A neural hub that coordinates learned and innate courtship behaviors

Article activity feed

1. 

   eLife

   Nov 17, 2021

   Author Response:

   Reviewer #1 (Public Review):

   The study aims to investigate the role of A11 neurons in courtship behavior and vocalizations. In particular, the authors determine the inputs/outpus of A11 neurons and uncover that the outputs are both dopamine and glutamate positive. They then lesion A11 cell bodies and terminals in the songbird song-motor nucleus HVC and find that these lesions affect song production, especially, though not exclusively, of courtship song. They also measure the location and movement of lesioned birds and find that birds with lesions of A11 cell bodies show less engagement with a female. Finally, they use fiber photometry to study the activity of A11 terminals in HVC during singing. While this is an interesting question supported by novel data, and I appreciate the diverse and creative approaches …

   Author Response:

   Reviewer #1 (Public Review):

   The study aims to investigate the role of A11 neurons in courtship behavior and vocalizations. In particular, the authors determine the inputs/outpus of A11 neurons and uncover that the outputs are both dopamine and glutamate positive. They then lesion A11 cell bodies and terminals in the songbird song-motor nucleus HVC and find that these lesions affect song production, especially, though not exclusively, of courtship song. They also measure the location and movement of lesioned birds and find that birds with lesions of A11 cell bodies show less engagement with a female. Finally, they use fiber photometry to study the activity of A11 terminals in HVC during singing. While this is an interesting question supported by novel data, and I appreciate the diverse and creative approaches employed in this study, the role of A11 in courtship behavior appears complicated and does not easily fit into the framework proposed by the authors. In particular, the authors argue that A11 is important for coordinating innate and learned aspects of courtship, however, their data fall short of supporting this idea.

   Strengths This is an impressive data set with considerable attention to detail.

   The tracing and histology data identify some novel connections not previously described in songbirds as well as the potential of A11 neurons to co-release of glutamate and dopamine.

   Photometry provides real-time monitoring of A11 and HVC neuron activity during singing.

   In principle, targeting both HVC terminals and A11 cell bodies has the potential to lend insight into the role of HVC terminals vs. the role of projections to other areas (see below for caveats).

   We appreciate the reviewer’s efforts and attention in evaluating our manuscript. We are grateful that the reviewer recognizes strengths in our study, which we agree provides novel insights into the brain circuits that enable a fully integrated courtship display comprising learned and innate behaviors.

   Weaknesses

   1. While I find the overall question and the data interesting, I am not convinced that they demonstrate that A11 is important for "coordinating innate and learned aspects of courtship". In general, birds with A11 lesions appear less motivated to perform female-directed song, however, it's not clear that this is a consequence of a lack of coordination between innate/learned aspects of behavior. Rather, perhaps A11 neurons are important to instigate or drive courtship behavior, or to relay signals from the POA or other regions important for courtship. Because the lesions abolish behavior, it is difficult to discern the role of these neurons in courtship.

   We agree that discerning the precise role of A11 is tricky. It could be acting to gate a (motivational) drive from another source, providing a primary source of this drive, and/or performing a more intricate role in coordinating the various aspects of the courtship display. The reviewer is correct that the current experiments do not allow us to clearly distinguish between these possibilities, and we have revised the manuscript accordingly, first by replacing “coordinate” with “gate” in the title and introduction and including a more thorough treatment of gating and other possible roles for A11 in lines 258-262 of the discussion. That said, we lean towards the latter possibility - a coordinating role for A11 - because of its location immediately proximal to regions that drive learned (HVC) and innate (ICo, RPgc) aspects of behavior and because A11 neurons can contain synthetic enzymes for a fast acting neurotransmitter (glutamate) in addition to DA. But, ultimately, we acknowledge that future experiments are needed to more completely answer this question.

   In addition, I disagree with the innate vs. learned distinction as recent data indicate that introductory notes, which the authors treat as innate, are actually learned (e.g. Kalra et al., 2021). Further, there is also no quantification of the effects of lesions on female-directed calls and little analysis of the activity during call production. This would seem to further complicate the overall interpretation. Overall, it's difficult to make sense of how A11 activity relates to vocalizations, especially given the innate/learned framework that they focus on.

