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  1. Author Response

    Reviewer 2

    Mouse olfactory neurons express one single type of odorant receptor (OR) out of ~1000 possible choices, and the neurons expressing the same type of OR project their axons to two or a few glomeruli in the olfactory bulb (OB). The goal of this work was to identify glomeruli that are activated by the lowest concentration of one given odorant (this would be the primary odorant for the glomerulus). A panel of 185 odorants that cover a wide range of chemical structures was designed for this purpose. The authors imaged the dorsal regions of the 8 OBs from 4 transgenic mice that express the Ca2+ reporter GCaMP6 in the mature olfactory neurons while they were exposed to the odorants delivered in vapor phase. In this way, the authors were able to identify glomeruli that were responsive to odorants at very low concentrations (estimated to be in the picomolar to nanomolar range). They also show that while the spatial representation of odorant chemicals in the bulb is sparse, rather than clearly delimited (except for that of amines and carboxylic acids), glomeruli recognizing structurally related odorants are co-tuned.

    The experiments are well executed and the images of the activated glomeruli in the OBs are impressive. These results show that olfactory neurons (and their cognate ORs) can be high affinity and selective receptors. These qualities cannot be easily detected when using conventional heterologous expression experiments or ex vivo assays, where responses are usually observed in the range of micromolar concentration of the odorants. The results reveal important aspects of odorant decoding in living mice and suggest that odorant concentrations that are effectively processed by the olfactory system are much lower than the ones usually considered. This high-resolution approach also facilitates the analysis of how odorant chemical structure is spatially represented in the OB.

    There are a few points that the authors might want to consider:

    Although it is assumed that each one of the glomeruli represents one OR type, the exact identity of the ORs that correlate with each of the 26 glomeruli remains unknown. Could the authors identify which ORs correspond to the 26 glomeruli based on the glomerular OB map determined by spatial transcriptomics (for example in https://www.nature.com/articles/s41593-022-01030-8) and on the position coordinates of the 26 glomeruli shown in Table S2? It would be nice to see whether the ORs sequences cluster in a way that correlates with co-tuning of the responses to structurally related odorants.

    We agree that it would be nice to relate the functionally-identified glomeruli to known ORs. Unfortunately this is difficult with current resources. The two recent glomerular maps of OR identity that are derived from spatial transcriptomics (the Wang et al., paper to which the reviewer refers, as well as Zhu et al (https://www.biorxiv.org/content/10.1101/2021.09.13.460128v1), on which we are contributing authors) do not provide sufficient precision in glomerular location to match to functionally-identified glomeruli solely on the basis of position. More fundamentally, the spatial ‘jitter’ in glomerular position from animal to animal, coupled with the interspersed nature of glomeruli with different odorant tuning properties, likely make it impossible to align functional maps to spatial transcriptomic maps derived in separate animals at the level of single ORs/glomeruli; though this approach could narrow the field of candidate ORs to a relatively small number (i.e., 10 – 20 ORs). We have added text to this effect in the Discussion (lines 574 - 82).

    We do think that the use of functionally-identified glomeruli could be paired with tagging of candidate ORs as a means of further deorphanization and in vivo characterization of OR response properties. To emphasize this point, we have added text pointing out that several of our identified glomeruli match well with position and ‘best’ odorants for a few ORs that have been previously mapped to dorsal glomeruli – namely, M72 (Olfr160), MOR204-34 (Olfr510; see Oka et al., 2006), and Olfr1377 (from the recent Zhu et al. paper). This text is in lines 253 - 259.

    Despite the fact that each OR has two glomeruli per bulb (one lateral and one medial), for most of the odorants, only one activated glomerulus per bulb was observed (ex. Figures 1 and 2). Is the other one always out of the field of vision (dorsal surface of OB), or is it not activated? This should be explained in the text.

    We thank the reviewer for raising this point. In general, only one glomerulus of the pair of OR-cognate glomeruli is visible on the dorsal surface – with the exception of the TAAR glomeruli, in which both the medial and lateral glomeruli are often dorsal, as shown by earlier studies. Consistent with this, we did observe paired glomeruli selectively activated by certain amines (other amines appeared to activate multiple TAARs and so evoked multi-glomerular maps). We agree that it is helpful to report this, and have added a supplementary Figure (Figure 2 –figure supplement 2) showing putative paired TAAR glomeruli; the Figure also shows that the ‘medial’ and ‘lateral’ glomerulus of each pair have near-identical response spectra, consistent with their being linked to the same TAAR. We have also added text addressing these points in the Results (lines 260-267).

