This is a website where people can build their own community and find friends! I feel like most websites nowadays have a feature for you to interact with other users, built into a function they built. I heard that interaction plays a very important role in keeping users active and attracting new users.

Janie gets jealous of a woman that was with Tea Cake and when she finds them alone she wonders if there is something going on behind Janie’s back. Tea Cake and Janie talk it out in order to understand and they eventually get closer than before.

Janie starts to get jealous because tea cake starts to spend less time with her

Janie never felt jealous with Logan or Joe because she didn’t feel love with them. She now has a reason to be jealous because she truly wants Tea Cake.

This is bad practice because the font is small which can make it difficult for visually impaired people to read the text.

This is a good practice as the font is bold and easy to read.

This is a good practice because a recommended audio option provides visually impaired people with an alternative option to interact with the content.

This is a good practice as it makes content accessible to people in various languages.

This is a good practice as they additional accessibility information and help for those who need it

I agree that most things are done by a small percentage of people. I see many examples in real life, not just crowdsourcing. No matter what you do, there is always a small percentage of people who are good, famous, and doing the most work in every field. This is a trend that we are used to, and I think it is also really helpful to think about it in a way so that we realize the more good you are the more 'responsibility' you have,

Janie finally feels truly happy and free with her new relationship with Tea Cake that is based on love and equality instead of control.

She wants freedom but her parters like controlling her

My understanding is that Janie is living the “rich life” since being with Tea Cake

It looks like figure 1A has been transformed and compressed, hard to understand what is going on there. I think figure1C and figure 1D were accidentally added there?

She is feeling overwhelmed about the things in her past

CBC includes a dedicated accessibility page linked at the bottom, demonstrating a commitment to inclusive web design. This supports the Robust principle of POUR, signalling that the site is designed with assistive technologies users in mind. A visible accessibility page allows user to understand what accomodations are available and how to access them.

The "Shorts" feature do not display closed captions by default, requiring users to manually enable them. People who are deaf or have a hard time hearing depend on captions to access video content equally. So leaving captions off by default violates the Robust principle of POUR, as content should be accessible for all users and assistive technologies without needing extra steps to enable them.

The "ALL LOCALS" section organizes content based on region in a clear predictable way with simple geographical labels. This supports the Understandable principle of POUR, as users can predict what content they can find without confusion. This may be beneficial for users with cognitive disabilities or those using screen readers, since the structure is logical and consistent.

The Quick Links section allows user direct, and clear labelled shortcuts to popular content, which reduces the number of steps needed to navigate the website. This also supports the Operable Principle of POUR, making navigation more efficient for users relying on keyboard navigation or assistive technologies.

The search function is placed at the top, and accessible without requiring a mouse as users can tab to it using just a keyboard. This supports the operable principle of POUR, requiring all functionality be available without mouse interactions for users with motor impairments

“Some interactive elements on the page appear visually similar, which may make navigation harder for users with cognitive or visual impairments. Greater visual distinction between sections and links could improve usability.

The consistent navigation structure helps users predict where information is located across the site. Consistency is an important accessibility practice because it reduces confusion and cognitive load.

The link text clearly explains its purpose instead of using vague phrases like ‘click here.’

The strong contrast between the text and background improves readability for users with visual impairments or colour blindness. This aligns with WCAG accessibility standards.

This page uses a clear heading hierarchy that helps users understand the organization of content.

La crítica al “vibe coding” y el uso de la inteligencia artificial para aprender es un tema importante. Algunas personas critican que las personas confien habilidades a la inteligencia artificial, especialmente en tareas como programar o escribir. Esta crítica es válida porque depender completamente de la inteligencia artificial puede impedir que las personas desarrollen una comprensión real sobre lo que están haciendo.

Sin embargo, el texto parece asumir que usar la inteligencia artificial necesariamente reemplaza el aprendizaje, cuando en realidad puede servir como un apoyo educativo. El problema no es la herramienta en sí, sino cómo se utiliza. Si se utiliza de manera pasiva, puede ser perjudicial. Pero si se utiliza de manera activa, puede ser muy útil.

Empresas como GitHub han reconocido que herramientas como Copilot pueden aumentar la productividad, pero también pueden llevar a aceptar errores o código inseguro si el usuario no tiene conocimientos previos. Esto demuestra que la inteligencia artificial no sustituye la necesidad de aprender, sino que hace que el pensamiento crítico sea aún más importante.

La fuente de esta información es el estudio de investigación de GitHub sobre Copilot. También se puede encontrar más información en un artículo público de GitHub sobre productividad y Copilot. https://github.blog/news-insights/research/research-quantifying-github-copilots-impact-on-developer-productivity-and-happiness/

En esta parte del texto se plantea que la escritura debilitó la memoria humana porque permitió almacenar información fuera de la mente. Sin embargo, me parece que esta postura reduce la memoria únicamente a la capacidad de retener datos. En realidad, las tecnologías han transformado históricamente la manera en que pensamos y organizamos el conocimiento. Un estudio de la revista Science mostró que las personas no necesariamente recuerdan menos por usar internet, sino que recuerdan más fácilmente dónde encontrar la información. Esto demuestra que la tecnología modifica nuestras estrategias cognitivas más de lo que simplemente “atrofia” capacidades. Además, sin herramientas externas como libros, archivos o bases de datos, gran parte del conocimiento científico contemporáneo sería imposible de sostener. Link del artículo: https://www.harvardmagazine.com/2011/10/how-the-web-affects-memory

The statement implies both toothbrush types are the same in use but does not address whether there is any real environmental or performance advantage.

The claim is vague and does not show how bamboo toothbrushes specifically improve cleaning compared to normal toothbrushes.

The article does not provide evidence to prove that bamboo toothbrushes clean teeth as effectively as plastic toothbrushes.

The claim may be misleading because bamboo toothbrushes still contain plastic components such as nylon bristles.

The statement may encourage consumers to see bamboo toothbrushes as a fully sustainable alternative without discussing their environmental limitations.

The article presents bamboo as sustainable, but it does not discuss transportation emissions or manufacturing processes that may also affect environmental impact.

Although marketed as fully biodegradable, the use of animal-based bristles may raise ethical concerns for vegan or environmentally conscious consumers.

This statement reveals a possible contradiction in the eco-friendly marketing claim because the environmental benefits depend heavily on proper disposal methods.

This claim may be misleading because the article does not provide scientific data or certification to prove the environmental impact comparison.

The claim may be misleading because the toothbrush bristles are still made from nylon and are not fully biodegradable.

This is crisp, clear, and I've observed it myself. Good to come back to

https://en.wikipedia.org/wiki/Daydream_(Wallace_Collection_song)

Users have the option to access information on this page in French, which is an important accessibility feature for Canada’s French-speaking population. This supports the perceivable principle because information is available in more than one language, allowing users to access content in the language they understand best. It also supports the operable principle, since the language-switching option is clearly visible on the homepage and can be accessed easily without requiring complex navigation.

The website uses photos to support short descriptions. This improves accessibility as it helps uses visually interpret content. The photos help replace larger descriptions of information through a visual feature. This supports the perceivable principle. This is also a good practice because it enhances clarity and improves the overall user engagement.

This is a weaker accessibility feature because advertisements and sidebar content can interrupt the reading flow. The article is about an emergency wildfire alert, so users may be trying to access important information quickly. Extra visual clutter can make the page harder to navigate, especially for users with attention difficulties or those using smaller screens.

This image caption is a good accessibility practice because it explains the meaning of the image instead of leaving users to interpret the visual on their own. Captions and text alternatives are important because users who cannot see the image still need access to the information it communicates. W3C accessibility guidance emphasizes text alternatives, captions, headings, and clear structure as key parts of accessible content.

The font-size controls support accessibility because they let users adjust the reading experience. This connects to Module 2’s point that not everyone can read small text easily. Giving users control over text size helps make the page more Perceivable, especially for people with low vision or eye strain.

This is a good accessibility feature because the video is supported by written text. This connects to the Perceivable principle because users who cannot hear the video, cannot watch it, or have limited bandwidth can still understand the key information. Module 2 emphasizes that videos should include captions or transcripts so information is not only available through audio or visuals.

This is a good example of the Understandable principle from Module 2. The headline is direct, specific, and tells readers the main issue right away. This matters because users should not have to search through the page to understand the purpose of the article, especially when the topic is an emergency alert.

Negative as it is a very broad statement therefore even with the clear language could still be a bit more specific to allow users to know why this is the best option.

The pictures are also positive as they show you exactly what you are booking and where u will be heading

Positive example wording is simple but still descriptive. With the clear language being used helps users understand the content without any confusion

Helpful as gives users clear advice instead of just giving location along with a link to also book the shuttles so u dont have to go looking even more

Positive accessibility feature because of clear headings separating different attractions you can visit. Helping users find what they are looking for quickly

Low Colour Contrast: The orange header, grey metadata text, and beige background create a colour palette with very weak contrast ratios. For users with low vision, colour‑contrast sensitivity, cataracts, or age‑related vision changes, these colours blend together, making it difficult to distinguish text from the background. Even users without diagnosed visual impairments may struggle to read the smaller grey text because it does not meet WCAG’s recommended contrast ratio of 4.5:1 for normal‑sized fonts. The lack of contrast also removes visual hierarchy meaning nothing stands out, so users cannot quickly identify navigation, headings, or interactive elements. This violates the Perceivable principle discussed in Module 2, which requires that information be presented in ways users can clearly perceive. The essential guideline to “distinguish the foreground from the background” is not met here, because the colour choices cause the interface to visually flatten, increasing effort and reducing readability for a wide range of users.

?

Positive example because of the clear categories which allows users to find what they need quickly.

focus

测度 是对大小的度量，F → R将事件输入映射到实数域数值 输入是事件A，输出为非负值，可以为正无穷

σ代数 需要对并集封闭，即任意个事件的并集要在集合F中，进一步引出全集Ω 也要作为F的元素之一 需要对补集封闭 空集也要为F的元素之一

需要封闭因为需要能进行可数无限次交并运算 不能是不可数无限是因为每一个事件的概率会直接为0，因为有不可数个

σ代数最终是为了能够做极限、积分等操作

并集封闭

补集封闭

Merci pour ce riche argumentaire !

Cette rubrique disparaîtra évidemment de l’appel. Dans le texte définitif, il faudra ajouter les informations habituelles contenues dans les appels de la revue (voir par exemple https://journals.openedition.org/revuehn/4267).

2015, n’est-ce pas ?

Les questions me semblent très pertinentes. Cependant, j’ai l’impression qu’elles pourraient être simplifiées : les précisions paraissent redondantes, tout en présentant des variations qui ne sont pas tout à fait compréhensibles. La première phrase de chaque question pourrait mieux faire ressortir le thème ; par exemple la question 6 porte sur la collaboration.

Répétition de « libre accès » ou deux idées différentes ?

L’édition est toujours numérique, c’est la publication qui peut avoir plusieurs supports, n’est-ce pas ?

Reformuler pour clarifier cette idée.

Je ne comprends pas ce dernier membre de phrase, trop allusif.

Cette phrase invite – à juste titre – à considérer les pratiques éditoriales en humanités numériques comme un domaine plus large que l’édition savante numérique. Serait-il pertinent de prendre en compte cet élargissement dans le titre complet et dans la portée de l’appel, pour l’ouvrir à d’éventuelles réflexions sur d’autres objets que l’édition de textes ?

1. In this webpage, it can be seen that some of the content is hard to perceive and isn't really that robust. There's simplicity in how the images are presented, but little accessibility is offered, and adaptability isn't ensured.

1. Many of these products that have a video explaining how it works or why you should buy it have closed captioning, although it's mostly AI-generated.

1. Many of the alt tag descriptions for these products exceed 125 characters. Also, if you were to click on one of these items, you would see that the normal descriptions are also extremely long.

1. In addition to the webpage complexity, scattered amongst this page are different rows for different genres of items without a clear contrast with the background.

1. There's a lot of webpage complexity here with a mix of colours that might cause cognitive overload or be difficult to distinguish for users who are colourblind.

apendice

The animals could probably sense that the storm was on its way.

Tea cake is acting like her ex husband’s. Just because he is jealous, does not mean he should be hitting her.

Ms. Tucker is racist to her own kind

Mrs turner is racist and love light skinned people. I wonder why when she is black herself.

Search bar used to easily find specific desired information. Improves efficiency of navigation and helpful to those using assistive technologies. The search bar is easily identifiable in the top right corner, can be enlarged with the feature on the website as well. Promotes perceivability and operability of the website.