   We thank the reviewers for drawing our attention to the recent Kalra 2021 paper, which we now cite while also making sure to emphasize that introductory notes may have learned features (lines 194-195 and 278-279). However, even that recent study concluded that males raised without a tutor or tutored on recorded songs that lack introductory notes altogether still developed songs that include introductory notes. Nonetheless, we include citation of this recent study and qualify our characterization of introductory notes as being shaped by innate predispositions and experience. Furthermore, we conducted additional analyses to quantify female-directed calling before and after 6-OHDA lesions in either HVC or A11 (results can be found in lines 164-165 and Figure 4C). In line with the divergent effects of these two types of lesions on the production of introductory notes, lesions in HVC did not affect female-directed calling whereas lesions in A11 largely abolished these vocalizations. While we acknowledge that the fiber photometry data on female-directed calling was limited, it nonetheless reinforces the conclusion that A11 transmits information to HVC about innate vocalizations, and it also transmits information to HVC about introductory notes. Along with the loss of introductory note production following A11 lesions, we do believe that our findings support the idea that A11’s role is essential to female-directed vocalizations generally, regardless of whether they are learned or innate, and of of somehow enabling the transition from production of female-directed calling and introductory notes to motifs. We have done our best to draw out these points in the revised discussion.

   1. The HVC lesions appear to create damage/necrosis (Fig 3-suppl 2) and this raises the question of the degree to which the HVC lesion effects are the result of dopamine/glutamate depletion or local damage. In particular, it is surprising that syllable structure and stereotypy show such a dramatic breakdown with HVC A11 input lesions and effectively no change with lesions of the cell bodies, even though both treatments lead to effectively similar reductions in song production.

   We appreciate that 6-OHDA lesions are not highly specific and can introduce unwanted effects on non-TH+ cells and processes. To further quantify the effects of 6-OHDA lesions on HVC cells, we conducted additional 6-OHDA injections in HVC and TUNEL staining studies in addition to the preliminary efforts we had made in the original manuscript. Quantification of these data confirmed our original impression that 6-OHDA treatment in HVC increased HVC cell death (these data are shown in Figure 3-figure supplement 2J, K). To further address this issue, we also added an analysis of song structure when D1 receptor blockers were dialyzed into HVC. No changes in song morphology were detected, similar to the lack of effects on song morphology following A11 cell body lesions (Figure 3 - figure supplement 3). Taken together, these additional experiments and analyses indicate that the changes in song morphology following 6-OHDA treatment in HVC may arise from local damage to HVC cell bodies. In contrast, the reduction in singing following A11 terminal or cell body lesions is likely to reflect diminished DA signaling from A11. However, as the reviewer notes, our primary finding is the differential effects on female-directed singing, and the distinction between more purely singing-related effects following 6-OHDA treatment in HVC and a broad effect on all courtship behaviors following 6-OHDA treatment in A11.

   1. If the idea is that A11 is important for coordinating innate and learned movements, it seems that a detailed analysis of the movements would be important. As is, the movement data provide further support of a decrease in either the motivation or ability to perform female-directed song, but they do not speak to a more specific role for A11 in coordinating innate and learned movements.

   We maintain that we did provide a detailed analysis of a number of important nonsong behaviors, including changes in head orientation and translational movements that the male makes towards the female, both of which are major appetitive features of courtship in songbirds and other vertebrates. We also appreciate that these analyses do not allow us to say much about precisely how movements are being coordinated during courtship, and we have changed language throughout the manuscript to emphasize a gating rather than coordinating role for A11. Furthermore, in response to the reviewer’s concern, we performed additional analyses of the male’s movements during courtship, including beak wipes, vertical changes in posture (“standing tall”), which are finer components of female-directed displays. Notably, this new analysis reveals that all of these behavioral components are abolished by A11 cell body lesions, but not by A11 terminal lesions in HVC (lines 168-190 and Figure 4I, J). We appreciate the reviewer’s suggestion, as we believe these additional analyses strengthen our core finding, namely that A11 functions as a hub to gate, recruit and possibly coordinate innate and learned movements to generate a complete courtship display. These different roles are more fully considered in the revised discussion (lines 256-262).

   Reviewer #2 (Public Review):

   Ben-Tov et al. investigate function of midbrain region A11 and provide evidence that it plays a role in promoting and coordinating a variety of motor responses to sexually or socially salient stimuli. They show lesions of A11 cell bodies abolish female directed calling, orienting and singing, while lesions of terminals in the song premotor nucleus HVC prevent female directed singing, but leave female directed calling and orienting intact. Together with anatomical data indicating projections from A11 to multiple downstream targets associated with song (HVC), calling (DM/ICO) and locomotion, these data support the authors' idea that A11 forms a 'hub' that drives and 'coordinates' multiple different aspects of behavioral responses to social (here female/sexual) stimuli. The results are intriguing and begin to reveal how a single social context can elicit and coordinate multiple coordinated responses. However, as outlined below, I think that some of the specific stronger claims would benefit from additional data, discussion or moderation.

