1. Author Response:

    Reviewer #2 (Public Review):

    1. Presentation, analysis, and discussion of calcium imaging results

    a) As the authors correctly pointed out, having a water control is indeed essential for interpreting calcium imaging results. As such, I would recommend having a water control panel (currently in Figure S3) in the main figure.

    Thank you for this suggestion. We now provide a new figure, which shows the change in fluorescence of lratd2a right dHb neurons first exposed to water (control) and then to cadaverine or alarm substance. (refer to Figure 2A-D).

    b) The current presentation and analysis of calcium imaging data in Figure 2B does not seem appropriate and can be improved - since the dynamics of olfactory responses are likely highly variable across neurons and fish, rather than comparing responses across time, it would be better to compare the summed response over a longer time window (as already done in Figure S2, but also including water flow control data). Do also mention the time window over which the calcium responses were integrated.

    As noted above, we have added a new figure to represent the change in fluorescence of lratd2a right dHb neurons averaged over a 5 min time window. This trace also includes the standard error of the mean (shaded) to represent the variability in responses among all neurons that were imaged (refer to Figure 2A- D).

    c) Discussion Line 393: "From calcium imaging, we validated that the right dHb appears more responsive than the left when larval zebrafish are exposed to aversive odors such as cadaverine or chondroitin sulfate" - this conclusion cannot be drawn from the existing presented data, unless calcium imaging was also performed in the left habenula.

    Thank you for pointing out this error. Indeed, we were unable to monitor calcium signaling in the left dHb owing to barely detectable levels of GCaMP labeling in the lratd2a cells on the left. We have now corrected this and added the following sentence to the Results:

    “We monitored the responses of individual cells in the right dHb, as GCaMP6f labeling was weakly or not detected in neurons on the left (data not shown).”

    d) It would be good to include in the methods section more detail on how the odor was delivered, volume delivered etc, and whether control experiments were done on the same day / clutch of fish etc.

    We added the requested information in the “Calcium imaging in larval zebrafish” section of the Materials and Methods.

    1. Presentation, analysis, and discussion of c-Fos results and comparison with calcium imaging

    a) Figure 2C-D: The difference / overlap between blue and brown are difficult to make out in the images, especially at this resolution and magnification. Is there a way to specifically quantify the % of lratd2a neurons that are activated by c-fos, rather than just neurons in the dorsal habenula as a whole? This would be necessary to support the claim in line 278: "Thus, the lratd2a subpopulation in the right dHb responds to cadaverine in both larvae and adults".

    We have shown magnified images of double-labeling with fos and lratd2a probes in Figure 2E to G’ to help with visualization of the overlap in colorimetric double in situ hybridization. We quantified the % of the lratd2a expression domain where fos is expressed and provided this information in the results (page 7, lines 128-129).

    b) In larvae (using calcium imaging), the effects of cadaverine to chondroitin sulfate were compared, whereas in adults (using c-Fos), the comparison is between cadaverine and alarm substance. Is there a reason why the alarm substance was not used in larvae, or chondroitin on adult fish? Perhaps the authors can elaborate on their rationale.

    We were basing our experimental approach on results that had been published by others. For example, Krishnan et al. (2014) had shown that dHb neurons respond to chondroitin sulfate in 6-9 day old larvae. Previous studies had reported that the earliest responses to alarm substance can be seen around 42 days post fertilization in zebrafish (Waldman, 1982) and 48-57 days post-hatching in fathead minnows (Carreau-Green et al., 2008). Jetti et al. (2014) examined the response to alarm substance at 25 dpf zebrafish.

    1. Presentation, analysis, and discussion of behavioral results a) The presentation of the alarm substance behavior results could be improved. The authors could include the words "alarm substance" somewhere in the panels so it is clear to the readers that they are looking at responses to that rather than to cadaverine which is described in the preceding panels. Similarly, to avoid confusion and to facilitate comparison, the same parameters should be presented for Figures 3-5 (currently distance in top is not shown in Figure 3 or 5, onset of fast swim and interval time not shown in Figures 4-5).

    As suggested, we added the words “Cadaverine” and “Alarm substance” to Figures 3, 4, 5 and Supplementary figure 4. We also now show the same parameters for the response to alarm substance in BoTx-GFP and intersectional BoTx-GFP transgenic lines, and in tcf7l2 and bsx mutants (refer to Figures 3, 4, 5 and Supplementary figure 4).

    b) Does cadaverine induce changes in swim speed and other kinematic parameters? Similarly, does the alarm substance induce avoidance of one side of the tank like cadaverine? It can be difficult for the reader to compare the effects of genetic manipulations on responses to both odorants since different behavioral parameters are being quantified, hence some means of direct comparison could be helpful.

