Author Response

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

Public Reviews:

Reviewer #1 (Public Review):

In this manuscript, Lee et al. compared encoding of odor identity and value by calcium signaling from neurons in the ventral pallidum (VP) in comparison to D1 and D2 neurons in the olfactory tubercle (OT).

Strengths:

They utilize a strong comparative approach, which allows the comparison of signals in two directly connected regions. First, they demonstrate that both D1 and D2 OT neurons project strongly to the VP, but not the VTA or other examined regions, in contrast to accumbal D1 neurons which project strongly to the VTA as well as the VP. They examine single unit calcium activity in a robust olfactory cue conditioning paradigm that allows them to differentiate encoding of olfactory identity versus value, by incorporating two different sucrose, neutral and air puff cues with different chemical characteristics. They then use multiple analytical approaches to demonstrate strong, low-dimensional encoding of cue value in the VP, and more robust, high-dimensional encoding of odor identity by both D1 and D2 OT neurons, though D1 OT neurons are still somewhat modulated by reward contingency/value. Finally, they utilize a modified conditioning paradigm that dissociates reward probability and lick vigor to demonstrate that VP encoding of cue value is not dependent on encoding of lick vigor during sucrose cues, and that separable populations of VP neurons encode cue value/sucrose probability and lick vigor.

Weaknesses:

The conclusions of the data are mostly well supported by the analyses, but the statistical analysis is somewhat limited and needs to be clarified and extended.

(1) The manuscript includes limited direct statistical comparison of the neural populations, and many of the comparisons between the subregions are descriptive, including descriptions of the percentage of neurons having specific response types, or differences in effect sizes or differing "levels" of significance. An additional direct comparison of data from each subpopulation would help to confirm whether the differences reported are statistically meaningful.

Response: We thank the reviewer for their helpful suggestions. As the reviewer noted, the first version of our manuscript had limited direct comparisons of single-neuron metrics across subpopulations. These analyses were also limited to the supplementary figures: 1) {SK vs. XK} and {SK vs. ST} decoder auROC (S10F), 2) Valence scores (S10G), and 3) S-cue confusion after MNR classification (S11D). We have now included the following statistical comparisons of single-neuron metrics across subpopulation: 1) % of neurons that respond to both S cues (Tables S10, S11), 2) % of neurons that have auROC >0.75 for {SK vs. XK}, {SK vs. PK}, and {SK vs. ST} (Tables S12-S17), 3) response magnitudes to S cues (Table S38), and 4) valence scores (Tables S44-46).

(2) When hypothesis tests are conducted between the neural populations, it is not clear whether the authors have accounted for the random effect of the subject, or whether individual units were treated as fully independent. For instance, pairwise differences are reported in Figures 4I, 5G/I/L, and others, but the statistical methods are unclear. Assessment of the statistics is further limited by the lack of reporting of degrees of freedom. If the individual neurons are treated as independent in these analyses, it could increase the likelihood of

Response: We have clarified when statistical analyses are comparing individual neurons vs. simultaneously recorded populations. Per the reviewer’s recommendation, we have also incorporated linear mixed-effects models when statistically analyzing individual neurons. Lastly, to further clarify the statistical analyses used, we have added multiple supplementary tables that better describe the statistical tests used and the relevant outputs.

Reviewer #2 (Public Review):

Summary:

This work is interesting since the authors provide an in vivo analysis into how odor-associations may change as represented at the level of olfactory tubercle (presynaptic) and next at the level of the ventral pallidum (postsynaptic). First the authors start-off with a seemingly careful characterization of the anterograde and retrograde connectivity of dopamine 1 receptor (D1) and dopamine 2 receptor (D2) expressing medium spiny neurons in the olfactory tubercle and neurons in the ventral pallidum. From this work they claim that regardless of D1 or D2 expression, tubercle neurons mainly project to the lateral portion of the ventral pallidum. Next, to compare how odor-associated neuronal activity in the ventral pallidum and the olfactory tubercle (D1 vs D2 MSNs) transforms across association learning, the authors performed 2photon calcium imaging while mice engaged in a lick / no-lick task wherein two odors are associated with reward, two odors are associated with no outcome, and two odors are associated with an air puff.