    A global analysis summarizing how the results could be extrapolated to the whole OB would be helpful and informative. For example, what is the total number of glomeruli in the mouse OB? What percentage of these were accessible for imaging in the experiments (1004 per bulb)? Primary odorants were identified for 26 glomeruli in the accessible region (dorsal OB), but according to Figure S3C, 288 glomeruli responded to only one odorant at low concentrations.

    We agree that it would be helpful to report such an estimated extrapolation of results to the remainder of the OB. As for an estimation of the fraction of glomeruli/ORs/TAARs visible in our imaging experiments, using the Zhu et al. paper as a reference, in which ORs/TAARs were directly measured from explants of the approximate imaging surface, we arrive at a low-end estimate of ~150 glomeruli (the Zhu et al. study detected 121 ORs and 9 TAARs from the functional-imaging area; assuming that all 15 TAARs project dorsally and that each of the paired TAAR glomeruli are visible on the dorsal surface, we arrive at ~120 OR-glomeruli + (15 x 2 TAAR glomeruli) = 150 glomeruli). One might reasonably increase this estimate by 10% to account for a failure to detect certain low-abundance ORs by Zhu et al. The number of odorant-responsive glomeruli across our 8 OBs ranged from 103 – 142 per OB (median, 126), suggesting that our odorant panel is able to probe a large majority of the dorsal-projecting OR/TAAR repertoire (75 – 90%), and to functionally identify approximately 15% of visible glomeruli. We have added these estimates to the Text (lines 117 - 122).

    To clarify about the other numbers mentioned by the reviewer, 1004 refers to the total number of imaged glomeruli across 8 OBs, as specified in the main Text and the Figure S3 legend. Likewise, 288 refers to the total number of glomeruli responding to only a single odorant across all 8 OBs.

    Would be good to summarize briefly in the text (Page 7 line 192), which were the stringent criteria used to select the glomeruli/diagnostic odorant pairs, even though it is in the methods. It would make it easier for the reader, and would also make clear why only 26 glomeruli out of the 288 were selected as good glomerulus/diagnostic odorant pairs. How many of the 185 odorants are diagnostic odorants for the imaged glomeruli? How many odorants are not diagnostic odorants for the imaged region, and could therefore be likely to act so for the glomeruli in the regions that were not accessible? And so on.

    We have clarified these criteria for choosing the 26 identified glomeruli, explicitly describing them in the main section of the Results (lines 225 - 232), and also clarified the reporting of the numbers of odorants that serve as diagnostic odorants using these criteria (41 odorants) (lines 238-239).

    The authors find that the glomerular sensitivities to different odorant structure classes are not clearly spatially discrete, but are overlapping and interdigitated. Are they temporally discrete instead? Could this question be addressed?

    Unfortunately the relatively slow kinetics of the GCaMP6s reporter is poorly suited to discern temporal differences in responses across glomeruli. However we agree that attempting to do so with faster reporters would be very interesting, especially since much earlier work from this laboratory has noted marked differences in response dynamics as a function of glomerular location and odorant identity; we have mentioned this as a possibility in new text in the Discussion (line 566-567).

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  2. Evaluation Summary:

    This is an important paper querying odor responses in the olfactory bulb at low concentrations. Classical studies have revealed a 'combinatorial code' for odorant recognition, with individual odorants represented by combinations of broadly tuned and low-affinity olfactory receptors. Here, the authors perform a large-scale analysis of odor responses across glomeruli and surprisingly observe that odorant receptors instead generally display remarkably narrow tuning profiles.

    (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 #1 and Reviewer #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    Burton, Wachowiak, and colleagues use an established olfactory bulb imaging preparation to perform a large-scale analysis of odor responses across the dorsal olfactory bulb. The strengths of the study involve a large odor set, and a broad odor concentration range. The major finding from this work is that most olfactory glomeruli display remarkably sharp tuning. These observations are surprising and important, given that the canonical model in the field is that odors are instead represented by combinations of low-affinity olfactory receptors. A minor issue is that responses were only analyzed in a fraction of glomeruli which were surgically accessible due to their dorsal location. Nevertheless, it seems safe to draw general conclusions given the large number of OR and TAAR glomeruli imaged. Imaging approaches seem expertly performed and conclusions appear solid.