Multiple links listed on the home page to easily access specific content. Descriptive link text can help the users understand the purpose of each link without having to browse through each sections full content which saves time and improves navigation efficiency.

Opposite color gradient used to enhance and define important text. This makes text more visually appealing as well as more clear and organized. Supports digital hygiene as the website is well organized and not heavily worded.

These are examples of assistive devices and shortcuts. Users are able to quickly skip to content as a shortcut to the content to promote efficiency. Not only, there is an option to change the text size, color, and translate to promote accessibility for all individuals needs. This supports the principle of operability and promotes robustness as the website considers diverse users and their specific needs.

The headings are clearly organized and describe the content under each one. This enables viewers of the website to easily navigate and find the desired content efficiently. The headings support the concept of perceivable and understandable.

eLife Assessment

This valuable study presents a tool that uses brain anatomy to predict the layout and size of early visual maps, and it is strengthened by the use of a large and diverse collection of scans to examine differences across people and groups. The evidence is solid for the general usefulness of the approach, but incomplete for some of the broader claims about prediction accuracy and use across data sets, particularly for estimates of map size and for showing that the model improves on repeated functional measurements. This paper is likely to be of significant interest to visual perception researchers, especially those who use fMRI.

Reviewer #1 (Public review):

Summary:

This paper describes a deep learning toolbox that can be used to automatically estimate functional topographic maps directly from human brain anatomy. Building on the first author's earlier work, which demonstrated the feasibility of using deep learning for this purpose, the new version of the toolbox now requires only a single anatomical MRI scan to generate predictions, eliminating the need for a myelin scan. This represents a significant practical improvement.

Strengths:

Having such a toolbox is very useful, since manual annotation and delineation of functional visual field maps is a laborious process that also requires deep expertise. The toolbox can save researchers substantial amounts of time and money, and also allows less experienced researchers to now perform this type of analysis. Notably, for certain participants and patients, the time they are able to reside in the scanner might be limited. Being able to focus on the primary research question, rather than the essential yet basic topographic information, could boost data quality and evaluation and might limit the number of participants that need to be included.

Weaknesses:

In the paper, the authors compare the performance of their new version to two previous approaches. Figure 2b shows that the new toolbox performs similarly to the previous deep-learning-based toolbox, but requires only an anatomical scan, which is a significant improvement. They also compare it to an older method that uses an atlas without requiring deep learning. For eccentricity and pRF size predictions, both deep-learning methods perform better than the older approach. For polar angle, a critical parameter for delineating visual field maps, the gain is substantially less. Moreover, the comparison to the atlas method (Benson2014) is not entirely fair, as, to our knowledge, there is also a more advanced atlas version that uses Bayesian fitting methods and already performs better than the old method. To better understand the gain of using deep learning, it would be beneficial if the authors also made the comparison to this more recent atlas-based approach. Moreover, it would be useful to know the correlations for the representative participant. Some examples of relatively "bad" maps would also be useful to have (and could be provided as supplementary information).

Figure 2b shows that the toolbox is quite good at estimating eccentricity and polar angle parameters, but less good at estimating the population receptive field (pRF) size. I will return to this latter point.

An interesting feature is that while the toolbox is trained on a specific data set (HCP), it can, "out-of-the-box", be applied to different existing data sets, without the need to retrain the model. This is quite important for the general utility of the method. The results for this are shown in Figure 3. Again, in panel b, it can be seen that the toolbox does a good job at estimating eccentricity and polar angle values, but performs rather poorly for pRF size: the deepRetinotopy toolbox has a strong tendency to only estimate very small pRFs, particularly when applying it across different datasets. For this reason, at the moment, these estimates appear hardly useful. It would be very helpful for readers if the authors could clarify or elaborate on this point, particularly regarding the limitations of pRF size predictions. They explain that this could be due to the use of different types of stimuli, but even within the same (HCP) dataset, the predictions primarily suggest tiny pRFs, even though the training dataset also contains larger ones (which can be better seen in supplementary Figure 4). Showing the predictions for higher-order brain areas, which have larger pRFs on average, could serve a similar evaluation purpose. Presumably, the underlying reasons are complex and could relate to the use of different stimuli, different analysis toolboxes, and how the deep learning model is currently being trained. Possibly, the abundance of small pRFs at lower eccentricity in the training set (which is usually the case in any empirical analysis) has given the model a very strong bias toward predicting small pRFs.

There would be various ways to verify which of these components is critical. For example, the model could be trained only on the bar stimuli of the HCP dataset, or the pRFs for all stimuli and datasets could be estimated using the same software tool. The latter seems important. For example, Supplementary Figure 4 indicates a high correlation between the Stanford and NYU cohorts that have used the same stimulus and analysis package, despite having different resolutions and scanners. Further investigation into the underlying reasons for these discrepancies would strengthen the paper. It would also provide valuable guidance for users of the toolbox on which toolbox predictions to trust and which not, as well as how well the model generalizes to other stimulus types, scanners, and image resolutions.

An aspect that is not directly apparent from the title, abstract, and introduction is that the deepRetinotopy toolbox does not by itself produce estimates of visual area labels or boundaries. It predicts only polar angle and eccentricity values. To predict labels and boundaries, the authors combine the toolbox with an atlas (the aforementioned Bayesian atlas). For visual areas V1 - V3, it does a very good job, in that the predictions are as good as the empirical ones. Notably, the authors indicate that the predictions for V2 and, in particular, V3 are worse than for V1, but Figure 4 clearly shows that predictions are as good as the empirical ones. More cannot be expected from a model that is trained on such empirical data.

Irrespective of the limitations with respect to predicting pRF size, the toolbox opens up functionally oriented analyses of very large cohorts of healthy participants, of which only anatomical data is available. The authors present an example of this by confirming the existence of differences in horizontal and vertical asymmetries in the field maps of the visual cortex of children and adults. While Figure 5 confirms the existence of differences, the analysis could be expanded to provide deeper insights, such as normalized developmental trajectories for both asymmetries, given the size of the dataset. This would better highlight the true power of their approach.

While the authors address limitations with respect to studying experience-dependent atypical functional organization, they do not address how the deepRetinotopy toolbox would handle (acquired) brain lesions. Addressing this, even if only speculative, would be welcome. Another welcome addition would be to see the predictions for additional brain areas, even if those would (presumably) be worse at present. Such information would nevertheless be essential for users considering applying this toolbox. Moreover, this could be a valuable resource serving as a benchmark for future iterations of either deepRetinotopy or other approaches.

Reviewer #2 (Public review):

Summary:

The authors introduce the deepRetinotopy toolbox, a deep learning-based software package that allows for user-friendly automatic delineation of visual areas based on anatomical (T1-weighted) MRI scans. This is an important evolution over a prior published version of the software, which required myelin maps additionally. The new version will hence allow many more users to obtain high-fidelity field-map delineations based on existing data or using standard protocols, providing a huge advance to the field. The authors exploited this strength and mapped visual field maps (for areas V1-V3) in 11060 human MRI scans covering different age classes to quantify changes of retinotopic organization across age groups, showing that previously functionally identified imbalances of early visual cortex field maps can now be identified on the basis of anatomical scans alone.

Strengths:

Overall, this is a tremendously important methodological contribution of primarily high practical and applied value. It allows functional imaging labs to delineate human cortical visual field maps with confirmed high fidelity using anatomical T1-weighted scans only. This will save expensive functional imaging and time-consuming analyses that were previously required to achieve nearly the same result and far better results than prior model-based approaches offered.

Also, the quantification of the accumulated very large dataset is meticulous and provides impressively detailed results of the field map changes for areas V1-V3 as a function of age.

Weaknesses:

(1) The weak point of the contribution is the choice to limit anatomical quality assessments and error quantifications to just three early regions, V1-V3, even though the deepRetinotopy toolbox can delineate over 20 regions (including parietal, ventral, and lateral regions, such as IPS0-5, hV4, VO1-2, V3A, PHC1-2, LO1-2, and TO1-2).

(2) The limit is fine for their large-scale application of the toolbox to age groups, as here, a clear hypothesis on early cortex variability was tested.

(3) However, the introduction of the toolbox itself warrants quality assessments and comparisons to prior models and ground truth beyond V1-V3, just like the authors did in their prior publication of the predecessor model.

(4) This is important as the vast majority of applications of this toolbox will likely go beyond V1-V3 to delineate dorsal, ventral, and lateral regions.

(5) For the present paper, this will require only 1 or 2 additional figures, or extending their present figures 2 and 4 along the lines of their previous figure 7 (Ribeiro et al 2021), which included error measures for high-level regions. Ideally, you provide sub-graphs separately for early visual, dorsal, ventral, and lateral regions.

(6) Going beyond V1-V3 is important for several reasons: first, future studies applying the software beyond V3 will need quantification for reassurance and justification. Second, for the sake of transparency, even if results are noisy or on par with prior models. Third, as a benchmark or reference point for future approaches.

Reviewer #3 (Public review):

Summary:

This valuable study presents a tool that uses brain anatomy to predict the layout and size of early visual maps, and it is strengthened by testing across a large and diverse collection of scans. The work will be useful for researchers who want to estimate likely visual map layout from standard anatomical scans and to relate anatomical differences to differences in visual organization across groups. The evidence is solid for the general usefulness of the approach, but incomplete for broader claims about prediction accuracy and use across datasets, particularly for estimates of map size and for showing that the model improves on repeated functional measurements.

Strengths:

The paper addresses a useful and important problem: estimating early visual map organization from anatomical measurements alone. Tools that predict these types of functional data from anatomical measurements were introduced more than a decade ago by Benson and colleagues, and the present authors have significantly extended that work. That is a real strength of the manuscript, because there is genuine value in having a practical tool that can estimate likely visual organization from standard anatomical scans.

Another major strength is the rigorous cross-dataset benchmarking and the accumulation of multiple datasets. The authors assembled a large and diverse set of scans and assessed model performance across different scanners, field strengths, and visual stimuli, which gives the reader a much better sense of how broadly the approach may apply. The retrospective analysis of more than 11,000 scans is especially notable and creates an unusual opportunity to ask how anatomical variation may relate to population differences in visual organization.

I also think the paper does a good job of showing why such a tool could matter in practice. A complete tool could be used in several ways. First, it could help users identify the locations of activations measured in other experiments with respect to the typical V1-V3 maps. Second, maps measured from an individual subject or patient could be compared with the predictions from the tool to ask whether they differ meaningfully from a standard anatomy-based map. Third, the tool can be used, as the authors have done here, to examine differences in anatomy across populations and interpret these differences with respect to retinotopic maps. Of these uses, the first already seems well supported by the current presentation.

Weaknesses:

(1) Quantification of the Analysis

My main concern is that the analysis relies heavily on global summary measures such as correlation and Dice score. Those measures are useful, but the paper would be more informative if it also quantified boundary differences in millimeters, especially for comparisons such as the V1/V2 boundary in Figure 2. That kind of analysis would help readers understand how large the errors are in physically meaningful terms.

(2) Model fitting methods

I also think the discussion of prediction failures for pRF size should be more explicit. The mismatch is likely influenced by the fact that the training data and several evaluation datasets were fit with different models and different analysis software. In particular, the network was trained on non-linear size estimates from the HCP data, while the comparison datasets were derived using other packages and, in some cases, different model assumptions. That likely contributes to the spread in Figure 3b and should be discussed more directly. It is important to discuss that the pRF parameters were derived using different software tools.

- HCP dataset (training data): analyzePRF (Compressive Spatial Summation model)

- NYU dataset: vistasoft

- Stanford dataset: vistasoft

- New Zealand dataset: SamSrf

- CHN dataset: Custom MATLAB software

(3) Clarifying Model Accuracy

If deepRetinotopy generates a true "noise-removed" representation of functional mapping based on anatomy, then fitting it to one fMRI measurement should predict a second, independent fMRI run better than the noisy data from the first run does.

The authors possess the exact data for this test. For the HCP dataset, the empirical fMRI data were explicitly separated into two halves: "fit 2" (the first half of the fMRI runs) and "fit 3" (the second half). They correlated these two halves to establish a "noise ceiling," the maximum possible reliability of the data. Looking at their results in Figure 2b, the correlation of the deepRetinotopy predictions falls below this noise ceiling. This means that the noisy functional Half 1 actually predicts functional Half 2 better than the anatomical model does.

The authors should state this explicitly. A side-by-side plot of Half 1 predicting Half 2 versus deepRetinotopy predicting Half 2 would show that the anatomical model regularizes map location well, but misses reliable subject-specific variation that anatomy alone cannot capture.