    As we had described in the discussion (page 14, lines 279-280): “Despite both being aversive cues (Hussain et al., 2013; Mathuru et al., 2012), cadaverine and alarm substance elicit different behavioral responses by adult zebrafish.” Alarm substance triggers immediate (within 1 min) erratic behavior such as rapid swimming, darting and prolonged freezing. Thus, it is not feasible to measure the same behavioral responses to the two aversive cues.

    c) The effect of cadaverine on control groups seems to be quite variable. In Figures 3 and 4 the avoidance effect persists the entire duration of the experiment. In Figures 5 and S4 the effect is only significant in 2 time bins. The authors' conclusions are still valid since the correct comparisons are indeed to their respective sibling controls, however it does make it a bit difficult to compare results across genotypes. For example, non-botox-expressing lratd2a:QF2 fish appear to have about the same degree of cadaverine avoidance as lratd2a:QF2, scl5a7a:Cre, QUAS:Botox fish. Similar to point (b), are there other parameters that can be measured that are more consistent in controls across genotypes? Or at the least, some discussion of the behavioral variability in the text.

    As correctly pointed out by the reviewer, different fish with different genetic backgrounds demonstrate different degrees in their response to odorants. However, behavioral measurements were reproducible over 2 to 3 trials testing the same groups of fish on different days. We now show the aversion index for all individuals tested for the response to cadaverine in a new figure (refer to Supplementary figure 7).

    d) tcf7l2 mutants (like bsx mutants) also have a significantly lower swim speed than controls, this is also worth mentioning / discussing in the text.

    We have now mentioned this in the results section of the main text (Page 10, lines 202-203).

    e) The link between habenular LR asymmetry and aversive behavior is indeed interesting - in the discussion, one proposal was that this asymmetry could promote directed turning and escape. From the existing data (particularly for the lratd2a:QF2, scl5a7a:Cre, QUAS:Botox fish), is there any evidence of differences in turning behavior (LR asymmetry, or probability of turns in general)?

    We did not observe any correlation between habenular L-R asymmetry and the direction of turning in response to alarm substance, although this is an interesting point. We added a sentence to the discussion (page 16, lines 323-324) to reference a recent study on the neural basis of this lateralized behavior.

    f) As a related point, it is not clear to me that one would expect an enhancement of cadaverine avoidance in bsx mutants, especially if the argument is that asymmetry is important for aversive behavior. Perhaps the discussion on this point could be framed less as a negative result but as a notable observation.

    We agree with the reviewer’s interpretation and have added the following sentences to the Results (page 11, lines 218-220): “Despite the symmetric activation of dHb neurons, bsx homozygotes and heterozygotes both showed reduced responsiveness to cadaverine,” and to the Discussion (page 15, lines 303-304): “We did not observe enhanced or prolonged aversion to cadaverine in bsxm1376 homozygotes relative to controls.”

    1. Statistical analyses: Unless data is normally-distributed, non-parametric tests should be used to compare on calcium and behavioral imaging data (such as Kruskal-Wallis for time course of the calcium / behavioral data, Wilcoxon Rank-Sum Test for others).

    As correctly pointed out by the reviewer, we have corrected our statistical analyses (refer to Materials and Methods and the Figure legends). We used two-way ANOVA followed by Bonferroni's post hoc test and an unpaired t-test for analyzing calcium imaging data. For analyzing the response to cadaverine within groups, we used the Wilcoxon signed-rank test and cited publications using comparable approaches (Koide et al., 2009; Wakisaka et al., 2017). For analyzing the data between groups, we used two-way ANOVA followed by Bonferroni's post hoc test.

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  2. Evaluation Summary:

    The three reviewers have appreciated the novelty and originality of the study, but note that improved visualization, quantifications and statistical analyses will be necessary to fully support the conclusions of the manuscript. Without performing these quantifications and statistical tests for all figures as detailed below, the magnitude and significance of reported effects are not clear, nor do they take into account the variability of the measures and the dependence of some of the measures.

    (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 #2 and Reviewer #3 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    The habenula is a remarkable asymmetrical structure in the vertebrate brain whose integrative functions are diverse and not well understood. The authors have identified an interesting subset of dorsal hanenular neurons expressing lratd2a and localized only on the right side of the body that receive inputs from the olfactory bulb, project to the ventral interpeduncular nucleus (vIPN). By combining sophisticated genetics, calcium imaging and behavior in adult zebrafish, the authors argue that an asymmetric dorsal habenula - ventral interpeduncular nucleus pathway is involved in processing aversive cues and mediates avoidance responses to olfactory cues.

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  4. Reviewer #2 (Public Review):

    In this manuscript, Choi et al combine new intersectional genetics and CRISPR-mediated knock-in strategies to dissect the role of an interesting population of lecithin retinol 23 acyltransferase domain containing 2a (lratd2a)-expressing cholinergic neurons in the right dorsal habenula. They show that these neurons are responsive to aversive odorant cues such as cadaverine, and that silencing these neurons specifically reduces avoidance behavior to this odorant. The authors also use mutant lines to explore the role of habenular asymmetry in aversive responses, which provide additional supporting evidence for the hemispheric specialization of behaviors. However, given the pleiotropic effects of such mutations, the results from these mutants are more difficult to interpret.