This manuscript builds off of prior work by several groups indicating that the olfactory tubercle neurons form flexible learned associations to odors by looking at outputs into the pallidum (but without looking specifically at palladial neurons that truly get input from tubercle I should highlight) and with that, this work is novel. We appreciated the use of a straight-forward odoroutcome behavioral paradigm and the careful computational methods and analyses utilized to disentangle the contributions of single neurons vs population level responses to behavior. With one exception from the Murthy lab, 2P imaging in the tubercle is a new frontier and that is appreciated - as is the 2P imaging in the pallidum which was well-supported by the histology. The anatomical work is also well presented.

Overall the approach and methods are superb. The issues come when considering how the authors present the story and what conclusions are made from these data. Several key points before going into specifics about each are: 1) The authors can not conclude that their results are contradictory to prior results, 2) The authors over-interpret the results and do not discuss several key methodological issues. We were concerned with the ability to make strong claims regarding the circuitry presented, especially given how much the presented claims contradict prior work. There were also issues with the interpretability of neuronal encoding of value vs valence based on the present behavior (in which a distinction between the air puff and neutral trial types was not clear) and the imaging methodology (in which the neuronal populations analyzed were not clearly defined). In addition to toning down and rectifying some of the language and interpretations, we suggest including a study limitations section where these methodological and interpretation issues are discussed. Over-interpreting and playing up the significance of this work is unnecessary, especially given eLife's new review and publication policy. Readers should be given a sufficiently detailed and nuanced presentation of these thought-provoking results, and from there allowed to interpret the results as they want.

Strengths:

State-of-the-art approaches (as detailed above)

Possible conceptual innovation in terms of looking into output from the olfactory tubercle which has yet to be investigated in this avenue.

Weaknesses:

On the first point regarding the authors repeated and unsupported claims that their results are contradictory. There are papers by numerous groups, in respected journals including this one, all together which used 5 different methods (cfos, photometry, 2P, units, fMRI), in animals ranging from humans to mice, which support that tubercle neurons reflect the emotional association of an odor, whether spontaneous or learned. With that, it is on the authors to not claim that their results contradict as if the other papers are suspect, but instead, from our standpoint it is on the authors to explain how and why their results differ from these other papers versus just simply saying they found something different [which at present is framed in a way that is 'correct' due to primacy if nothing else].

Response: We acknowledge that the first version of the manuscript contained unnecessary disagreeing language. We do not think that our results are broadly in disagreement with the existing literature, but we do come to different conclusions about what the OT is representing. Namely, our comparison of valence encoding in OT to that in the VP strongly indicates that the anteromedial OT has a less robust representation of valence, and we argue that this reflects either an intermediate form of valence representation or potentially might not be important for valence representation at all. We have toned down our conclusions, made clear that we are only recording from one domain of the OT, limited our speculation to the discussion and added a “speculations” section.

Second, onto the points of interpretation of results, there are several specific areas where this should be rectified. As is, the authors overinterpret their results and draw too far-reaching conclusions. This needs to be corrected.

In particular, the claims that D1 and D2 neurons of the olfactory tubercle nearly exclusively send projections to the ventral pallidum must be interpreted with caution given that the authors injected an anterograde AAV into the anteromedial olfactory tubercle, and did not examine the projections from either the posterior or lateral portions of the olfactory tubercle. This is especially significant since the retrograde tracing performed from the ventral pallidum indicates that the lateral olfactory tubercle, not the medial olfactory tubercle, primarily projects to the ventral pallidum (Fig 1D-F), however this may be due to leakage into the nucleus accumbens, as seen in the supplementary figure, S1G.

Response: We thank the reviewer for the point of caution. We have now made it clear that our conclusions are limited to the anteromedial portion of the OT, and other areas may have other projections.

The same caution must be advised when interpreting the retrograde tracing performed in Fig 1G-I, since the neuronal tracer used and the laterality and rostral-caudal injection site within the VTA could result in different projection patterns and under- or over-labelling. Additionally, the metric used, %Fiber Density (Figure 1C), as in the percentage of 16-bit pixels within the region of interest with an intensity greater than 200, is semi-quantitative, and is more applicable for examining axonal fibers that pass through a region rather than the synaptic terminals (like with a synaptophysin fusion protein-based tracing paradigm) found within a region (puncta). The statements made in contrast to prior studies should therefore be softened, and these concerns should be addressed in the introduction, discussion, and the limitations section if added.