   The authors also provide compelling support for the idea that A11 plays a differential role in female-directed versus undirected song. This is especially underpinned by the observations that 1) A11 afferent activity in HVC appears to differ between directed and undirected signing, with increases in activity preceding song motifs only during directed song, and 2) lesions of A11 cell bodies or inputs to HVC have a dramatic suppressive effect on directed singing, but can leave undirected song largely unchanged. These observations that A11 differentially contributes to socially elicited versus spontaneous singing seem especially interesting and merit further highlighting and discussion as one of the especially striking aspects of the study that seems distinct from the thesis of a role in coordinating learned and unlearned behaviors.

   We appreciate the reviewer’s efforts and attention in evaluating our manuscript. We are grateful that the reviewer recognizes strengths in our study, which we agree provides novel insights into the differential contribution of A11 to socially elicited versus spontaneous singing. We also agree that this point should be highlighted and we expanded our treatment of this point in the discussion section of the revised manuscript (lines 296-309).

   Specific comments

   A central idea around which the results are discussed is that A11 plays a particular role in coordinating learned versus innate behaviors. I have several questions around this thesis where further guidance from the authors about both technical points and interpretation would be helpful.

   First is the question of how specific are the manipulations and conclusions to A11 itself versus other neighboring midbrain dopaminergic regions within which it is embedded. The authors show histology of lesions, injection sites and retrograde labelling in supplementary figures, but do not provide enough guidance for me to understand the strength of the argument that manipulations are restricted to A11 and/or its afferents. Can the boundaries between A11 and neighboring regions be better demarcated? What are the neighboring regions to which there might have been spillover? For lesions of A11 axons within HVC, wouldn't 6-OHDA also damage any other dopaminergic afferent to HVC, including those coming from regions such as VTA? Some discussion of these and related points regarding the specificity of manipulations to A11 would be helpful, especially in light of the literature that points to potential roles of neighboring dopaminergic regions in contributing to motivated behaviors and song more specifically.

   We appreciate that the definition and boundaries of A11 might be confusing. We demarcated A11 and neighboring regions in the relevant figures to better define A11’s boundaries. The reviewer is correct in surmising that the VTA is fairly close to A11 and hence a reasonable concern is that 6-OHDA treatment in A11 could spill over to the VTA and possibly the SNc. To address the concern that 6-OHDA lesions in HVC might cause cell damage to other DA sources to HVC, we quantified the number of VTA/SNc cells following HVC DA lesions. This additional analysis, provided in Figure 3-figure supplement 1D-F, shows that the number of VTA/SNc cells following 6-OHDA injections into either A11 or HVC is comparable to that of intact birds. These additional analyses support the conclusion that the behavioral deficits that emerge following 6-OHDA treatments reflect damage to A11 or A11 terminals in HVC.

   These points also relate to the general question of what is meant by A11 being a 'hub for coordination of learned and innate courtship behaviors'. Ultimately, it seems likely that many regions must work together to orchestrate these behaviors, and it is not clear from the present results how much I should view A11 as having a more specific role than other neighboring dopaminergic regions (or hypothalamic regions such as POA) that are interconnected and seem likely to also play critical roles. As the authors note, many of the relevant structures, including A11 and song system structures, are recurrently connected, further complicating interpretation of any one area as a hub. In this respect, I am not sure how much the authors are intending to argue that A11 is both necessary and sufficient for driving each of the studied behaviors in a courtship context, and it would be helpful to discuss this more specifically - does 'coordination' as used here imply that A11 is capable of triggering these behaviors - an interesting possibility raised by the current results but that does not yet seem to be demonstrated - or something else?

   As we noted in our response to a similar point made by the first reviewer, we agree that discerning the precise role of A11 is tricky. As we commented in that earlier response, A11 could gating a (motivational) drive from another source, providing a primary source of this drive, and/or performing a more intricate role in coordinating the various aspects of the courtship display. We agree that the current experiments do not allow us to make a clear distinction between these possibilities, and we have revised the manuscript accordingly, including a more thorough treatment of these various roles for A11 in the discussion (lines 256-262). That said, we lean towards the latter possibility - a coordinating role for A11 - because of its location immediately proximal to regions that drive learned (HVC) and innate (ICo, RPgc) aspects of behavior and because A11 neurons can release a fast acting neurotransmitter (glutamate) in addition to DA. But, ultimately, we acknowledge that future experiments are needed to more completely answer this question. In the revised manuscript, we emphasize a gating role for A11 in the title and introduction, and then in the discussion expand to encompass the possibility of a coordinating or timing role for A11.