(4) The Hemodynamic Response Function

The assumptions used to generate the original empirical maps are permanently baked into the deep learning model. However, the authors explicitly mention the hemodynamic response function (HRF) only once, noting in the Methods that the modeled time series was "convolved with a canonical hemodynamic response function."

Beyond this single mention, there is no direct discussion of how the assumption of a single canonical HRF across all 161 HCP training subjects might have systematically impacted or biased the network's predictions. The authors address cross-dataset differences broadly under the umbrella of "experimental design" and "fMRI preprocessing pipeline" biases, but the HRF is a core biological property that mediates the connection between the anatomy and the data. The authors should explicitly discuss how this canonical assumption limits or biases the resulting deepRetinotopy network.

(5) Scoping the Input Data and Normative Use

The authors use FreeSurfer to generate a mean curvature map for the entire midthickness cortical surface. This full-hemisphere curvature map is resampled to a standard template surface space (32k_fs_LR), acting as the data frame that feeds input features into the neural network. However, while the network receives the full geometric structure of the hemisphere, it is explicitly trained to predict retinotopic parameters only within a restricted posterior ROI, based on the Wang et al. atlas and containing roughly 3,200 vertices per hemisphere.

A useful experiment to try, and perhaps the authors have already considered this, would be to restrict the input features exclusively to the posterior vertices. Including all anterior vertices may make it harder for the network to fit the localized visual data. A brief commentary on why the full hemisphere was retained as input could be highly informative for researchers adapting this geometric deep learning pipeline.

Author response:

Public Reviews:

Reviewer #1 (Public review):

In the paper, the authors compare the performance of their new version to two previous approaches. Figure 2b shows that the new toolbox performs similarly to the previous deep-learning-based toolbox, but requires only an anatomical scan, which is a significant improvement. They also compare it to an older method that uses an atlas without requiring deep learning. For eccentricity and pRF size predictions, both deep-learning methods perform better than the older approach. For polar angle, a critical parameter for delineating visual field maps, the gain is substantially less. Moreover, the comparison to the atlas method (Benson2014) is not entirely fair, as, to our knowledge, there is also a more advanced atlas version that uses Bayesian fitting methods and already performs better than the old method. To better understand the gain of using deep learning, it would be beneficial if the authors also made the comparison to this more recent atlas-based approach. Moreover, it would be useful to know the correlations for the representative participant. Some examples of relatively "bad" maps would also be useful to have (and could be provided as supplementary information).

We thank the reviewer for their constructive feedback. We plan to expand our benchmarking section to include the Bayesian model comparison. Note, however, that the additional accuracy gain afforded with the Bayesian model of retinotopy (Benson and Winawer, 2018) results from combining anatomical data with retinotopic maps estimated with a few minutes of functional data. The Bayesian model of retinotopy without such functional data is equivalent to Benson14. We plan to report the correlations (between predicted and empirical maps) for the representative participant shown in Figure 2 and include an additional supplementary figure showing retinotopic map predictions for a participant whose predictions deviate the most from empirical maps, as suggested by the reviewer.

Figure 2b shows that the toolbox is quite good at estimating eccentricity and polar angle parameters, but less good at estimating the population receptive field (pRF) size. I will return to this latter point.

An interesting feature is that while the toolbox is trained on a specific data set (HCP), it can, "out-of-the-box", be applied to different existing data sets, without the need to retrain the model. This is quite important for the general utility of the method. The results for this are shown in Figure 3. Again, in panel b, it can be seen that the toolbox does a good job at estimating eccentricity and polar angle values, but performs rather poorly for pRF size: the deepRetinotopy toolbox has a strong tendency to only estimate very small pRFs, particularly when applying it across different datasets. For this reason, at the moment, these estimates appear hardly useful. It would be very helpful for readers if the authors could clarify or elaborate on this point, particularly regarding the limitations of pRF size predictions. They explain that this could be due to the use of different types of stimuli, but even within the same (HCP) dataset, the predictions primarily suggest tiny pRFs, even though the training dataset also contains larger ones (which can be better seen in supplementary Figure 4). Showing the predictions for higher-order brain areas, which have larger pRFs on average, could serve a similar evaluation purpose. Presumably, the underlying reasons are complex and could relate to the use of different stimuli, different analysis toolboxes, and how the deep learning model is currently being trained. Possibly, the abundance of small pRFs at lower eccentricity in the training set (which is usually the case in any empirical analysis) has given the model a very strong bias toward predicting small pRFs.

There would be various ways to verify which of these components is critical. For example, the model could be trained only on the bar stimuli of the HCP dataset, or the pRFs for all stimuli and datasets could be estimated using the same software tool. The latter seems important. For example, Supplementary Figure 4 indicates a high correlation between the Stanford and NYU cohorts that have used the same stimulus and analysis package, despite having different resolutions and scanners. Further investigation into the underlying reasons for these discrepancies would strengthen the paper. It would also provide valuable guidance for users of the toolbox on which toolbox predictions to trust and which not, as well as how well the model generalizes to other stimulus types, scanners, and image resolutions.

We will expand our discussion of the limitations of pRF size prediction, highlighting that differences in visual stimuli, analysis toolboxes used to estimate pRF parameters from empirical data, and the current training of deepRetinotopy affect prediction accuracy. As the reviewer pointed out, the underlying reasons are complex, and it is difficult to isolate all the potential contributing factors. However, in addition to our expanded discussion, we also intend to present results from additional experiments that assess the impact of different loss functions on the range of predicted pRF sizes (to explain how training may partly account for the differences observed in the HCP dataset). We will also perform pRF fitting on at least one dataset using the same software/encoding model as in the HCP dataset (the training data) to illustrate that the lower performance in pRF size prediction in out-of-distribution datasets is also partly explained by differences in how the empirical maps were obtained.

An aspect that is not directly apparent from the title, abstract, and introduction is that the deepRetinotopy toolbox does not by itself produce estimates of visual area labels or boundaries. It predicts only polar angle and eccentricity values. To predict labels and boundaries, the authors combine the toolbox with an atlas (the aforementioned Bayesian atlas). For visual areas V1 - V3, it does a very good job, in that the predictions are as good as the empirical ones. Notably, the authors indicate that the predictions for V2 and, in particular, V3 are worse than for V1, but Figure 4 clearly shows that predictions are as good as the empirical ones. More cannot be expected from a model that is trained on such empirical data.

We will edit the introduction and abstract to make it clearer that the deepRetinotopy toolbox does not yet produce estimates of visual boundaries on its own.

Irrespective of the limitations with respect to predicting pRF size, the toolbox opens up functionally oriented analyses of very large cohorts of healthy participants, of which only anatomical data is available. The authors present an example of this by confirming the existence of differences in horizontal and vertical asymmetries in the field maps of the visual cortex of children and adults. While Figure 5 confirms the existence of differences, the analysis could be expanded to provide deeper insights, such as normalized developmental trajectories for both asymmetries, given the size of the dataset. This would better highlight the true power of their approach.

Although providing insights into developmental trajectories for horizontal and vertical asymmetries is beyond the scope of the current work, as it would require aggregating datasets such that individuals’ age span a larger range (ABCD dataset only contains individuals between 9-11 years old and the HCP Young Adult dataset between 22-36 years old), we plan to provide some complementary analyses (differences across ages and sex within the ABCD dataset).

While the authors address limitations with respect to studying experience-dependent atypical functional organization, they do not address how the deepRetinotopy toolbox would handle (acquired) brain lesions. Addressing this, even if only speculative, would be welcome. Another welcome addition would be to see the predictions for additional brain areas, even if those would (presumably) be worse at present. Such information would nevertheless be essential for users considering applying this toolbox. Moreover, this could be a valuable resource serving as a benchmark for future iterations of either deepRetinotopy or other approaches.

We plan to expand and report performance evaluation across other visual areas (using Wang atlas’ parcels) to serve as a benchmarking resource. Moreover, we will expand our discussion on how deepRetinotopy would handle brain lesions.

Reviewer #2 (Public review):

(1) The weak point of the contribution is the choice to limit anatomical quality assessments and error quantifications to just three early regions, V1-V3, even though the deepRetinotopy toolbox can delineate over 20 regions (including parietal, ventral, and lateral regions, such as IPS0-5, hV4, VO1-2, V3A, PHC1-2, LO1-2, and TO1-2).

(2) The limit is fine for their large-scale application of the toolbox to age groups, as here, a clear hypothesis on early cortex variability was tested.

(3) However, the introduction of the toolbox itself warrants quality assessments and comparisons to prior models and ground truth beyond V1-V3, just like the authors did in their prior publication of the predecessor model.

(4) This is important as the vast majority of applications of this toolbox will likely go beyond V1-V3 to delineate dorsal, ventral, and lateral regions.

(5) For the present paper, this will require only 1 or 2 additional figures, or extending their present figures 2 and 4 along the lines of their previous figure 7 (Ribeiro et al 2021), which included error measures for high-level regions. Ideally, you provide sub-graphs separately for early visual, dorsal, ventral, and lateral regions.

(6) Going beyond V1-V3 is important for several reasons: first, future studies applying the software beyond V3 will need quantification for reassurance and justification. Second, for the sake of transparency, even if results are noisy or on par with prior models. Third, as a benchmark or reference point for future approaches.

We thank the reviewer for their constructive feedback, and we agree that expanding our performance assessment beyond V1-3 would be a valuable benchmarking resource. Thus, we plan to evaluate retinotopic map prediction accuracy across visual areas defined by the Wang atlas’ parcels, expanding on the results reported in Figure 2, and provide it as a supplementary figure. However, performance estimation ultimately depends on the quality of the dataset used for evaluation. The empirical maps, although treated as ground truth, may themselves misrepresent the underlying retinotopic organization. As a matter of fact, the quality of the empirical data (HCP dataset and others) is indeed lowest in some of the higher-order visual areas.

It may be unclear from the text that the deepRetinotopy toolbox does not yet produce estimates of visual boundaries on its own. Accordingly, we illustrate how deepRetinotopy toolbox’s predictions can be combined with another tool [the Ba yesian model of retinotopy from Benson and Winawer (2018)] to obtain visual area boundaries automatically. We will edit the introduction and abstract to make it clearer. Given the availability of empirical labels (currently only for V1-3) and the segmentation tool (which was only assessed for V1-3), we cannot expand Figure 4 to other visual areas as suggested.

Reviewer #3 (Public review):

Quantification of the Analysis: My main concern is that the analysis relies heavily on global summary measures such as correlation and Dice score. Those measures are useful, but the paper would be more informative if it also quantified boundary differences in millimeters, especially for comparisons such as the V1/V2 boundary in Figure 2. That kind of analysis would help readers understand how large the errors are in physically meaningful terms.

We thank the reviewer for their constructive feedback. Following the reviewer’s suggestion, we plan to expand our segmentation evaluation to quantify the extent to which boundary predictions from deepRetinotopy’s maps deviate from those from empirical maps, in millimetres.

Model fitting methods: I also think the discussion of prediction failures for pRF size should be more explicit. The mismatch is likely influenced by the fact that the training data and several evaluation datasets were fit with different models and different analysis software. In particular, the network was trained on non-linear size estimates from the HCP data, while the comparison datasets were derived using other packages and, in some cases, different model assumptions. That likely contributes to the spread in Figure 3b and should be discussed more directly. It is important to discuss that the pRF parameters were derived using different software tools.

We will expand our discussion of the limitations of pRF size prediction, highlighting that differences in visual stimuli, different encoding models for estimating pRF parameters from empirical data, and the current training of deepRetinotopy affect prediction accuracy. In addition to our expanded discussion, we intend to also present results from additional experiments that assess the impact of those factors on pRF size prediction performance.

Clarifying Model Accuracy: If deepRetinotopy generates a true "noise-removed" representation of functional mapping based on anatomy, then fitting it to one fMRI measurement should predict a second, independent fMRI run better than the noisy data from the first run does.

The authors possess the exact data for this test. For the HCP dataset, the empirical fMRI data were explicitly separated into two halves: "fit 2" (the first half of the fMRI runs) and "fit 3" (the second half). They correlated these two halves to establish a "noise ceiling," the maximum possible reliability of the data. Looking at their results in Figure 2b, the correlation of the deepRetinotopy predictions falls below this noise ceiling. This means that the noisy functional Half 1 actually predicts functional Half 2 better than the anatomical model does.

The authors should state this explicitly. A side-by-side plot of Half 1 predicting Half 2 versus deepRetinotopy predicting Half 2 would show that the anatomical model regularizes map location well, but misses reliable subject-specific variation that anatomy alone cannot capture.