Response: We have added statements to address these limitations.

The other major concern is whether the behavioral data generated is indicative of the full spectrum of valence. The authors appropriately state that the mice "perceive" the air puff, yet based on their data the mice did not clearly experience the puff-associated odor as emotionally aversive (viz., negative valence). The way the authors describe these results, it seems they agree with this. With that, the authors can't say the puff is aversive without data to show such - that is an assumption which, while seemingly intuitive, is not supported by the data unfortunately. To elaborate more since this is important to the messaging of the paper: The authors utilized a simple behavioral design, wherein two molecular classes of odors were included in either a sucrose rewarded, neutral no outcome, or air puff punished trial type. The odor-outcome pairs were switched after three days, allowing the authors to compare neuronal responses on the basis of odor identity and the later associated outcome. While the mice showed clear learning of the rewarded trial types by an increase in anticipatory licking during the odor, they did not show any significant changes in behavior that indicated learning of the air puff trial type (change in running velocity or % maximal eye size), especially in contrast to the neutral trial type. This brings up the concern that either the odor-air puff aversive associations (to odors) were not learned, or that the neutral trial types, in which a reward was omitted, were just as aversive as the air puff to the rear, despite the lack of startle response - perhaps due to stimulus generalization between neutral and air puff odor. The possibility of lack of learning is addressed in the paragraph starting at line 578, but does not account for the possibility that the lack of reward is also sufficiently punishing. The authors also address the possibility that laterality in the VP contributed to the lack of neural responsivity observed, but should also include a statement regarding laterality in the olfactory tubercle, as described in https://doi.org/10.7554/eLife.25423 and https://doi.org/10.1523/JNEUROSCI.0073-15.2015, since the effects of modulating the lateral portion of the olfactory tubercle are not yet reported. Lastly, use of the term "reward processing" should be avoided/omitted since the authors did not specifically study the processing of reinforcers.

Response: As the reviewer points out, we tried to be cautious interpreting the “aversive” odor response, and focused mainly on the reward association. This was discussed in the discussion. We don’t see the need to further add a redundent statement to a “limitations section”. We have also added a note about the previously identified laterality of the OT, which might account for lack of aversive responsive neurons in the OT. The reviewer makes an interesting suggestion that behavioral responses to airpuff-associated odors are not significantly different from un-associated because the lack of reward in this context is already aversive. We note that the walking velocity between reward- and puff-associated odor is significantly different, but not that to unassociated. This is in agreement with the suggestion, and we have added a statement to reflect this.

Also, I would appreciate justification of the term "value". How specifically does the assay used assess value versus a more simplistic learned association which influences perceived hedonics or valence of the odors.

Response: We have removed the term “value” with the exception of areas where we cite the work of others. We acknowledge that the word value is complicated in the incentive learning field and appreciate the suggestion. Our experimental design was meant to investigate learned association for positive and negative stimuli, thus valence is more appropriate and we have used this term.

More information is needed regarding how neurons are identified day-to-day, both in textual additions to the Methods and also in terms of elaborating more in the results and/or figure legends about what neurons are included:

(a) The ROI maps for identifying/indicating cells in the FOVs are nice to see and at the same time raise some concerns about how cells are identified and/or borders for those specific ROIs drawn. For instance, Figure 4, A & D, ROI #13 (cell #13) between those two panels is VERY different in shape/size. Also see ROIs 15 and 4. Why was an ROI map not made on day 1 and then that same map applied and registered to frames from consecutive imaging days in that same mouse? As it is new ROIs are drawn, smaller for some "cells" and larger for others. And at least in ROI #13 above, one ROI is about twice as large as the other. This inconsistency in the work flow and definition of the ROIs is needing to be addressed in Methods. Also, the authors should address if and how this could possibly impact their results.