   One additional question regarding the framework for interpreting the function of A11 as coordinating 'learned and innate' courtship behaviors, is for some further clarification and citations regarding what is learned versus innate, especially as it relates to song. The authors characterize introductory notes as 'innate', but previous work from Rajan and colleagues has demonstrated that aspects of introductory notes including acoustic structure and patterning are influenced by learning, and I am not sure what the literature says about orienting and calling to females.

   We thank the reviewer for drawing our attention to this recent study from the Rajan group which indeed concluded that some aspects of the introductory notes are learned. We also note that this study showed that juvenile males tutored on song playbacks that lacked introductory notes or that were raised without a tutor still produced introductory notes. Nonetheless, we include a citation of this recent study and qualify our characterization of introductory notes as being shaped by innate predispositions as well as through experience and learning (lines 194-195 and 278-279). Furthermore, our original analyses of birds with 6-OHDA treatment in HVC revealed that introductory note morphology was unchanged, whereas syllable morphology was degraded. Therefore, even if certain features of introductory notes are influenced by tutor experience, they apparently do not depend on HVC in the same manner as do the learned syllables in the motif. Lastly, we conducted additional analyses to quantify female-directed calling and other movements, before and after 6-OHDA lesions in either HVC or A11. In line with the divergent effects of 6-OHDA treatment in these two regions on the production of introductory notes, lesions in HVC did not affect female-directed calling, beak wipes or changes in male’s posture, whereas lesions in A11 largely abolished all of these behaviors (Figure 4C, I, J). While we agree with the reviewer that a distinction between innate and learned behaviors may not be straightforward, the more fundamental observation is that we can dissociate different aspects of the courtship display and that A11 is situated in a position to drive, gate or coordinate a unified display that involves a variety of learned and innate vocal and non-vocal movements.

   I also would find it helpful to have some further clarification in this context about what it means to coordinate learned and innate aspects of song. The authors indicate that undirected song is largely unaffected by A11 lesions while directed song is largely eliminated, leaving only innate calls or introductory notes. I think it would be helpful to see here a more complete characterization of the nature of vocalizations that remain following A11 lesions in the female directed context. While I understand that no recognizable 'learned motifs' are produced, it is unclear from the example that is shown how much the residual vocalizations should be construed as 'severely disrupted songs' versus strings of calls that resemble innate calls that were present prior to lesions, versus 'normal' patterns of introductory notes that resemble in acoustic structure what the birds produced prior to lesions, but that never proceed to song motifs, etc. A better understanding of the nature of these residual vocalizations might also help to interpret what A11 is doing. Do these birds seem motivated to 'sing' in terms of their posture? Do the authors think that HVC is engaged or that the same residual vocalizations would be produced in a bird that had HVC lesions? How do the authors interpret these data in terms of how learned and unlearned vocalizations are normally coordinated in the context of directed singing?

   We performed additional analyses of the male’s vocalizations and movements during courtship, including female-directed calls, beak wipes, vertical changes in posture (“standing tall”), all of which are components of female-directed courtship displays. Notably, this new analysis reveals that all of these behavioral components are abolished by A11 cell body lesions, but not by A11 terminal lesions in HVC (lines 168-190 and Figure 4C, I, J). Along with our prior report that males with A11 cell body lesions do not sing female-directed motifs, the additional analysis indicates that these males produce little or no female-directed vocalizations or non-vocal behaviors of any kind.

   We previously reported that males with A11 terminal lesions produced only introductory notes but not motifs but realize that this observation would benefit from more quantification. As noted in the previous response, we previously established that introductory note morphology was unchanged by 6-OHDA treatment in HVC (Figure 4 - figure supplement 1A-D). To extend this analysis further in this revised manuscript, we built on the observation that males with 6-OHDA treatment in HVC produce only introductory notes to females, with no song motif, whereas they produce a series of introductory notes followed by motifs comprising distorted syllables when alone (Figure 3K, Figure 3 - figure supplement 2, Figure 4B). To confirm that the directed introductory notes and undirected syllables were indeed distinct vocalizations, we computed their durations and spectral similarity scores (using Sound Analysis Pro). The introductory notes produced during directed conditions differed markedly in their durations from distorted syllables produced during undirected conditions, and these two types of vocalizations had very low similarity scores, indicating that they were cleanly separable vocal behaviors (Figure 4 - figure supplement E, F). Given that introductory notes are unchanged by 6-OHDA treatment in HVC, these analyses support the idea that males treated in this manner can still produce motifs, albeit distorted ones, when alone but not when in the company of a female.