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  4. Reviewer #2 (Public Review):

    Mouse olfactory neurons express one single type of odorant receptor (OR) out of ~1000 possible choices, and the neurons expressing the same type of OR project their axons to two or a few glomeruli in the olfactory bulb (OB). The goal of this work was to identify glomeruli that are activated by the lowest concentration of one given odorant (this would be the primary odorant for the glomerulus). A panel of 185 odorants that cover a wide range of chemical structures was designed for this purpose. The authors imaged the dorsal regions of the 8 OBs from 4 transgenic mice that express the Ca2+ reporter GCaMP6 in the mature olfactory neurons while they were exposed to the odorants delivered in vapor phase. In this way, the authors were able to identify glomeruli that were responsive to odorants at very low concentrations (estimated to be in the picomolar to nanomolar range). They also show that while the spatial representation of odorant chemicals in the bulb is sparse, rather than clearly delimited (except for that of amines and carboxylic acids), glomeruli recognizing structurally related odorants are co-tuned.

    The experiments are well executed and the images of the activated glomeruli in the OBs are impressive. These results show that olfactory neurons (and their cognate ORs) can be high affinity and selective receptors. These qualities cannot be easily detected when using conventional heterologous expression experiments or ex vivo assays, where responses are usually observed in the range of micromolar concentration of the odorants. The results reveal important aspects of odorant decoding in living mice and suggest that odorant concentrations that are effectively processed by the olfactory system are much lower than the ones usually considered. This high-resolution approach also facilitates the analysis of how odorant chemical structure is spatially represented in the OB.

    There are a few points that the authors might want to consider:
    Although it is assumed that each one of the glomeruli represents one OR type, the exact identity of the ORs that correlate with each of the 26 glomeruli remains unknown. Could the authors identify which ORs correspond to the 26 glomeruli based on the glomerular OB map determined by spatial transcriptomics (for example in https://www.nature.com/articles/s41593-022-01030-8) and on the position coordinates of the 26 glomeruli shown in Table S2? It would be nice to see whether the ORs sequences cluster in a way that correlates with co-tuning of the responses to structurally related odorants.

    Despite the fact that each OR has two glomeruli per bulb (one lateral and one medial), for most of the odorants, only one activated glomerulus per bulb was observed (ex. Figures 1 and 2). Is the other one always out of the field of vision (dorsal surface of OB), or is it not activated? This should be explained in the text.

    A global analysis summarizing how the results could be extrapolated to the whole OB would be helpful and informative. For example, what is the total number of glomeruli in the mouse OB? What percentage of these were accessible for imaging in the experiments (1004 per bulb)? Primary odorants were identified for 26 glomeruli in the accessible region (dorsal OB), but according to Figure S3C, 288 glomeruli responded to only one odorant at low concentrations. Would be good to summarize briefly in the text (Page 7 line 192), which were the stringent criteria used to select the glomeruli/diagnostic odorant pairs, even though it is in the methods. It would make it easier for the reader, and would also make clear why only 26 glomeruli out of the 288 were selected as good glomerulus/diagnostic odorant pairs. How many of the 185 odorants are diagnostic odorants for the imaged glomeruli? How many odorants are not diagnostic odorants for the imaged region, and could therefore be likely to act so for the glomeruli in the regions that were not accessible? And so on.

    The authors find that the glomerular sensitivities to different odorant structure classes are not clearly spatially discrete, but are overlapping and interdigitated. Are they temporally discrete instead? Could this question be addressed?

    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    This is an important paper querying odor responses in the olfactory bulb at low concentrations. The main conclusion is that at low concentrations the dimensionality of odor responses rises, and responses become very sparse, sparse enough that a subset of odors can be identified that deterministically activate a single characteristic glomerulus. The observation that one can precisely match odors to specific glomeruli - as has been done previously in flies - has substantial implications for the ways in which receptors interact with odors, and for future experiments querying odor coding. From a technical perspective this paper is excellent, but the ways in which glomerular responses are computed, some of the conclusions related to sparseness and dimensionality, and the model used to compare descriptions of odor space require some revision. My sense is that after appropriate revision this will be an excellent addition to the literature, and provide us with valuable tools for querying odor processing.

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