Response: We have added details and clarified the methods section to make this more clear. We note that we extracted calcium transients from the raw data with the the widely used Constrained Nonnegative Matrix Factorization (CNMF) algorithm. This processing algorithm simultaneously identifies spatial and temporal components using modeled kinetics of calcium transients and pre-trained CNN classifiers. Using 2-photon microscopy the optical resolution in the z plane is narrow and we may not always capture components of a neuron that look like “neurons”, but all ROIs were confirmed manually to ensure they were not artifacts.

(b) Also, more details are needed in results and/or figure legends regarding the changes in cell numbers over days that are directly compared in the results. Some days there are 10% or more or less cells. Why? It is not the same population being compared in this case and so some Discussion of this is needed.

Response: The shapes of the spatial components can vary across days due to nonrigid motion in the brain and/or miniscule differences in the imaging angle across days. Although we visually verified that we are imaging approximately the same z plane across days, we cannot (and do not) claim to image identical populations of neurons across days.

Reviewer #3 (Public Review):

Summary:

This manuscript describes a study of the olfactory tubercle in the context of reward representation in the brain. The authors do so by studying the responses of OT neurons to odors with various reward contingencies and compare systematically to the ventral pallidum. Through careful tracing, they present convincing anatomical evidence that the projection from the olfactory tubercle is restricted to the lateral portion of the ventral pallidum.

Using a clever behavioral paradigm, the authors then investigate how D1 receptor- vs. D2 receptor-expressing neurons of the OT respond to odors as mice learn different contingencies. The authors find that, while the D1-expressing OT neurons are modulated marginally more by the rewarded odor than the D2-expressing OT neurons as mice learn the contingencies, this modulation is significantly less than is observed for the ventral pallidum. In addition, neither of the OT neuron classes shows significant modulation by the reward itself. In contrast, the OT neurons contained information that could distinguish odor identities. These observations have led the authors to conclude that the primary feature represented in the OT is not reward.

Strengths:

The highly localized projection pattern from olfactory tubercle to ventral pallidum is a valuable finding and suggests that studying this connection may give unique insights into the transformation of odor by reward association.

Comparison of olfactory tubercle vs. ventral pallidum is a good strategy to further clarify the olfactory tubercle's position in value representation in the brain.

Weaknesses:

The authors' interpretation of the physiologic results - that a novel framework is needed to interpret the OT's role - requires more careful treatment.

Response: We thank the reviewer for their recommendation. We have toned down the conclusiveness of our language in the discussion. Additionally, we have removed several speculative sentences from the concluding paragraph.

Reviewer recommendations for Authors:

We thank the reviewers for this helpful list of recommended changes to the manuscript.
Regrettably, a few of the recommendations were overlooked in the revision, as indicated below.
We do agree with the suggestions and plan to add appropriate changes to the version of record.

Reviewer #1 (Recommendations For The Authors):

If the comparisons mentioned in point 2 in the public review do not account for the lack of independence of individual neurons, I suggest the authors do so by either running linear mixed effects models with a random effect for subject, or one-way ANOVAs with a random effect of subject, where appropriate. The authors could also run analyses on summarized individual subject data (averages, % of neurons, etc.), though the authors would lose substantial power when assessing whether average changes differ between subjects in each recording group.

We have clarified when statistical analyses are comparing individual neurons vs. simultaneously recorded populations. Per the reviewer’s recommendation, we have also incorporated linear mixed-effects models when statistically analyzing individual neurons. Lastly, to further clarify the statistical analyses used, we have added supplementary tables for every statistical test that better describe the parameters used and the relevant outputs.

Reviewer #2 (Recommendations For The Authors):

Of minor note, there are some symbols/special characters that did not translate in the figure caption for Figure 6C, repeated text between lines 700-705 and 707-712, and some other small grammatical errors. Additionally, the source of the anterograde tracing virus (AAV9-phSyn1FLEX-tdTomato-T2A-SypEGFP-WPRE) needs to be stated.

Thank you for pointing these out. We have added description to the figure legend, and deleted the repeated lines and fixed grammatical errors. During the revision, we Regrettably overlooked the request to provide the source for the AAV9-phSyn1-FLEX-tdTomato-T2A-SypEGFP-WPRE. We agree that this small detail is important and will add it before publication of the version of record. This viral vector was purchased from The Salk Institute GT3 Core.