We will expand our benchmarking session to make these comparisons (“Half 1 predicting Half 2 versus deepRetinotopy predicting Half 2”) more explicit. It is important to highlight that there is more subject-specific variation that is currently not captured by our model, and it can also serve as a benchmarking resource for future model versions and newer approaches.

The Hemodynamic Response Function: The assumptions used to generate the original empirical maps are permanently baked into the deep learning model. However, the authors explicitly mention the hemodynamic response function (HRF) only once, noting in the Methods that the modeled time series was "convolved with a canonical hemodynamic response function."

Beyond this single mention, there is no direct discussion of how the assumption of a single canonical HRF across all 161 HCP training subjects might have systematically impacted or biased the network's predictions. The authors address cross-dataset differences broadly under the umbrella of "experimental design" and "fMRI preprocessing pipeline" biases, but the HRF is a core biological property that mediates the connection between the anatomy and the data. The authors should explicitly discuss how this canonical assumption limits or biases the resulting deepRetinotopy network.

As Reviewers 3 and 1 have noted, the observed limitations in pRF size prediction stem from multiple underlying factors. One of those factors is indeed the HRF assumed in the encoding models. We will expand our discussion about factors that may introduce biases into deepRetinotopy predictions, including the HRF.

Scoping the Input Data and Normative Use: The authors use FreeSurfer to generate a mean curvature map for the entire midthickness cortical surface. This full-hemisphere curvature map is resampled to a standard template surface space (32k_fs_LR), acting as the data frame that feeds input features into the neural network. However, while the network receives the full geometric structure of the hemisphere, it is explicitly trained to predict retinotopic parameters only within a restricted posterior ROI, based on the Wang et al. atlas and containing roughly 3,200 vertices per hemisphere.

A useful experiment to try, and perhaps the authors have already considered this, would be to restrict the input features exclusively to the posterior vertices. Including all anterior vertices may make it harder for the network to fit the localized visual data. A brief commentary on why the full hemisphere was retained as input could be highly informative for researchers adapting this geometric deep learning pipeline.

Thanks for this suggestion. We have not performed a systematic evaluation of using ROIs that span a larger portion of the cortex (including the full hemisphere). It is a great idea to do so and report it in our manuscript to inform other researchers interested in adapting our pipeline. We intend to also update our toolbox by retraining our models to take all posterior vertices as suggested, which would improve the coverage of current predictions.

The website appears to be optimized for both desktop and mobile devices, which reflects the “Robust” principle of accessibility by ensuring content remains functional and adaptable across different technologies and screen sizes.

This webpage demonstrates the “Understandable” principle because the headings, buttons, and layout are simple and predictable, helping users quickly understand how to interact with the site.

The website relies heavily on large visual images to promote products. While this creates an appealing design, it may create accessibility challenges if images do not include descriptive alt text for screen reader users.

The navigation menu is organized clearly and consistently, which supports the “Operable” principle by helping users move through the website more easily and efficiently.

This is a good example of the POUR principle “Perceivable” because the website uses clear colour contrast and readable text that makes the content easier to view for users with visual impairments.

too fluffy

Interesting decision to end a paragraph on a quote. Not analyzing a quote usually leaves an ambiguous result. Why include a quote at the end of the paragraph here?

Utilizing differnt forms of primary source research within the same paragraph seeks to convince the reader from multiple perspectives.

Classic 3-2-1-2-3 sentence structure in essays

Not overanalyzing, you are simply stating what you do know and what you don't know. Being honest about the extant of the research.

adds humanization? Idk nice writerly move but i don't think im trying to humanize a lot in my own essay

Good hook

Congratulations and welcome to the club! Definitely the machine of a serious writer or novelist. These were the workhorses of newspapers and magazines through the 70s and 80s. In my mind, it's the last truly great manual typewriter ever manufactured.

Well known users of the Olympia SG3 included: Ingeborg Bachmann, Jimmy Breslin, Paddy Chayefsky, Philip K. Dick, Harlan Ellison, Michael Ende, Howard Fast, Jim Lehrer, Elmore Leonard, William E. Leuchtenburg, Terrence McNally, James Michener, Dudley Randall, and Wallace Stegner

Robert Redford used one in the movie ALL THE PRESIDENT'S MEN.

If you need a manual: https://site.xavier.edu/polt/typewriters/tw-manuals.html

Ribbon is still easily found: https://site.xavier.edu/polt/typewriters/tw-faq.html#q1

The Olympia SG3 uses 1/2" wide (12.7mm) typewriter ribbon, which has been standardized as DIN2103, in combination with the Group 1 spool, designated as DIN 32755. (Doesn't need eyelets.)

Other useful resources available at: https://boffosocko.com/research/typewriter-collection/

reply to u/Prudent_Highway_1855 at https://www.reddit.com/r/typewriters/comments/1tdy2eu/my_first_typewriter/

eLife Assessment

This is an important and rigorous study that addresses the question of what determines the spatial organization of endocytic zones at synapses. The authors use compelling approaches, in both Drosophila and rodent model systems, to define the role of activity and active zone structure on the organization of the peri-active zone. While the findings are primarily negative, they are carefully executed and contribute to the field by refining existing models of presynaptic organization.

Reviewer #1 (Public review):

Summary:

In this manuscript, Emperador-Melero et al. seek to determine whether recruitment of endocytic machinery to the periactive zone is activity-dependent or tethered to delivery of active zone machinery. They use genetic knockouts and pharmacological block in two model synapses - cultured mouse hippocampal neurons and Drosophila neuromuscular junctions - to determine how well endocytic machinery localizes after chronic inhibition or acute depolarization by super-resolution imaging. They find acute depolarization in both models have minimal to no effect on the localization of endocytic machinery at the periactive zone, suggesting that these proteins are constitutively maintained rather than upregulated in response to evoked activity. Interestingly, chronic inhibition slightly increases endocytic machinery levels, implying a potential homeostatic upregulation in preparation for rebound depolarization. Using genetic knockouts, the authors show that localization of endocytic machinery to periactive zones occurs independently of proper active zone assembly, even in the absence of upstream organizers like Liprin-α.

Overall, they propose that the constitutive deployment of endocytic machinery reflects its critical role in facilitating rapid and reliable membrane internalization during synaptic functions beyond classical endocytosis, such as regulation of the exocytic fusion pore and dense-core vesicle fusion. Although many experiments reveal limited changes in the localization or abundance of endocytic machinery, the findings are thorough, and data substantially supports a model in which endocytic components are organized through a pathway distinct from that of the active zone. This work advances our understanding of synaptic dynamics by supporting a model in which endocytic machinery is constitutively recruited and regulated by distinct upstream organizers compared to active zone proteins. It also highlights the utility of super-resolution imaging across diverse synapse types to uncover functionally conserved elements of synaptic biology.

Strengths:

The study's technical strengths, particularly the use of super-resolution microscopy and rigorous image analyses developed by the group, bolster their findings.

Weaknesses:

One limitation, acknowledged by the authors, is the persistence of spontaneous activity at these synapses, which could still impact the organization of these regions.

Comments on revisions:

The authors have addressed all of my previous comments.

Reviewer #2 (Public review):

Summary:

This study examines whether the localization of endocytic proteins to presynaptic periactive zones depends on synaptic activity or active zone scaffolds. Using genetic and pharmacological perturbations in both Drosophila and mouse neurons, the authors show that key endocytic proteins remain localized to periactive zones even when evoked release or active zone architecture is disrupted. While the findings are largely negative, the study is methodologically solid and provides useful constraints for current models of synaptic vesicle recycling.

Strengths:

The experimental design is careful and systematic, spanning both fly and mammalian systems. The use of advanced genetic models, including Liprin-α quadruple knockout mice, is a notable strength. High-resolution imaging approaches (STED, Airyscan) are appropriately applied to assess nanoscale organization. The study clarifies that strict activity dependence of endocytic recruitment may not be a general principle.

Weaknesses (largely addressed in revision):

Several initial concerns have been satisfactorily addressed in the revised manuscript. In particular, the inclusion of EndoA/Dap160 experiments and the expanded discussion improve the work. Some limitations remain, including the reliance on Tetanus toxin at the Drosophila NMJ, which does not fully abolish presynaptic fusion, and the still limited insight into the mechanistic basis of periactive zone organization. The biological interpretation of small changes in protein levels upon silencing also remains somewhat unclear.

Comments on revisions:

I thank the authors for the careful revision of the manuscript. The additional experiments, in particular the inclusion of EndoA and Dap160 at the Drosophila NMJ, as well as the extended discussion of limitations, are appreciated and address important points raised in the first round.

While the principal conclusions of the study remain unchanged, and the manuscript is still largely based on negative results, I find that the authors now present these data in a more balanced and transparent manner. The discussion of activity-dependence is improved and more nuanced, especially with regard to possible contributions of spontaneous release and homeostatic effects.

In my opinion, despite the mostly negative nature of the findings, the work provides a valuable and relevant contribution, as it defines important constraints on current models of periactive zone organization. The study is technically strong, carefully executed, and systematically performed across different model systems.

Overall, the revised manuscript is clearly improved and represents a solid and well-executed piece of work that will be of interest to the field.

Reviewer #3 (Public review):

Summary:

This study examines how synaptic endocytic zones are positioned using a combination of cultured neurons and the Drosophila neuromuscular junction. The authors test whether neuronal activity, active zone assembly, or liprin-α function is required to localize endocytic zone markers, including Dynamin, Amphiphysin, Nervous Wreck, PIPK1γ, and AP-180. None of the manipulations tested caused a coordinated disruption in the localization or abundance of these markers, leading to the conclusion that endocytic zones form independently of synaptic activity and active zone scaffolds.

Strengths:

The work is systematic and carefully executed, using multiple manipulations and two complementary model systems. The authors consistently examine multiple molecular markers, strengthening the interpretation that endocytic zone positioning is robust to changes in activity and structural assembly.

Weaknesses:

The main limitation is that the study does not test whether the methods used are sensitive enough to detect subtle functional disruption, and no condition tested produces clear disorganization of the endocytic zone. As a result, the conclusion that these zones assemble independently is supported by negative data, without a strong positive control for disassembly or mislocalization.

This paper addresses a longstanding question in synaptic biology and provides a well-supported boundary on the types of mechanisms that are likely to govern endocytic zone localization. The conclusions are well justified by the data, though additional evidence would be needed to define the assembly mechanism itself.

Comments on revisions:

The authors responded to the initial review with care. They both revised the manuscript and conducted new experiments to address each reviewer's concern. The responses to the review were effective, and I think that the revised manuscript provides significant new insights. In my view, it does not require additional revisions.

Author response:

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

We thank the reviewers for their careful consideration of our work and constructive comments. We are glad that reviewers appreciated the rigor and value of our work. In response to the reviewer comments we have made the following changes:

(1) Addition of new experiments on EndoA localization at the Drosophila NMJ (Fig. 2).

(2) Addition of new experiments on Dap160 localization at the Drosophila NMJ (Fig. 2).

(3) Addition of new experiments to validate Dynamin, Dap160 and EndoA antibodies (Fig. 2 – figure supplement 1).

(4) Assessment of the activity-dependence of EndoA and Dap160 localization at the Drosophila NMJ (Fig. 3).

(5) Assessment of the liprin-dependence of EndoA and Dap160 localization at the Drosophila NMJ (Fig. 8).

(6) Addition of a limitations section to the discussion to directly address that spontaneous release was not fully ablated in our studies and might contribute to recruitment.

(7) Addition of an outlook to the same section on what experimental avenues could address the limitations in the future.

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this manuscript, Emperador-Melero et al. seek to determine whether recruitment of endocytic machinery to the periactive zone is activity-dependent or tethered to delivery of active zone machinery. They use genetic knockouts and pharmacological block in two model synapses - cultured mouse hippocampal neurons and Drosophila neuromuscular junctions - to determine how well endocytic machinery localizes after chronic inhibition or acute depolarization by super-resolution imaging. They find that acute depolarization in both models has minimal to no effect on the localization of endocytic machinery at the periactive zone, suggesting that these proteins are constitutively maintained rather than upregulated in response to transient activity. Interestingly, chronic inhibition slightly increases endocytic machinery levels, implying a potential homeostatic upregulation in preparation for rebound depolarization. Using genetic knockouts, the authors show that localization of endocytic machinery to periactive zones occurs independently of proper active zone assembly, even in the absence of upstream organizers like Liprin-α. Overall, they propose that the constitutive deployment of endocytic machinery reflects its critical role in facilitating rapid and reliable membrane internalization during synaptic functions beyond classical endocytosis, such as regulation of the exocytic fusion pore and dense-core vesicle fusion. Although many experiments reveal limited changes in the localization or abundance of endocytic machinery, the findings are thorough, and data substantially support a model in which endocytic components are organized through a pathway distinct from that of the active zone. This work advances our understanding of synaptic dynamics by supporting a model in which endocytic machinery is constitutively recruited and regulated by distinct upstream organizers compared to active zone proteins. It also highlights the utility of super-resolution imaging across diverse synapse types to uncover functionally conserved elements of synaptic biology.