   These questions relate in part to that of how much is the trigger to sing eliminated by A11 afferent lesions versus the ability to produce the relevant song output? It seems like there may still be a trigger to sing - short latency vocal response to female - but inability to produce motif. One point that may be interesting to note in this regard is that this seems somewhat opposite of observations made in other contexts about the effects of directed versus undirected context on song - for example, juveniles can produce better song when it is directed (Kojima), and deafened birds that are beginning to exhibit song deterioration can exhibit normalization of song structure during directed conditions (Nordeen).

   We agree with the reviewer’s point that birds with 6-OHDA lesions in HVC may still be triggered to sing, but are unable to produce a motif, given that they still produce introductory notes and seem to have the right posture, orientation and proximity to the female. We appreciate the reviewer’s comment regarding changes in song that can be elicited by females in either juvenile males or adult males that are deaf, although these additional contexts fall outside of the current study, which focused on adult male finches with normal hearing.

   Reviewer #3 (Public Review):

   The authors use a combination of quantitative acoustic and other behavioral analyses to evaluate the role of the midbrain dopaminergic area A11 in the production of female-directed song in adult male zebra finches. They show that female-directed courtship displays, which consist of song and the production of female-directed displacement behaviors, are dependent on A11 because targeted chemical lesions of this structure, using 6-hydroxydopamine (6-OHDA), permanently (i.e. for at least several months) eliminate both the vocal and non-vocal elements of this behavior. Destruction of A11 axons that directly target HVC, by administering 6-OHDA into HVC, only eliminates female-directed singing without causing any change in the other observed female-directed behaviors. Because these same lesions only temporarily (5-10 days) abolish undirected song, these findings suggest that A11 is not directly involved in song production but acts instead as a gate for the production of female directed courtship behaviors. The authors follow these lesion studies with fiber photometry-based calcium imaging of A11 axons that target HVC to show that A11 activation patterns precede activity in HVC during female-directed singing and that calcium elevation is primarily elevated during the production of the many introductory notes (a component of song that is primarily observed during female-directed singing) that precede the production of the learned song motif. These findings suggest that A11 inputs to HVC likely play a role in triggering and/or activating HVC to synchronize the production of introductory notes (which are likely produced by midbrain circuits) with the learned song component that immediately follows them. In contrast, activation of A11 axons during undirected song (which contain few to no introductory notes) do not precede HVC activation patterns. Consistent with the rapid transmission of A11 neurons, the authors also confirm, as has been suggested for A11 in mammals, that A11 dopaminergic neurons co-release glutamate.

   The findings of this study are of significant interest to our understanding of the neural mechanisms by which these complex behaviors are synchronized and open up a new way of thinking about how learned behavioral motifs can be synchronized with non-learned (e.g. female displacement behavior) behaviors. The study is rigorous, with many different experimental approaches being used to examine the proposed hypotheses, and the findings are convincing. Particularly impressive is the complete elimination of female-directed courtship behaviors following targeted elimination of A11. The primary weaknesses of the manuscript lie (1) in the way they present their anatomical findings and (2) how the authors discuss their findings in the discussion. In the discussion, which is very short (~750 words), the authors miss the opportunity to draw parallels with similar studies in drosophila (they only provide a cursory statement with a few references). In the discussion, the authors propose a model that seems quite oversimplified and lacks, in fact, many of the anatomical connectivity that they show in the first part of their study (for example A11 is only shown having a unidirectional connection to ICo/DM when in fact the connections are bidirectional). The model is also presented in simple hierarchical fashion with many connections omitted. Perhaps these omissions were made to simplify the model but in my opinion such simplification possibly misrepresents the actual mechanisms involved in the coordinated control of courtship song.

   We thank the reviewer for their careful reading of the manuscript and his supportive and constructive comments. We agree that the loss of all female-directed behaviors (which we now extend to female-directed calling and other non-vocal behaviors, such as beakwipes and postural changes) following A11 cell body lesions is especially intriguing. Further, the different effects of A11 cell body lesions and A11 terminal lesions in HVC, along with the connectivity of A11, indicate that A11 acts via a range of downstream sites to gate these various female-directed behaviors. We have done our best to address the two primary weaknesses identified by the reviewer. First, we have done our best to provide a more detailed accounting of the anatomical findings. Second, we have expanded the discussion to address parallels with other studies, as in the fly, and to provide a more nuanced and complete consideration of how A11 may function to facilitate male courtship behaviors.

