response:

We thank the reviewers for their thoughtful reviews. We believe that we can address these comments through revisions within the manuscript (writing/analysis) or as matters of clarification. In this preliminary response, we focus on a few aspects of the reviewer comments.

Experimental design

We will ensure that the rationale for our use of 10-minute analytic periods is clear. These 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) during playback stimulation, and these could differ across neurochemicals of interest, we decided to collect, analyze, and report in two periods. Our results suggest that this was appropriate, comparing values across the two stimulus periods and the pre-stimulus control. 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 called these periods “Stim 1” and “Stim 2”, but each used the same examplar sequences in the same order.

For behavioral analyses, observation periods were much shorter than 10 mins, but the main purpose of behavioral analyses was to relate to the neurochemical data. As a result, we matched the temporal features of the behavioral and neurochemical analyses. We will ensure that this is clearly described in the revision. 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.

One reviewer expressed concern that we did not utilize a “control” playback stimulus, suggesting white noise as the control. We gave extensive consideration to this in our design. We concluded, based on our previous work, that white noise is not 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 disappear in the absence of experience.

One reviewer comments that our p90-p180 mice are “old”. This is not the case. 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.

Data and statistical analyses

Two reviewers express 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 processing. Similar to calcium imaging or many electrophysiological recordings, the information is based on a comparison to baseline values. We will consider supplying concentration values in supplemental material.

Two reviewers comment on correlations we presented, with different perspectives. We will review our correlation analyses to determine if these are appropriate and what should be reported.

Although Reviewer 2 raises several valid issues that we will address 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
• clarify or modify box-plot representations of data
• point to our methods that describe corrections for multiple comparisons
• clarify sample size concerns
• address questions of correlation between neurochemicals and behavior

Factual Corrections

Two reviewer comments and an associated editorial comment suggest that statistical power is lacking. The reviewer comments are incorrect. If the editorial suggestion is based on those comments, we challenge that as well.

Reviewer 1 states that normalization “creates a baseline period with minimal variation…that could inflate statistical power.” We believe that this statement is incorrect. We will justify elsewhere the rationale for using normalized neurochemical data, but the suggestion that this very common transformation alters statistical power is unwarranted.

Reviewer 2 states, in the 4th Recommendation for the Authors, that sample sizes are too small. The reviewer gives examples of sample sizes of 3, but that is incorrect. In revising figures, we will ensure that sample numbers appear clearly, but the reviewer’s claim that we used sample size of 3 is not correct. The minimum sample size is 5.

If these reviewer comments are the bases for the editorial recommendation that the manuscript may require additional power, we believe the recommendation is based on incorrect comments.