We thank the reviewer for the positive assessment of our study.

Strengths:

The study's technical strengths, particularly the use of super-resolution microscopy and rigorous image analyses developed by the group, bolster their findings.

We thank the reviewer for highlighting the technical strength of our work.

Weaknesses:

One notable limitation, however, is the absence of interrogation of endocytic proteins previously suggested to be recruited in an activity-dependent manner, in particular, endophilin.

We thank the reviewer for the suggestion. We have added experiments to assess the localization of two more proteins at Drosophila NMJs. These proteins are EndoA and Dap160, both of which have been reported to traffic between the synaptic vesicle cloud and the plasma membrane in response to stimulation [1-3]. In line with these studies, we observed that EndoA and Dap160 partially co-localize with a synaptic vesicle marker and with a periactive zone marker, indicating localization to both compartments (Fig. 2). However, neither high frequency stimulation nor expression of TeNT changed the levels or the distribution of these two proteins at the periactive zone (Fig. 3). Similarly, the deployment of these proteins at the periactive zone at the Drospophila NMJ was not dependent on the active zone scaffold Liprin-α (Fig. 8). Our data indicate that deployment of EndoA and Dap160 to the periactive zone does not require evoked synaptic activity.

We believe that there are multiple plausible explanations for our findings compared to previous work on Endophilin, which we discuss on lines 407-410: “Increased synaptic enrichment was also observed for Endophilin at nematode NMJs in mutants with disrupted exocytosis (Bai et al., 2010). We do not see such large shifts in Endophilin following similar manipulations, which might reflect distinct synaptic architectures in the C. elegans dorsal cord versus Drosophila NMJ terminals.” Further, this study finds that a plasma membrane-tethered Endophilin strongly colocalizes with endocytic machinery and largely rescues function. This suggests that the plasma membrane is the primary functional compartment for Endophilin. Together with our work, we conclude that these data suggest that Endophilin constitutively, but not completely, localizes to the periactive zone.

Reviewer #2 (Public review):

Summary:

This study examines whether the localization of endocytic proteins to presynaptic periactive zones depends on synaptic activity or active zone scaffolds. Using a combination of genetic and pharmacological perturbations in Drosophila and mouse neurons, the authors show that proteins such as Dynamin, Amphiphysin, AP-180, and others are still recruited to periactive zones even when evoked release or active zone architecture is disrupted. While the results are mostly negative, the study is methodologically solid and contributes to a more nuanced understanding of synaptic vesicle recycling machinery.

We thank the reviewer for deeming our work solid and for highlighting its importance for the field.

Strengths:

(1) The experimental design is careful and systematic, covering both fly and mammalian systems.

(2) The use of advanced genetic models (e.g., Liprin-α quadruple knockout mice) is a notable strength.

(3) High-resolution imaging (STED, Airyscan) is well used to assess spatial localization.

(4) The findings clarify that certain core assumptions - such as strict activity dependence of endocytic recruitment - may not hold universally.

We thank the reviewer for pointing out these strengths.

Weaknesses:

(1) The study would benefit from a clearer positive control to demonstrate activity-dependent recruitment (e.g., Endophilin).

We have added experiments to measure the localization of Endophilin, a protein previously reported to localize to the synaptic vesicle cloud [1], in Drosophila NMJs (Figs. 2 and 3). We observed that EndoA localized both to the synaptic vesicle cloud and to the periactive zone area. While stimulation did not enhance levels in either compartment, this outcome is not inconsistent with shuttling of protein between compartments during activity. Nevertheless, our data support a model in which EndoA, like the other tested endocytic proteins, is present at the periactive zone at rest.

(2) The reliance on Tetanus toxin in the Drosophila NMJ experiments in my eyes is a limitation, as it does not block all presynaptic fusion events; this should be discussed more directly.

We agree with the point of the reviewer. To more directly discuss it, we have included a “Limitations and Outlook” section in the revised version. We state that “conclusions that can be drawn on the roles of spontaneous release in periactive zone assembly remain limited” (lines 514-515). We further state that, while the manipulations that we included result in decreased spontaneous release, “it is possible that the remaining spontaneous release supports periactive zone assembly” (518-519) and that “Future studies might test manipulations with strong effects on miniature release including those affecting SNARE proteins and their regulators, with the caveat that these manipulations might have effects on upstream trafficking and in some cases on cell survival (Kaeser and Regehr, 2014; Santos et al., 2017).” (519-523).

(3) The potential role of Dynamin in organizing other periactive zone proteins is not addressed and could be an important next step.

We agree with the reviewer that this is an interesting possibility. On lines 454-455, we make the broad point that “interactions between endocytic proteins may further contribute to the anchoring of this apparatus”, and on lines 459-460, we specifically suggest a role for Dynamin by stating that “perturbing interactions between Dynamin-1 and Endophilin-A1 increases the distance between these proteins (Imoto et al., 2024), suggesting their binding has a scaffolding function.”

(4) Some small changes in protein levels upon silencing are reported; their biological meaning (e.g., compensation vs. variability) is not fully clarified.

These changes might include homeostatic adaptations. In the revised version of the manuscript, this is addressed on lines 135-137 and 405-407. We think it is overall difficult to assign biological meaning to small-magnitude changes, and chose to highlight the main point that there are no large-magnitude changes.

(5) While alternative organizing mechanisms (actin, lipids, adhesion molecules) are mentioned, a more forward-looking discussion of how to test these models would be helpful.

Following the reviewer’s suggestion, we have added an outlook section to the discussion where we provide suggestions for future studies (lines 510-543).

(6) The authors should consider including, or at least discussing, a well-established activity-dependent endocytic protein (e.g., Endophilin) as a positive control to help contextualize the negative findings.

We have included new experiments on EndoA at the fly neuromuscular junction (Fig. 2, Fig. 3, Fig. 8, Fig. 3 – figure supplement 1) and have added appropriate discussion of these findings as outlined above.

Reviewer #3 (Public review):

Summary:

This study examines how synaptic endocytic zones are positioned using a combination of cultured neurons and the Drosophila neuromuscular junction. The authors test whether neuronal activity, active zone assembly, or liprin-α function is required to localize endocytic zone markers, including Dynamin, Amphiphysin, Nervous Wreck, PIPK1γ, and AP-180. None of the manipulations tested caused a coordinated disruption in the localization or abundance of these markers, leading to the conclusion that endocytic zones form independently of synaptic activity and active zone scaffolds.

We thank the reviewer for reviewing our work.

Strengths:

The work is systematic and carefully executed, using multiple manipulations and two complementary model systems. The authors consistently examine multiple molecular markers, strengthening the interpretation that endocytic zone positioning is robust to changes in activity and structural assembly.

We thank the reviewer for pointing out these strengths.

Weaknesses:

The main limitation is that the study does not test whether the methods used are sensitive enough to detect subtle functional disruption, and no condition tested produces clear disorganization of the endocytic zone. As a result, the conclusion that these zones assemble independently is supported by negative data, without a strong positive control for disassembly or mislocalization.

We are confident that our methods are sensitive enough to detect changes within synaptic compartments. First, for mouse neurons assessed with STED microscopy, we have demonstrated that we can distinguish between the N- and the C-termini of the presynaptic protein Bassoon, which are positioned only a few tens of nanometers apart [4]. We have subsequently been consistently able to resolve the localization of pre- and postsynaptic proteins that also localize a few tens of nanometers apart and have established that genetic manipulations of active zone proteins induce detectable disruptions as assessed by STED microscopy [4-12]. Given that the periactive zone is larger than the distances that we can resolve, we are confident that we can detect changes in this area with enough sensitivity. Second, for Drosophila NMJs, we use a carefully validated workflow that allows assessing the distribution of periactive zone proteins and can detect subtle changes [13]. Unfortunately, there are no known manipulations that lead to periactive zone disassembly that could serve as a positive control, which reflects the little knowledge available in this field. We acknowledge that there may be subtle changes in protein localization that escape the resolution of our microscopy methods or experimental design, but this would not undermine the conclusion that the periactive zone remains assembled across the manipulations that we have tested. Overall, none of the manipulations we test induces a detectable disruption of the periactive zone. Naturally, we cannot exclude milder effects and have added a limitations section to discuss this possibility and some of the subtle changes we observe.

This paper addresses a longstanding question in synaptic biology and provides a well-supported boundary on the types of mechanisms that are likely to govern endocytic zone localization. The conclusions are well justified by the data, though additional evidence would be needed to define the assembly mechanism itself.

We thank the reviewer for the support of the conclusion of our study.

Recommendations for the authors:

Reviewing Editor Comments:

This is a rigorous study that, while presenting largely negative data, delimitates the processes that control peri-active zone organization. In addition to the interpretive and technical comments below, we encourage the authors to consider extending this study in two areas. First, examining the activity-dependence of Endophilin, and perhaps other factors, being recruited to the PAZ, where previous research has indicated a positive role for activity. Second, further characterization of the role of miniature release events in potentially contributing to PAZ organization. Overall, this was a rigorous and well-executed study.

We thank the reviewing editor for this positive assessment of our work.

Reviewer #1 (Recommendations for the authors):

(1) The rationale for comparing chronic inhibition to acute depolarization could be more clearly articulated. While this approach may be grounded in prior studies, the physiological consequences of chronic silencing differ markedly from those of transient activity, and these distinctions should be more explicitly addressed in the interpretation of results. For example, might lower intensity, chronic stimulation be a better comparison? Since fixation takes place immediately after stimulation, the time window to capture changes in protein recruitment may be curtailed.

We thank the reviewer for this comment. The introduction of the manuscript now includes a rationale on lines 110-112. By inhibiting evoked synaptic vesicle fusion throughout the lifespan of neurons, we assessed whether this process is necessary for periactive zone assembly and concluded that it is not a requirement. By acutely depolarizing neurons with 50 mM KCl or with a 40 Hz train of action potentials, we were able to test whether synaptic vesicle fusion triggers the rapid recruitment of endocytic proteins to the periactive zone and concluded that this is not the case for most of the endocytic proteins that we studied. While these results indicate that a constitutive pathway must exist to assemble the periactive zone, we remain agnostic as to whether stimulation paradigms not tested in our study can enhance the deployment of endocytic proteins, especially over long periods of time. This may be the case for low, chronic stimulation, as suggested by the reviewer. We clarify these limitations on a “limitations and outlook” section of the discussion (lines 510-543).

(2) Amphiphysin stood out as the only protein showing a notable change in opposite directions under either active zone protein knockout/blockers and Liprin-α knockout. Given the predominance of negative results, it would be valuable to devote more discussion to why Amphiphysin behaves differently. What functional role might it play in this context that sets it apart from other endocytic components?

As suggested by the reviewer, we have extended the discussion on Amphiphysin. One possibility why Amphiphysin may respond differently to different genetic manipulations or changes in stimulation is that different endocytic proteins might belong to different endocytic submachineries. This is addressed on lines 421-424. On lines 444-449, we further discuss the subtle decrease in the levels of Amphiphysin and AP-180 in Liprin-α mutants. We suggest that the actin cytoskeleton may be the link between the active zone and the endocytic apparatus, and that this link may be partially disrupted in Liprin-α mutants. Overall, we note that Amphiphysin is still localized to the periactive zone at rest, and hence that it fits with the overall model of constitutive deployment that we propose.

(3) The claim of activity-independence may need to be nuanced. Although the data suggest no recruitment in response to acute stimulation, the subtle changes following chronic inhibition complicate this interpretation, especially when considering redundancy. If activity-dependence is considered bidirectional, these findings might reflect a more complex regulatory mechanism. The interpretation in lines 188-190 more accurately captures this complexity than earlier generalizations.

We agree with the reviewer that the dependence on activity should be discussed in a nuanced fashion. We have scrutinized the manuscript on this point and state throughout that recruitment is independent of evoked activity and not necessarily of any kind of activity. We believe that this interpretation is accurate because evoked release of neurotransmitter was ablated by the pharmacological and genetic manipulations that we used. Furthermore, we have included a “Limitations of the study” section in the discussion where we openly address that spontaneous fusion of synaptic vesicles cannot be ruled out as a potential mechanism to sustain periactive zone assembly (lines 514-523). Finally, we have expanded on the complexity of periactive zone assembly relative to activity. In particular, homeostasis may contribute to increased levels of endocytic proteins upon chronic blockade of evoked transmission (lines 404-406).