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2. 

   eLife

   Nov 17, 2021

   Evaluation Summary:

   This paper will be interesting for understanding how the innate and learned components are synchronized and temporally coordinated in courtship behavior. This study gives the first insight into the midbrain dopaminergic region and its' role in courtship behavior.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

   Read the original source
3. 

   eLife

   Nov 17, 2021

   Reviewer #1 (Public Review):

   The study aims to investigate the role of A11 neurons in courtship behavior and vocalizations. In particular, the authors determine the inputs/outpus of A11 neurons and uncover that the outputs are both dopamine and glutamate positive. They then lesion A11 cell bodies and terminals in the songbird song-motor nucleus HVC and find that these lesions affect song production, especially, though not exclusively, of courtship song. They also measure the location and movement of lesioned birds and find that birds with lesions of A11 cell bodies show less engagement with a female. Finally, they use fiber photometry to study the activity of A11 terminals in HVC during singing. While this is an interesting question supported by novel data, and I appreciate the diverse and creative approaches employed in this study, the …

   Reviewer #1 (Public Review):

   The study aims to investigate the role of A11 neurons in courtship behavior and vocalizations. In particular, the authors determine the inputs/outpus of A11 neurons and uncover that the outputs are both dopamine and glutamate positive. They then lesion A11 cell bodies and terminals in the songbird song-motor nucleus HVC and find that these lesions affect song production, especially, though not exclusively, of courtship song. They also measure the location and movement of lesioned birds and find that birds with lesions of A11 cell bodies show less engagement with a female. Finally, they use fiber photometry to study the activity of A11 terminals in HVC during singing. While this is an interesting question supported by novel data, and I appreciate the diverse and creative approaches employed in this study, the role of A11 in courtship behavior appears complicated and does not easily fit into the framework proposed by the authors. In particular, the authors argue that A11 is important for coordinating innate and learned aspects of courtship, however, their data fall short of supporting this idea.

   Strengths
   This is an impressive data set with considerable attention to detail.

   The tracing and histology data identify some novel connections not previously described in songbirds as well as the potential of A11 neurons to co-release of glutamate and dopamine.

   Photometry provides real-time monitoring of A11 and HVC neuron activity during singing.

   In principle, targeting both HVC terminals and A11 cell bodies has the potential to lend insight into the role of HVC terminals vs. the role of projections to other areas (see below for caveats).

   Weaknesses

   1. While I find the overall question and the data interesting, I am not convinced that they demonstrate that A11 is important for "coordinating innate and learned aspects of courtship". In general, birds with A11 lesions appear less motivated to perform female-directed song, however, it's not clear that this is a consequence of a lack of coordination between innate/learned aspects of behavior. Rather, perhaps A11 neurons are important to instigate or drive courtship behavior, or to relay signals from the POA or other regions important for courtship. Because the lesions abolish behavior, it is difficult to discern the role of these neurons in courtship.

   In addition, I disagree with the innate vs. learned distinction as recent data indicate that introductory notes, which the authors treat as innate, are actually learned (e.g. Kalra et al., 2021). Further, there is also no quantification of the effects of lesions on female-directed calls and little analysis of the activity during call production. This would seem to further complicate the overall interpretation. Overall, it's difficult to make sense of how A11 activity relates to vocalizations, especially given the innate/learned framework that they focus on.

   1. The HVC lesions appear to create damage/necrosis (Fig 3-suppl 2) and this raises the question of the degree to which the HVC lesion effects are the result of dopamine/glutamate depletion or local damage. In particular, it is surprising that syllable structure and stereotypy show such a dramatic breakdown with HVC A11 input lesions and effectively no change with lesions of the cell bodies, even though both treatments lead to effectively similar reductions in song production.

   2. If the idea is that A11 is important for coordinating innate and learned movements, it seems that a detailed analysis of the movements would be important. As is, the movement data provide further support of a decrease in either the motivation or ability to perform female-directed song, but they do not speak to a more specific role for A11 in coordinating innate and learned movements.