Reviewer #3 (Recommendations For The Authors):

The authors' interpretation of the physiologic results - that a novel framework is needed to interpret the OT's role - requires more careful treatment. As the authors note, there is rewardcontingency modulation in OT, especially when D1 neurons are compared against D2, as shown in Fig. 3D,E, Fig. 4I, and Fig. F,J. Though small in effect size, presumably, these modulations cannot be explained by the odor identity. These observations, to this reviewer, suggest the D1 neurons of OT have a component of cue-reward representation. In other words, rather than developing an entirely new framework, an alternative possibility that D1 neurons of OT occupy an intermediate stage in associating cues with reward (i.e., under the same framework, but occupying a different position in the emergence of value representation) should be considered.

We thank the reviewer for this thoughtful comment. We have eliminated the statement that “novel framework is needed” and have been more conservative in our interpretations. We have also acknowledged that our results are not necessarily in conflict with existing literature, but we do draw different conclusions, namely that the anteromedial OT is not a robust valence encoding population in comparison to that in the VP. We appreciate the suggestion of the term “intermediate stage” in reward association and have now included this in the discussion. Lastly, we have limited broader speculation to a “speculation” section of the discussion.

Related to the above point, have the authors analyzed if the similarities in the chemical structures correspond to perceptual and neural similarities? In the data presented in Figure S4, there are greater similarities in the population patterns within the same rewarding condition than within chemical groups. A comparison of the reward vs. chemical group (a simpler version of Fig. 5B) may be beneficial and take full advantage of the experimental design.

This comparison already exists in 5B and lines 285-289 of results. In VP populations, the distribution was structured such that intervalence pairwise comparisons between sucrose-paired and not sucrose-paired odors (e.g. ||SK-PK|| and ||SK-XK||) were larger than intravalence pairwise comparisons (e.g. ||SK-ST||, or ||XK-XT||). OTD1 populations showed an intermediate trend where most intravalence pairwise distances were smaller than intervalence pairwise distances with the exception of ||SK-ST||.

Related to the point about chemical similarities - is the smaller effect size (amount of modulation associated with reward contingency) in this study, compared to the study by Martiros et al, explained by the similarities of odorants used?

This is an interesting point. Although the odorants we use are different from those in Martiros et al, we think it is unlikely to the basis of smaller effect size due to reward modulation. If OT represents odor in a population code, whereby identity is encoded in unique ensembles of activity, then variation in the expression of D1R between OT neurons could account for different effects in different ensembles. However, there is no evidence for such varied expression and it doesn’t seem like an ideal mechanism for the OT to broadly associate odor with reward. Moreover, we do not observe any differences in effect size of reward association between the different odorants used in our study. Rather, we think the difference between our findings is more likely to result from recording in different populations of neurons, which is addressed in lines 522-535.

Regarding the data presented in Fig. 3I - the rewarded odor responses (Sk) are compared against neutral ones (Xk responses), but an S vs. P comparison may be informative, too. Even though the authors mention that the effect of air puff is subtle, the behavioral data presented in Fig. 2F and G suggest that these serve as aversive stimuli. For example, on day 4, the first day after the reward contingency switch, the licking levels seem the lowest for the P odors.

We have added the S vs P comparison. Indeed, we had originally omitted this because the neural and behavioral response to puff cues was not robust. This is discussed in the discussion (lines 563-579), and our conclusions about aversive conditioning are cautious.

Regarding the data presented in Fig. 4G: it is difficult to interpret the data when the data for day 1 reward period and day 3 reward cue period are combined. Or do the authors mean day 1 S cue and day 3 S cue?

These data were based on an observation that some neurons in the VP only responded to sucrose (not odor) on day 1, but later became responsive to the associated odor on day 4. To quantify this, Fig. 4G shows the percentage of these neurons by reporting the percentage that were both responsive to sucrose (not odor) on day 1 and also rewarded odor on day 3. This is described in lines 260-274.

Figure 6 presentation would benefit from a revision. For example, it is unclear if the water port becomes available for the "N" odors with 100% or 50% chance of reward delivery, and if so, how that happens. There are some errors e.g., colormap used for panel G; odors listed may be wrong in line 752 etc. It was unfortunately not possible to understand what was presented.