(4) Given published work on endophilin's role in activity-dependent endocytic recruitment, adding endophilin (at least in the Drosophila NMJ experiments) would be highly informative.

We thank the reviewer for the suggestion. We have added experiments to assess the localization of two more proteins at Drosophila NMJs. These proteins are EndoA and Dap160, both of which have been reported to traffic between the synaptic vesicle cloud and the plasma membrane in response to stimulation [1-3]. In line with these studies, we observed that EndoA and Dap160 partially co-localize with a synaptic vesicle marker and with a periactive zone marker, indicating localization to both compartments (Fig. 2). However, neither high frequency stimulation nor expression of TeNT changed the levels or the distribution of these two proteins at the periactive zone (Fig. 3). Similarly, the deployment of these proteins at the periactive zone at the Drosophila NMJ was not dependent on the active zone scaffold Liprin-α (Fig. 8). Our data indicate that deployment of EndoA and Dap160 to the periactive zone does not require evoked synaptic activity.

We believe that there are multiple plausible explanations for these findings compared to previous work on Endophilin [3], which we discuss on lines 407-410:

“Increased synaptic enrichment was also observed for Endophilin at nematode NMJs in mutants with disrupted exocytosis (Bai et al.,2010). We do not see such large shifts in Endophilin following similar manipulations, which might reflect distinct synaptic architectures in the C. elegans dorsal cord vs Drosophila NMJ terminals.” Further, this study finds that a plasma membrane-tethered Endophilin strongly colocalizes with endocytic machinery and largely rescues function. This suggests that the plasma membrane is the primary functional compartment for Endophilin. Together, all data are compatible with a model in which Endophilin constitutively, but not completely, localizes to the periactive zone.

(5) Line 57 might have a typo in the citation.

We thank the reviewer for pointing this out. The citations now include: Bai et al., 2010; Jiang et al., 2024; Koh et al., 2007; Winther et al., 2013 and Winther et al. 2015. Please note that these two last citations are grouped as Winther et al. 2013, 2015 following our formatting style.

(6) Line 208 might be missing a citation that justifies parameters.

In the revision, this information is discussed on lines 222-224, where we cite our prior work describing these data: “Each unit is divided into ‘mesh’ and ‘core’ regions, where the periactive zone mesh is a ~175 nm wide area localized at ~330 nm from the center, and the ‘core’ region is the interior to this mesh (Del Signore et al., 2023)”.

Reviewer #2 (Recommendations for the authors):

(1) Please consider including, or at least discussing, a well-established activity-dependent endocytic protein (e.g., Endophilin) as a positive control to help contextualize the negative findings.

We thank the reviewer for the suggestion. We have added experiments to assess the localization of two more proteins at Drosophila NMJs. These proteins are EndoA and Dap160, both of which have been reported to traffic between the synaptic vesicle cloud and the plasma membrane in response to stimulation [1-3]. In line with these studies, we observed that EndoA and Dap160 partially co-localize with a synaptic vesicle marker and with a periactive zone marker, indicating localization to both compartments (Fig. 2). However, neither high frequency stimulation nor expression of TeNT changed the levels or the distribution of these two proteins at the periactive zone (Fig. 3). Similarly, the deployment of these proteins at the periactive zone at the Drosophila NMJ was not dependent on the active zone scaffold Liprin-α (Fig. 8). Our data indicate that deployment of EndoA and Dap160 to the periactive zone does not require evoked synaptic activity.

We believe that there are multiple plausible explanations for our findings compared to previous work on Endophilin [3], which we discuss on lines 407-410: “Increased synaptic enrichment was also observed for Endophilin at nematode NMJs in mutants with disrupted exocytosis (Bai et al.,2010). We do not see such large shifts in Endophilin following similar manipulations, which might reflect distinct synaptic architectures in the C. elegans dorsal cord vs Drosophila NMJ terminals.” Further, this study finds that a plasma membrane-tethered Endophilin strongly colocalizes with endocytic machinery and largely rescues function. This suggests that the plasma membrane is the primary functional compartment for Endophilin. Together, all data are consistent with a model in which Endophilin constitutively, but not completely, localizes to the periactive zone.

(2) Expand the discussion of TeNT's limitations-specifically that it does not block spontaneous fusion or alternative fusion pathways-and consider referencing more stringent tools (e.g., Botulinum toxins or SNARE mutants), even if they weren't used here.

Following the reviewer’s suggestion, we have included a “Limitations and Outlook” section in the revised version. We state that “conclusions that can be drawn on the roles of spontaneous release in periactive zone assembly remain limited” (lines 514-515). We further state that, while the manipulations that we included result in decreased spontaneous release, “it is possible that the remaining spontaneous release supports periactive zone assembly” (518-519) and that “Future studies might test manipulations with strong effects on miniature release including those affecting SNARE proteins and their regulators, with the caveat that these manipulations might have effects on upstream trafficking and in some cases on cell survival (Kaeser and Regehr, 2014; Santos et al., 2017)” (520-523).

(3) We encourage the authors to briefly discuss whether Dynamin might contribute to periactive zone structure beyond its role in membrane fission. Loss-of-function data could be particularly informative in future work.

We agree with the reviewer that this is an interesting possibility. On lines 454-455, we make the broad point that “interactions between endocytic proteins may further contribute to the anchoring of this apparatus”, and on lines 459-460, we specifically suggest a role for Dynamin by stating that “perturbing interactions between Dynamin-1 and Endophilin-A1 increases the distance between these proteins (Imoto et al., 2024), suggesting their binding has a scaffolding function.”

(4) Clarify the interpretation of increased endocytic protein levels upon chronic silencing - are these interpreted as homeostatic responses or experimental variability?

We suggest that these changes might include homeostatic adaptations. We note that this increase is of the same magnitude as the increase in active zone proteins following a similar pharmacological manipulation on lines 405-406, where we state that “a mechanism for this effect might be a homeostatic response (Wen and Turrigiano, 2024) similar in magnitude to the increase in active zone protein levels following activity blockade (Held et al., 2020).”

(5) The Discussion could be strengthened by sketching out more concrete experimental approaches to test candidate mechanisms (e.g., roles for actin, lipids, adhesion molecules) in organizing periactive zones.

The potential roles of the cell adhesion molecules (lines 430-440), cytoskeleton and lipids (442-452) are addressed in the discussion. Furthermore, following the reviewer’s suggestion, we have added the following statement (lines 541-543): “This work builds a foundation to assess alternative mechanisms and models of periactive zone assembly, including roles of the cytoskeleton, lipids, adhesion molecules, and intrinsic endocytic protein interactions”. We hope that the reviewer agrees that the discussion of our paper is not the right format to provide a concrete experimental plan for future work. In our view, the discussion should put the findings of our experiments in the context of the field.

Reviewer #3 (Recommendations for the authors):

(1) At a spine synapse, the endocytic zone is estimated to be between 100-200nm from the active zone. The focus of the author's analysis is largely outside of this region (0-150nm), raising the question of whether the area studied may be outside of the area affected by the manipulations made. While STED systems claim ~80 nm resolution, this is rarely achieved in practice, and the authors do not report the effective resolution of their system. Reporting the resolution achieved would address this issue. In addition, super-resolution imaging does not appear to have been used at the Drosophila NMJ. The authors should clarify whether resolution limitations influenced the choice of analysis region and whether their imaging approach is sufficient to detect changes in the endocytic zone.

We believe that it is unlikely that the relevant signals were missed. First, in mouse synapses, most signal corresponding to endocytic proteins was detected inside the selected region of interest. Our rationale to select the area was based on the fact that expanding the region analyzed would have reduced the sensitivity of our approach, as averaging over a larger area would dilute the signal. The resolution of our microscopy should not be a limitation either. In our previous work, we demonstrated that STED microscopy allows discriminating between the N- and the C-terminal termini of the presynaptic scaffold Bassoon, which are positioned only a few tens of nanometers apart [4]. This establishes that we can resolve differences at tens of nanometers in biological context, which is more relevant than the resolution measured with fluorescent beads (which we have repeatedly assessed to be ~80 nm laterally). Subsequently, we have also been consistently able to resolve the localization of pre- and postsynaptic proteins that also localize a few tens of nanometers apart [4-12]. Given that the periactive zone spans over a larger area than the distances that we can resolve experimentally in the examples above, we are confident that our measurements are sensitive enough to detect changes in this area.

Second, for Drosophila NMJs, the choice for the region of interest and the overall analysis was done following a workflow validated in our previous work [13]. This method analyzes both immediately adjacent and more distant regions from the active zone, and does not exclude any region based on distance from the active zone as described on lines 222-224: “Each unit is divided into ‘mesh’ and ‘core’ regions, where the periactive zone mesh is a ~175 nm wide area localized at ~330 nm from the center, and the ‘core’ region is the interior to this mesh (Del Signore et al., 2023).” In our previous study, we analyzed the distribution of periactive zone proteins at rest with STED microscopy and with Airyscan confocal microscopy. The resolution provided by Airyscan is reported to be ~175 nm in XY and ~400 nm in Z, which is sufficient to assess localization to the periactive zone compartment imaging methods and is not inferior to imaging methods previously used to report changes in the distribution of endocytic proteins; for examples, see [1,2]. In the revised manuscript, we have added new data measuring the levels and distribution of EndoA and Dap160 using STED microscopy (Figure 3 – figure supplement 1). The results acquired with STED microscopy and with Airyscan confocal microscopy are consistent with one another.

Overall, the accuracy of the imaging methods and analyses used in this study are sufficient to assess periactive zone structure given its size and organization.

(2) Interestingly, in a number of cases, the authors observe significant differences in endocytic markers (Figure 1q, 4k, 6k, 6r). However, little is made of these differences. The authors should provide more discussion of these changes and how they make sense of them alongside their claims of a lack of effect from their manipulations.

The reviewer raises a good point. We interpret these changes in two different ways. First, we suggest that changes observed in response to block of action potentials or disassembly of the active zone might be homeostatic. This is addressed on lines 135-137. Second, we discuss that the actin cytoskeleton may be the link between the active zone and the endocytic apparatus. Several active zone proteins interact with the actin cytoskeleton. One of them is Liprin-α. This interaction may explain the decrease in the level of Amphiphysin and AP-180 at the periactive zone in Liprin-α null neurons. This is addressed on lines 444-449. We hope that the reviewer agrees that overall, we should focus on the main conclusion that deployment of endocytic proteins persists over a number of manipulations and synapse types.

(3) The graphs in Figure 1c and 1g, 3g, 4c, 4e, 6c, and 6g do not appear to be identical. If the solid line represents the mean and the lighter color represents the distribution of these data, these data appear to be different from one another. It is surprising that these differences are not significant. What statistical tests were used to determine whether the differences in these graphs are not significant? Is the issue that a relatively now number of synapses were examined (30-60)? Did the authors conduct a power analysis?

We apologize if the display of our data and analyses was not clear. We do not perform statistical analyses on the line profiles. Instead, we perform it on two values that are extracted from line profiles. These values are (1) the distance between the peak intensity values of the protein of interest and the marker and (2) the peak intensity values. For example, in Figure 1, distances are quantified and statistically analyzed in panel j, and the peak levels are quantified and statistically analyzed in panel k. We have clarified this in the legend of current Figures 1, 4, 5, and 7.

(4) The authors clearly state that their experiments address the role of evoked activity in endocytic zone positioning, but they do not examine whether spontaneous vesicle fusion might play a role. Given the availability of Drosophila mutants that decrease (Doc2, Dunc-13) or increase (syt1) spontaneous release, this is a notable omission. Ideally, these mutants should be examined. And at a minimum, the authors should discuss whether spontaneous release could contribute to endocytic zone organization.