   Read the original source
4. 

   eLife

   Nov 17, 2021

   Reviewer #2 (Public Review):

   Ben-Tov et al. investigate function of midbrain region A11 and provide evidence that it plays a role in promoting and coordinating a variety of motor responses to sexually or socially salient stimuli. They show lesions of A11 cell bodies abolish female directed calling, orienting and singing, while lesions of terminals in the song premotor nucleus HVC prevent female directed singing, but leave female directed calling and orienting intact. Together with anatomical data indicating projections from A11 to multiple downstream targets associated with song (HVC), calling (DM/ICO) and locomotion, these data support the authors' idea that A11 forms a 'hub' that drives and 'coordinates' multiple different aspects of behavioral responses to social (here female/sexual) stimuli. The results are intriguing and begin to …

   Reviewer #2 (Public Review):

   Ben-Tov et al. investigate function of midbrain region A11 and provide evidence that it plays a role in promoting and coordinating a variety of motor responses to sexually or socially salient stimuli. They show lesions of A11 cell bodies abolish female directed calling, orienting and singing, while lesions of terminals in the song premotor nucleus HVC prevent female directed singing, but leave female directed calling and orienting intact. Together with anatomical data indicating projections from A11 to multiple downstream targets associated with song (HVC), calling (DM/ICO) and locomotion, these data support the authors' idea that A11 forms a 'hub' that drives and 'coordinates' multiple different aspects of behavioral responses to social (here female/sexual) stimuli. The results are intriguing and begin to reveal how a single social context can elicit and coordinate multiple coordinated responses. However, as outlined below, I think that some of the specific stronger claims would benefit from additional data, discussion or moderation.

   The authors also provide compelling support for the idea that A11 plays a differential role in female-directed versus undirected song. This is especially underpinned by the observations that 1) A11 afferent activity in HVC appears to differ between directed and undirected signing, with increases in activity preceding song motifs only during directed song, and 2) lesions of A11 cell bodies or inputs to HVC have a dramatic suppressive effect on directed singing, but can leave undirected song largely unchanged. These observations that A11 differentially contributes to socially elicited versus spontaneous singing seem especially interesting and merit further highlighting and discussion as one of the especially striking aspects of the study that seems distinct from the thesis of a role in coordinating learned and unlearned behaviors.

   ***
   Specific comments

   A central idea around which the results are discussed is that A11 plays a particular role in coordinating learned versus innate behaviors. I have several questions around this thesis where further guidance from the authors about both technical points and interpretation would be helpful.

   First is the question of how specific are the manipulations and conclusions to A11 itself versus other neighboring midbrain dopaminergic regions within which it is embedded. The authors show histology of lesions, injection sites and retrograde labelling in supplementary figures, but do not provide enough guidance for me to understand the strength of the argument that manipulations are restricted to A11 and/or its afferents. Can the boundaries between A11 and neighboring regions be better demarcated? What are the neighboring regions to which there might have been spillover? For lesions of A11 axons within HVC, wouldn't 6-OHDA also damage any other dopaminergic afferent to HVC, including those coming from regions such as VTA? Some discussion of these and related points regarding the specificity of manipulations to A11 would be helpful, especially in light of the literature that points to potential roles of neighboring dopaminergic regions in contributing to motivated behaviors and song more specifically

   These points also relate to the general question of what is meant by A11 being a 'hub for coordination of learned and innate courtship behaviors'. Ultimately, it seems likely that many regions must work together to orchestrate these behaviors, and it is not clear from the present results how much I should view A11 as having a more specific role than other neighboring dopaminergic regions (or hypothalamic regions such as POA) that are interconnected and seem likely to also play critical roles. As the authors note, many of the relevant structures, including A11 and song system structures, are recurrently connected, further complicating interpretation of any one area as a hub. In this respect, I am not sure how much the authors are intending to argue that A11 is both necessary and sufficient for driving each of the studied behaviors in a courtship context, and it would be helpful to discuss this more specifically - does 'coordination' as used here imply that A11 is capable of triggering these behaviors - an interesting possibility raised by the current results but that does not yet seem to be demonstrated - or something else?

   One additional question regarding the framework for interpreting the function of A11 as coordinating 'learned and innate' courtship behaviors, is for some further clarification and citations regarding what is learned versus innate, especially as it relates to song. The authors characterize introductory notes as 'innate', but previous work from Rajan and colleagues has demonstrated that aspects of introductory notes including acoustic structure and patterning are influenced by learning, and I am not sure what the literature says about orienting and calling to females.

   I also would find it helpful to have some further clarification in this context about what it means to coordinate learned and innate aspects of song. The authors indicate that undirected song is largely unaffected by A11 lesions while directed song is largely eliminated, leaving only innate calls or introductory notes. I think it would be helpful to see here a more complete characterization of the nature of vocalizations that remain following A11 lesions in the female directed context. While I understand that no recognizable 'learned motifs' are produced, it is unclear from the example that is shown how much the residual vocalizations should be construed as 'severely disrupted songs' versus strings of calls that resemble innate calls that were present prior to lesions, versus 'normal' patterns of introductory notes that resemble in acoustic structure what the birds produced prior to lesions, but that never proceed to song motifs, etc. A better understanding of the nature of these residual vocalizations might also help to interpret what A11 is doing. Do these birds seem motivated to 'sing' in terms of their posture? Do the authors think that HVC is engaged or that the same residual vocalizations would be produced in a bird that had HVC lesions? How do the authors interpret these data in terms of how learned and unlearned vocalizations are normally coordinated in the context of directed singing?