    The genetic manipulations and anatomical characterization are excellently done, behavioral results sound, and the manuscript well-written. I do not believe any additional experiments are needed for publication, but will make some recommendations in terms of data analysis and improving clarity in text and figures, to facilitate better comparisons across the different experiments and odorant cues (cadaverine vs alarm substance).

    1. Presentation, analysis, and discussion of calcium imaging results

    a) As the authors correctly pointed out, having a water control is indeed essential for interpreting calcium imaging results. As such, I would recommend having a water control panel (currently in Figure S3) in the main figure.

    b) The current presentation and analysis of calcium imaging data in Figure 2B does not seem appropriate and can be improved - since the dynamics of olfactory responses are likely highly variable across neurons and fish, rather than comparing responses across time, it would be better to compare the summed response over a longer time window (as already done in Figure S2, but also including water flow control data). Do also mention the time window over which the calcium responses were integrated.

    c) Discussion Line 393: "From calcium imaging, we validated that the right dHb appears more responsive than the left when larval zebrafish are exposed to aversive odors such as cadaverine or chondroitin sulfate" - this conclusion cannot be drawn from the existing presented data, unless calcium imaging was also performed in the left habenula.

    d) It would be good to include in the methods section more detail on how the odor was delivered, volume delivered etc, and whether control experiments were done on the same day / clutch of fish etc.

    2. Presentation, analysis, and discussion of c-Fos results and comparison with calcium imaging

    a) Figure 2C-D: The difference / overlap between blue and brown are difficult to make out in the images, especially at this resolution and magnification. Is there a way to specifically quantify the % of lratd2a neurons that are activated by c-fos, rather than just neurons in the dorsal habenula as a whole? This would be necessary to support the claim in line 278: "Thus, the lratd2a subpopulation in the right dHb responds to cadaverine in both larvae and adults".

    b) In larvae (using calcium imaging), the effects of cadaverine to chondroitin sulfate were compared, whereas in adults (using c-Fos), the comparison is between cadaverine and alarm substance. Is there a reason why the alarm substance was not used in larvae, or chondroitin on adult fish? Perhaps the authors can elaborate on their rationale.

    3. Presentation, analysis, and discussion of behavioral results

    a) The presentation of the alarm substance behavior results could be improved. The authors could include the words "alarm substance" somewhere in the panels so it is clear to the readers that they are looking at responses to that rather than to cadaverine which is described in the preceding panels. Similarly, to avoid confusion and to facilitate comparison, the same parameters should be presented for Figures 3-5 (currently distance in top is not shown in Figure 3 or 5, onset of fast swim and interval time not shown in Figures 4-5).

    b) Does cadaverine induce changes in swim speed and other kinematic parameters? Similarly, does the alarm substance induce avoidance of one side of the tank like cadaverine? It can be difficult for the reader to compare the effects of genetic manipulations on responses to both odorants since different behavioral parameters are being quantified, hence some means of direct comparison could be helpful.

    c) The effect of cadaverine on control groups seems to be quite variable. In Figures 3 and 4 the avoidance effect persists the entire duration of the experiment. In Figures 5 and S4 the effect is only significant in 2 time bins. The authors' conclusions are still valid since the correct comparisons are indeed to their respective sibling controls, however it does make it a bit difficult to compare results across genotypes. For example, non-botox-expressing lratd2a:QF2 fish appear to have about the same degree of cadaverine avoidance as lratd2a:QF2, scl5a7a:Cre, QUAS:Botox fish. Similar to point (b), are there other parameters that can be measured that are more consistent in controls across genotypes? Or at the least, some discussion of the behavioral variability in the text.

    d) tcf7l2 mutants (like bsx mutants) also have a significantly lower swim speed than controls, this is also worth mentioning / discussing in the text.

    e) The link between habenular LR asymmetry and aversive behavior is indeed interesting - in the discussion, one proposal was that this asymmetry could promote directed turning and escape. From the existing data (particularly for the lratd2a:QF2, scl5a7a:Cre, QUAS:Botox fish), is there any evidence of differences in turning behavior (LR asymmetry, or probability of turns in general)?

    f) As a related point, it is not clear to me that one would expect an enhancement of cadaverine avoidance in bsx mutants, especially if the argument is that asymmetry is important for aversive behavior. Perhaps the discussion on this point could be framed less as a negative result but as a notable observation.

    4. Statistical analyses: Unless data is normally-distributed, non-parametric tests should be used on calcium and behavioral imaging data (e.g. Kruskal-Wallis for time course calcium / behavioral data, Wilcoxon Rank-Sum Test for others)

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  5. Reviewer #3 (Public Review):

    The authors use genetic approaches to label, monitor and manipulate a specific set of cholinergic neurons in the right dorsal habenula of zebrafish that connect to a territory of the interpeduncular nucleus. The original question and the combination of the approaches represent clear strengths of this study. The manuscript would benefit from a better description of the data, especially the calcium imaging experiment lack a bit of clarity. The conclusions provided by the authors are based on the data provided. I find the discussion session well thought as puts the study in the context of the published literature in a variety of model systems.

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