We agree with the reviewer that spontaneous fusion of synaptic vesicles may contribute to periactive zone organization. Many of the genetic manipulations that we used in mouse neurons result in a significant decrease in spontaneous release. This includes Ca_V2 triple knockouts with a ~60% decrease in spontaneous fusion [10], RIM+ELKS quadruple knockouts with a ~70% decrease in spontaneous fusion [9] and Liprin-α quadruple knockouts with a ~50% decrease in spontaneous fusion [7]. We cannot rule out that the spontaneous release that is left is sufficient to mediate assembly functions. The conclusive way to address this possibility is using a manipulation that ablates spontaneous release without altering other pathways. However, to our knowledge, this is not available. The manipulations suggested by the reviewer might suffer from similar limitations, as they would change the frequency of spontaneous release without fully ablating it, and they would also affect evoked release. We have included a limitations section in the discussion where we address this (lines 514-523), specifically stating “conclusions that can be drawn on the roles of spontaneous release in periactive zone assembly remain limited. While many of the manipulations used here, including Ca_V2 knockout (Held et al., 2020), RIM+ELKS knockout (Tan et al., 2022; Wang et al., 2016) and Liprin-α knockout (Emperador-Melero et al., 2024) in hippocampal neurons, and TeNT expression in fly NMJs (Sweeney et al.,1995) , result in 50% to 70% decreased spontaneous release rates, it is possible that the remaining spontaneous release supports periactive zone assembly. Future studies might test manipulations with strong effects on miniature release including those affecting SNARE proteins and their regulators, with the caveat that these manipulations might have effects on upstream trafficking and in some cases on cell survival (Kaeser and Regehr, 2014; Santos et al., 2017).” We hope that the reviewer agrees that assessing these mutants should be a topic of future studies, given that we already test many mutants in the paper.

(5) In Figures 1 and 6, the authors assess presynaptic protein localization in cultured neurons, but it is unclear whether these are synaptic sites. Many presynaptic proteins traffic together and can accumulate at sites lacking postsynaptic specializations. The authors should validate that the observed spatial organization occurs at bona fide synapses, ideally by co-labeling with postsynaptic markers as done in Figure 4. If methods like these were used, providing more details on how synapses were identified and selected would be useful to the reader.

While we understand the reviewer’s point, we are confident that the structures analyzed are bona fide synapses for three reasons, as we have established before across many papers [4-8,10-12,17].

The diameter of the structures detected using the synaptic vesicle marker Synaptophysin aligns much more closely with the size of the large vesicle clusters found at presynaptic terminals than with that of a few transport vesicles.

In side-view synapses, the bar-like distribution of the active zone marker (Bassoon or Munc13-1) at one edge of the vesicle cloud indicates that active zone proteins are organized at one edge of the vesicle cluster—consistent with the architecture of synapses.

Synaptophysin is one of our key markers for detecting synapses. In our cultures, most of the Synaptophysin signal colocalizes with postsynaptic markers (either PSD-95 or Gephyrin), as we have established across many studies [4,7-12]. This indicates that the markers used here are sufficient to select synapses. Furthermore, the frequency at which synapses were identified using an active zone marker as the second marker was similar to that observed when using a postsynaptic marker, suggesting that we were not randomly including unrelated structures.

(6) Many of the images, particularly of the Drosophila NMJ, are of low quality and are shown in very small images. In addition, the quality of the images throughout the paper makes it difficult to assess the author's analysis and results. The authors should provide larger, higher-quality images that show examples of the means for each of the examples shown. This is an issue for most of the figures, but is particularly prominent in the dNMJ. A minor additional point is that the authors should be clear whether the dNMJ images are collected at super-resolution or using a conventional microscope.

We believe that the quality of our images is sufficient for the assessments made for the following reasons:

These images were acquired with enough spatial resolution to assess levels at the PAZ as discussed in response to this reviewer’s first comment. In our previous work, we used images acquired at the same resolution and presented in the same manner for both mouse hippocampal synapses [6,7] and Drosophila NMJs [13,18]. In those previous studies, we drew conclusions at a similar level of detail as in the current study.

In our view, our representative images are not inferior in quality to other papers in the field addressing similar questions [1,2,19,20].

We have selected sample images based on the quantified mean values per condition. Hence, we strived to select panels that are objectively representative regarding the quantified parameters.

We have specified microscopy methods in the figure legends. Specifically, for Drosophila NMJs, we used Airyscan confocal microscopy and STED microscopy. For each experiment, it is now stated which microscopy method was used in the corresponding legend.

References:

(1) Winther, Å. M. E. et al. An Endocytic Scaffolding Protein together with Synapsin Regulates Synaptic Vesicle Clustering in the Drosophila Neuromuscular Junction. J Neurosci 35, 14756–14770 (2015).

(2) Winther, Å. M. E. et al. The dynamin-binding domains of Dap160/intersectin affect bulk membrane retrieval in synapses. J Cell Sci 126, 1021–1031 (2013).

(3) Bai, J., Hu, Z., Dittman, J. S., Pym, E. C. G. & Kaplan, J. M. Endophilin functions as a membrane-bending molecule and is delivered to endocytic zones by exocytosis. Cell 143, 430–441 (2010).

(4) Wong, M. Y. et al. Liprin-alpha3 controls vesicle docking and exocytosis at the active zone of hippocampal synapses. Proc Natl Acad Sci U S A 115, 2234–2239 (2018).

(5) Emperador-Melero, J., de Nola, G. & Kaeser, P. S. Intact synapse structure and function after combined knockout of PTPδ, PTPσ, and LAR. Elife 10, (2021).

(6) Emperador-Melero, J. et al. PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure. Nat Commun 12, 3057 (2021).

(7) Emperador-Melero, J. et al. Distinct active zone protein machineries mediate Ca2+ channel clustering and vesicle priming at hippocampal synapses. Nature Neuroscience 2024 1–15 (2024) doi:10.1038/s41593-024-01720-5.

(8) Tan, C., Wang, S. S. H., de Nola, G. & Kaeser, P. S. Rebuilding essential active zone functions within a synapse. Neuron 110, 1498-1515.e8 (2022).

(9) Wang, S. S. H. et al. Fusion Competent Synaptic Vesicles Persist upon Active Zone Disruption and Loss of Vesicle Docking. Neuron 91, 777–791 (2016).

(10) Held, R. G. et al. Synapse and Active Zone Assembly in the Absence of Presynaptic Ca(2+) Channels and Ca(2+) Entry. Neuron 107, 667-683.e9 (2020).

(11) Chin, M. & Kaeser, P. S. The intracellular C-terminus confers compartment-specific targeting of voltage-gated calcium channels. Cell Rep 43, 114428 (2024).

(12) Nyitrai, H., Wang, S. S. H. & Kaeser, P. S. ELKS1 Captures Rab6-Marked Vesicular Cargo in Presynaptic Nerve Terminals. Cell Rep 31, 107712 (2020).

(13) Del Signore, S. J., Mitzner, M. G., Silveira, A. M., Fai, T. G. & Rodal, A. A. An approach for quantitative mapping of synaptic periactive zone architecture and organization. Mol Biol Cell 34, (2023).

(14) Sweeney, S. T., Broadie, K., Keane, J., Niemann, H. & O’Kane, C. J. Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14, 341–351 (1995).

(15) Kaeser, P. S. & Regehr, W. G. Molecular mechanisms for synchronous, asynchronous, and spontaneous neurotransmitter release. Annu Rev Physiol 76, 333–363 (2014).

(16) Santos, T. C., Wierda, K., Broeke, J. H., Toonen, R. F. & Verhage, M. Early Golgi Abnormalities and Neurodegeneration upon Loss of Presynaptic Proteins Munc18-1, Syntaxin-1, or SNAP-25. Journal of Neuroscience 37, 4525–4539 (2017).

(17) de Jong, A. P. H. et al. RIM C2B Domains Target Presynaptic Active Zone Functions to PIP2-Containing Membranes. Neuron 98, 335-349.e7 (2018).

(18) Del Signore, S. J. et al. An autoinhibitory clamp of actin assembly constrains and directs synaptic endocytosis. Elife 10, (2021).

(19) Imoto, Y. et al. Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis. EMBO Journal https://doi.org/10.1038/S44318-024-00145-X

(20) Imoto, Y. et al. Dynamin is primed at endocytic sites for ultrafast endocytosis. Neuron 110, 2815-2835.e13 (2022).

eLife Assessment

This potentially useful manuscript addresses the 3D chromatin architecture in monocytes from a few patients with alcohol-associated hepatitis and its relationship to enhanced transcription of innate immune genes. While the concept and methodological approach are interesting in principle, the evidence is incomplete as a result of insufficient sample sizes as well as other substantive analytical concerns.

Reviewer #3 (Public review):

In this manuscript, the authors use HiC to study the 3D genome of CD14+ CD16+ monocytes from the blood of healthy and those from patients with Alcohol-associated Hepatitis.

Overall, the authors perform a cursory analysis of the HiC data and conclude that there are a large number of changes in 3D genome architecture between healthy and AH patient monocytes. They highlight some specific examples that are linked to changes in gene expression. The analysis is of such a preliminary nature that I would usually expect to see the data from all figures in just one or two figures.

In addition, I have a number of concerns regarding the experimental design and the depth of the analyses performed that I think must be addressed.

(1) There is a myriad of literature that describes the existence of cell-type-specific 3D genome architecture. In this manuscript, there is an assumption by the authors that the CD14+ CD16+ monocytes represent the same population from both the healthy and diseased patients. Therefore, the authors conclude that the differences they see in the HiC data are due to disease-related changes in the equivalent cell types. However, I am concerned that the AH patient monocytes may have differentiated due to their environment so that they are in fact akin to a different cell type and the 3D genome changes they describe reflect this. This is supported by published articles, for example: Dhanda et al., Intermediate Monocytes in Acute Alcoholic Hepatitis Are Functionally Activated and Induce IL-17 Expression in CD4+ T Cells. J Immunol (2019) 203 (12): 3190-3198, in which they show an increased frequency of CD14+ CD16+ intermediate monocytes in AH patients that are functionally distinct.

I suggest that if the authors would like to study the specific effects of AH on 3D genome architecture then they should carefully FACsort the equivalent monocyte populations from the healthy and AH patients.

(2) The analysis of the HiC data is quite preliminary. In the 3D genome field, it is usual to report the different scales of genome architecture, for example, compartments, topologically associated domains (TADs) and loops. I think that reporting this information and how it changes in AH patients in the appropriate cell types would be of great interest to the field.

Comments on revisions:

In the revision the authors did not respond to my concerns which I believe still remain valid and compromise the author's conclusions of AH-specific effects on genome architecture.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors investigate the relationship between 3D chromatin architecture and innate immune gene regulation in monocytes from patients with alcohol-associated hepatitis (AH). Using Hi-C technology, they attempt to identify structural changes in the genome that correlate with altered gene expression. Their central claim is that genome restructuring contributes to the hyper-inflammatory phenotype associated with AH.

Strengths:

(1) The manuscript employs Hi-C technology, which, in principle, is a powerful approach for studying genome organization.

(2) The focus on disease-relevant genes, particularly innate immune loci, provides a contextually important angle for understanding AH.

Weaknesses:

(1) Sample Size: The study relies on an exceptionally small cohort (4 AH patients and 4 healthy controls), rendering the results statistically underpowered and highly susceptible to variability.

(2) Hi-C Resolution unpaired to RNA seq: The data are presented at a resolution of 100kb, which is insufficient to uncover meaningful chromatin interactions at the level of individual genes. This data is unpaired.

(3) Functional Validation: The manuscript lacks experiments to directly link changes in chromatin architecture with gene expression or monocyte function, leaving the claims speculative.

(4) Data Integration: The lack of Hi-C with ATAC and RNA-seq data handicaps the analysis and really makes it superficial. In short, it does not convincingly demonstrate a functional relationship.

(5) Confounding Factors: The manuscript neglects critical confounding variables such as comorbidities, medications, and lifestyle factors, which could influence chromatin structure and gene expression independently of AH.

Appraisal of the Aims and Results:

The manuscript sets out to establish a connection between chromatin architecture and AH pathology. However, the study fails to achieve its stated aims due to inadequate methods and insufficient data. The conclusions drawn from the Hi-C analyses alone are poorly supported, and the lack of functional validation undermines the credibility of the proposed mechanisms. Overall, the results do not provide compelling evidence to substantiate the authors' claims.

Impact on the Field and Utility to the Community:

The work, in its current form, is unlikely to have a meaningful impact on the field. The limited scope, methodological shortcomings, and lack of robust data significantly diminish its potential utility. Without addressing these critical gaps, the study does not offer new insights into the role of genome architecture in AH or provide useful methodologies or datasets for the community.

Additional Context:

The manuscript would benefit from a more comprehensive analysis of potential mechanisms underlying the observed changes, including the interplay between chromatin architecture and epigenetic modifications. Furthermore, longitudinal studies or therapeutic interventions could provide insights into the dynamic aspects of genome restructuring in AH. These considerations are entirely absent from the current study.