   These questions relate in part to that of how much is the trigger to sing eliminated by A11 afferent lesions versus the ability to produce the relevant song output? It seems like there may still be a trigger to sing - short latency vocal response to female - but inability to produce motif. One point that may be interesting to note in this regard is that this seems somewhat opposite of observations made in other contexts about the effects of directed versus undirected context on song - for example, juveniles can produce better song when it is directed (Kojima), and deafened birds that are beginning to exhibit song deterioration can exhibit normalization of song structure during directed conditions (Nordeen).

   Read the original source
5. 

   eLife

   Nov 17, 2021

   Reviewer #3 (Public Review):

   The authors use a combination of quantitative acoustic and other behavioral analyses to evaluate the role of the midbrain dopaminergic area A11 in the production of female-directed song in adult male zebra finches. They show that female-directed courtship displays, which consist of song and the production of female-directed displacement behaviors, are dependent on A11 because targeted chemical lesions of this structure, using 6-hydroxydopamine (6-OHDA), permanently (i.e. for at least several months) eliminate both the vocal and non-vocal elements of this behavior. Destruction of A11 axons that directly target HVC, by administering 6-OHDA into HVC, only eliminates female-directed singing without causing any change in the other observed female-directed behaviors. Because these same lesions only temporarily …

   Reviewer #3 (Public Review):

   The authors use a combination of quantitative acoustic and other behavioral analyses to evaluate the role of the midbrain dopaminergic area A11 in the production of female-directed song in adult male zebra finches. They show that female-directed courtship displays, which consist of song and the production of female-directed displacement behaviors, are dependent on A11 because targeted chemical lesions of this structure, using 6-hydroxydopamine (6-OHDA), permanently (i.e. for at least several months) eliminate both the vocal and non-vocal elements of this behavior. Destruction of A11 axons that directly target HVC, by administering 6-OHDA into HVC, only eliminates female-directed singing without causing any change in the other observed female-directed behaviors. Because these same lesions only temporarily (5-10 days) abolish undirected song, these findings suggest that A11 is not directly involved in song production but acts instead as a gate for the production of female directed courtship behaviors. The authors follow these lesion studies with fiber photometry-based calcium imaging of A11 axons that target HVC to show that A11 activation patterns precede activity in HVC during female-directed singing and that calcium elevation is primarily elevated during the production of the many introductory notes (a component of song that is primarily observed during female-directed singing) that precede the production of the learned song motif. These findings suggest that A11 inputs to HVC likely play a role in triggering and/or activating HVC to synchronize the production of introductory notes (which are likely produced by midbrain circuits) with the learned song component that immediately follows them. In contrast, activation of A11 axons during undirected song (which contain few to no introductory notes) do not precede HVC activation patterns. Consistent with the rapid transmission of A11 neurons, the authors also confirm, as has been suggested for A11 in mammals, that A11 dopaminergic neurons co-release glutamate.

   The findings of this study are of significant interest to our understanding of the neural mechanisms by which these complex behaviors are synchronized and open up a new way of thinking about how learned behavioral motifs can be synchronized with non-learned (e.g. female displacement behavior) behaviors. The study is rigorous, with many different experimental approaches being used to examine the proposed hypotheses, and the findings are convincing. Particularly impressive is the complete elimination of female-directed courtship behaviors following targeted elimination of A11. The primary weaknesses of the manuscript lie (1) in the way they present their anatomical findings and (2) how the authors discuss their findings in the discussion. In the discussion, which is very short (~750 words), the authors miss the opportunity to draw parallels with similar studies in drosophila (they only provide a cursory statement with a few references). In the discussion, the authors propose a model that seems quite oversimplified and lacks, in fact, many of the anatomical connectivity that they show in the first part of their study (for example A11 is only shown having a unidirectional connection to ICo/DM when in fact the connections are bidirectional). The model is also presented in simple hierarchical fashion with many connections omitted. Perhaps these omissions were made to simplify the model but in my opinion such simplification possibly misrepresents the actual mechanisms involved in the coordinated control of courtship song.

   Read the original source
6. 

   Version published to 10.1101/2021.09.09.459618 on bioRxiv

   Sep 10, 2021