We have added a schematic (Fig 6B) to better describe the movement of the port and details to the methods. The color scale was indeed inverted in panel G (now H), and it has been corrected. We have verified that the odors listed in the methods are correct. Although not included in the revision, in the version of record we will also add corresponding descriptors (e.g., LHi & Lx) to the odors in the methods for easier comparison.

Minor comments

For Figure 2H, an alternative description in the legend may be beneficial, as the phrasing is not intuitive. A suggested alternative is "licks in response to sugar-associated odors expressed as fraction of all odors".

We appreciate the suggestion and have changed this to “licks during either sucrose cue expressed as a fraction of all licks during any odor.”

Figure 2H: please explain the color code for crosses in the legend and the statistical comparison shown in the figure.

We have added a legend to explain the color code and included a statement about the statistics in the legend with a link to a supplemental table for statistical parameters.

Figure 3D: may contain mislabeling in the legend - the legend for 3D does not match the plot (legend refers to bar graph while plot shows line graphs)

Unclear what is meant. 3D legend says: “Percentage of total neurons that were significantly excited or inhibited by each odor (Bonferroni- adjusted FDR < 0.05) as a function of time relative to odor. Lines represent the mean across biological replicates and the shaded area reflects the mean ± SEM.” This is not a bar plot and is not referred to as one. 3E does show bar plots and is correctly described in the legend.

Figure 3M: uses letters to refer to cell populations that are identical to the roman numerals used in Fig 3 A-C as well as colours similar to the ones in Fig 3C. However, the cell groups are unrelated; splitting the figures or using a different nomenclature might help

We have adapted a different color code that we think makes this more distinct.

Figure 4I: statistical comparison shown in figure not explained (neither in main text nor legend)

We have added a statement about the statistical comparison and referenced a supplementary table.

Figure 5 D: color code appears to have a different range than the values shown (i.e. lower limit is 0.7 while the plot shows values below 0.7)

We confirm this is not a mistake but a stylistic choice. The displayed color scale does only show values to lower limit of 0.7, while the lower limit of values is 0.67. Although the color for 0.67 is not shown in the scale it is approximately the same as the lower limit. The values are reported for full transparency and accuracy.

Figure 5 G, I, & L: statistical comparison shown in figure not explained

The comparisons have been explained in supplemental tables (S22-29) and referenced in the legend.

Figure 5 I: meaning of symbols overlayed over bars not explained

“Markers represent the mean across biological replicates” has been added.

Figure 5 J&K: please state if error bars show SEM or SD; also please describe individual thinner lines in the legend

This has been added to describe 5I. The same format applies to J&K.

Figure 5L: please describe the individual crosses overlayed over bars in the legend

Described in 5I.

Figure S6A-C: please mention the odors used.

S6A-C shows kinetics for the odor a-terpinene, which is now indicated in the legend.

Line 129: mentions a 70 psi airpuff but methods say 75 psi - please clarify This has been corrected. 70 psi is the correct value.

Line 134 typo: SP should be PK

This has been corrected.

Line 428: typo; should be cluster 3, not 2

This has been corrected.

Line 474 (and figure 6O): please explain what "P" is

“P” is probability, used as P(S), as in probability of sucrose. This is defined in in line 466.

Line 692: please describe the staining protocol in the methods (rather than just listing the antibodies and concentrations)

We have added more details (lines 692-699).

Line 707-712: duplicate text (identical to Line 700-705)

This has been deleted.

Figures and data

OTD1 and OTD2 primarily project to the lateral portion of the VP.

Head-fixed 2-photon Ca2+ imaging of OTD1, OTD2, or VP neurons during 6-odor conditioning paradigm.

VP neurons encode reward-contingency more robustly than OTD1 or OTD2 neurons.

Sucrose responsive VP neurons become sucrose-cue responsive after pairing.

OT encodes odor identity in high-dimensional space and VP encodes reward-contingency in low-dimensional space.

Separate VP populations encode reward-contingency and licking vigor.

Head-fixed 2-photon Ca²⁺ imaging of OT_D1, OT_D2, or VP neurons during 6-odor conditioning paradigm.

VP neurons encode reward-contingency more robustly than OT_D1 or OT_D2 neurons.