Conclusion:

The manuscript does not achieve its stated goals and does not present sufficient evidence to support its conclusions. The limitations in sample size, resolution, and experimental rigor severely hinder its contribution to the field. Addressing these fundamental flaws will be essential for the work to be considered a meaningful addition to the literature.

Reviewer #2 (Public review):

Summary:

Dr. Adam Kim and collaborators study the changes in chromatin structure in monocytes obtained from alcohol-associated hepatitis (AH) when compared to healthy controls (HC). Through the usage of high throughput chromatin conformation capture technology (Hi-C), they collected data on contact frequencies between both contiguous and distal DNA windows (100 kB each); mainly within the same chromosome. From the analyses of those data in the two cohorts under analysis, authors describe frequent pairs of regions subject to significant changes in contact frequency across cohorts. Their accumulation onto specific regions of the genome -referred to as hotspots- motivated authors to narrow down their analyses to these disease-associated regions, in many of which, authors claim, a number of key innate immune genes can be found. Ultimately, the authors try to draw a link between the changes observed in chromatin architecture in some of these hotspots and the differential co-expression of the genes lying within those regions, as ascertained in previous single-cell transcriptomic analyses.

Strengths:

The main strength of this paper lies in the generation of Hi-C data from patients, a valuable asset that, as the authors emphasize, offers critical insights into the role of chromatin architecture dysregulation in the pathogenesis of alcohol-associated hepatitis (AH). If confirmed, the reported findings have the potential to highlight an important, yet overlooked, aspect of cellular dysregulation-chromatin conformation changes - not only in AH but potentially in other immune-related conditions with a component of pathological inflammation.

Weaknesses:

In what I regard as the two most important weaknesses of the work, I feel that they are more methodological than conceptual. The first of these issues concerns the perhaps insufficient level of description provided on the definition of some key types of genomic regions, such as topologically associated domains, DNA hotspots, or even DNA loci showing significant changes in contact frequency between AH and HC. In spite of the importance of these concepts in the paper, no operational, explicit description of how are they defined, from a statistical point of view, is provided in the current version of the manuscript.

Without these definitions, some of the claims that authors make in their work become hard to sustain. Some examples are the claim that randomizing samples does not lead to significant differences between cohorts; the claim that most of the changes in contact frequency happen locally; or the claim that most changes do not alter the structure of TADs, but appear either within, or between TADs. In my viewpoint, specific descriptions and implementation of proper tests to check these hypotheses and back up the mentioned specific claims, along with the inclusion of explicit results on these matters, would contribute very significantly to strengthening the overall message of the paper.

The second notable weakness of the study pertains to the characterization of the changes observed around immune genes in relation to genome-wide expectations. Although the authors suggest that certain hotspots contain a high number of immune-related genes, no enrichment analysis is provided to verify whether these regions indeed harbor a higher concentration of such genes compared to other genomic areas. It would be important for readers to be promptly informed if no such enrichment is observed, for in that case, the presence of some immune genes within these hotspots would carry more limited implications.

Additionally, the criteria used to define a hotspot are not clearly outlined, making it difficult to assess whether the changes in contact frequencies around the immune genes highlighted in figures 5-8 are truly more pronounced than what would be expected genome-wide.

Reviewer #3 (Public review):

In this manuscript, the authors use HiC to study the 3D genome of CD14+ CD16+ monocytes from the blood of healthy and those from patients with Alcohol-associated Hepatitis.

Overall, the authors perform a cursory analysis of the HiC data and conclude that there are a large number of changes in 3D genome architecture between healthy and AH patient monocytes. They highlight some specific examples that are linked to changes in gene expression. The analysis is of such a preliminary nature that I would usually expect to see the data from all figures in just one or two figures.

In addition, I have a number of concerns regarding the experimental design and the depth of the analyses performed that I think must be addressed.

(1) There is a myriad of literature that describes the existence of cell type-specific 3D genome architecture. In this manuscript, there is an assumption by the authors that the CD14+ CD16+ monocytes represent the same population from both healthy and diseased patients. Therefore, the authors conclude that the differences they see in the HiC data are due to disease-related changes in the equivalent cell types. However, I am concerned that the AH patient monocytes may have differentiated due to their environment so that they are in fact akin to a different cell type and the 3D genome changes they describe reflect this. This is supported by published articles for example: Dhanda et al., Intermediate Monocytes in Acute Alcoholic Hepatitis Are Functionally Activated and Induce IL-17 Expression in CD4+ T Cells. J Immunol (2019) 203 (12): 3190-3198, in which they show an increased frequency of CD14+ CD16+ intermediate monocytes in AH patients that are functionally distinct.

I suggest that if the authors would like to study the specific effects of AH on 3D genome architecture then they should carefully FACsort the equivalent monocyte populations from the healthy and AH patients.

(2) The analysis of the HiC data is quite preliminary. In the 3D genome field, it is usual to report the different scales of genome architecture, for example, compartments, topologically associated domains (TADs), and loops. I think that reporting this information and how it changes in AH patients in the appropriate cell types would be of great interest to the field.

We thank the reviewers for their careful and thorough examination of our manuscript. We agree with all of their comments regarding the limitations of the study. Many of the criticisms focus on the small sample size of our study (n=4 for healthy controls and disease patients) in both Hi-C and single-cell RNA-seq experiments, and that these experiments are unpaired, or in other words, PBMCs came from different patients for each experiment.

Unfortunately, these experiments are fairly complicated to perform, requiring patient cells and very expensive deep sequencing. We are not currently in a position to be able to easily or cost effectively increase sample size. In the case of Hi-C, we still believe our study to be of value as Hi-C is not a commonly used technique to study disease effects on chromatin, and very few studies have employed a large enough sample size to perform statistical comparisons. Additionally, to analyze the data at a higher resolution would require deeper sequencing, and unfortunately we do not have the resources to sequence these libraries deeper. Regarding the single-cell RNA-seq data, this dataset was generated for an earlier study [1] focusing on gene expression responses to LPS, and we were unable to get PBMCs from exactly the same patients to perform the Hi-C study.

We disagree that our study has limited scientific value. Our study is the first to use Hi-C to show that the 3D genome architecture of primary monocytes is changed in a disease context. The only other study to follow a similar approach performed Hi-C in monocytes from 2 healthy and 2 Systemic lupus erythematosus (SLE) patients, and in their study the data from both patients were combined prior to comparison. No statistics were performed and their conclusion was no differences in genome architecture due to disease. They did find differences between primary monocytes and the THP1 monocytic cell line, but this lacked statistical analysis. Their conclusion was that inflammatory disease may not lead to genome wide changes in architecture. Our study, though a very different disease than SLE, shows statistically significant differences between AH and healthy controls. We believe our study lays the groundwork for how Hi-C can be used to study genome architecture in human disease, and the possible downstream effects.

Confounding Factors: The manuscript neglects critical confounding variables such as comorbidities, medications, and lifestyle factors, which could influence chromatin structure and gene expression independently of AH.

This is an interesting suggestion. This dataset only contains 4 AH patients, which we have included basic clinical data in Supplemental Table 1, including Age, HCA1c, Bilirubin, AST, ALT, Creatinine, Albumin, and MELD score. 3/4 of these patients are severe AH while 1 is moderate (AH2). Despite one patient being moderate, all four AH patients had similar correlations with each other, suggesting these disease specific differences we observed are not indicative of severity. More patient samples are needed to determine if genome architecture changes throughout disease progression. We have added this important discussion to the manuscript (page 12, lines 5-14).

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

The criteria used to determine which pairs of regions exhibit significant differences in contact frequency between alcohol-associated hepatitis (AH) and healthy controls (HC) are not disclosed. It would be beneficial for the authors to provide this information, including details such as the number of pairs tested, the nature of the statistical tests conducted, the method of multiple testing correction applied, as well as the significance thresholds used, and the number of loci-pairs below these thresholds for each chromosome. This information would greatly enhance the reader's understanding of the relevance of the reported findings.

Thank you for this comment, though we are not sure we totally understand. All of our statistics were performed using multiHiCcompare [2], where we input all 8 datasets (.hic files from Juicer), then measured statistical differences between defined groups (HC vs AH). For our randomization studies, we randomized the group comparisons, so each group contained a mix of HC and AH.

Second, a formal statistical definition of what constitutes a hotspot would be valuable for clarity.

Thank you for this suggestion. Initially, hotspots were defined as just regions of the genome with a high frequency of very significant differential contacts. We have defined a more formal definition of “hotspot” based on similar criteria. A hotspot is defined by both adjusted p value and frequency of locations. First, we filtered all pair-wise chromosomal interactions by a very, very stringent padj < 0.0000001 to focus on only the most changed coordinates (Supplemental Table 4). Then we looked for regions of the genome with a high frequency of these differential locations. Borders for each hotspot were determined more liberally by looking at the full list of differential spots (padj < 0.05). Then we used code to list genes within each interacting region. We have added these important details to the Methods (page 14, lines 11-14).

Third, a clear definition of the criteria used to identify different topologically associated domains (if these were indeed defined in the data and/or utilized in the analyses) would also be a helpful addition.

Thank you for this suggestion, we did not identify TADs or really utilize TADs in any of these analyses.

Likewise, several statements throughout the paper lack support from specific analyses, although it should be feasible to implement such analyses (or at least present them if they have already been conducted) to substantiate these claims:

If randomizing samples does not result in significant differences between (randomized) cohorts, it would be beneficial to provide insights into the number of loci pairs that exhibit differences in frequency when using both the actual and randomized cohorts.

Thank you for asking this question, as this is an important point. Using multiHiCcompare, if we compare WT (n=4) to AH (n=4), we get the results in the figures and supplementary data but if we randomize Group 1 (WT, WT, AH, AH) vs Group 2 (WT, WT, AH, AH), we get almost 0 significant changes in contact frequency. To show this more robustly, we performed 5 randomized comparisons and found far fewer changes in contact frequency between groups. This shows that these changes in contact frequency caused by disease are not random, but rather due to our real difference in AH. This point has been added to the Results (page 6, lines 15-17), and Methods (page 14, lines 16-21)

If most changes in contact frequency occur locally, it would be useful to visualize the relationship between effect sizes and/or significance levels for the observed differences in frequency in relation to the distance between the involved loci. Additionally, comparing these results to the average baseline contact intensities as a function of distance would be informative. This comparison could help determine whether the distance decay in effect size/significance for the differences between AH and HC is faster or slower than the decay rates for baseline contact frequencies.

This is a good suggestion. In our initial analysis, we made a number of figures relating chromosome positions, distance between loci, and statistics regarding the differential contact frequency. In the initial submission, we only showed Figure 3, which shows the logFC (log fold change) for the differential contact frequency by chromosomal position on both sides. To address this question, we have added a supplemental figure showing logFC as a function of the distance between two loci (new Supplemental Figure 3)

Similarly, the assertion that most changes do not affect the structure of topologically associated domains (TADs) but occur either within or between TADs should be supported by specific testing; otherwise, or else, removed.

Thank you, yes we have adjusted the language in the Discussion

Furthermore, the authors should clarify whether differences in chromatin conformation are more pronounced around immune genes compared to genome-wide expectations. If this is not the case, it would be helpful to quantify the intensity of these differences around the highlighted genes in relation to the rest of the genome. To achieve this, I would suggest the following:

Conduct enrichment analyses on the genes located within the most prominent hotspots to determine whether they are significantly enriched in immune genes (and, or, alternatively, in any other functional category).

Estimate the average absolute fold change in contact frequency within all topologically associated domains (TADs) identified in the study. This would allow for the identification of immune gene-containing TADs highlighted in Figures 5-8, providing readers with a quantitative understanding of how anomalously different these genomic regions are with regards to the magnitude of its alterations in AH, compared to the rest of the genome.

While some of the selected gene clusters appear to co-localize well with topologically associated domains (e.g., Figures 5A, 8A), others seemingly encompass either multiple TADs (Figure 6) or only portions of them (Figure 7). This should be clarified.

Thank you, this is a great suggestion. In order to be as unbiased as possible, we took all genes present in the regions with the highest significant changes in genome (Supplemental Table 4) that we used to identify the hotspots. And you are correct, we do in fact see enrichment of genes involved in innate immune signaling. This has been added to Results (page 7, lines 19-25) and Figure 4.

Finally, there are several minor issues concerning the figures that could be easily addressed to substantially enhance their readability:

Font sizes in most figures should be increased, particularly for some axis labels and tick marks. This issue affects most figures; for instance, in Figure 4, it hinders the reader's ability to interpret the ranges of the data presented.

Thank you, the figures have been adjusted

Figures 5 to 8 (panels A and B) would benefit significantly from a more consistent format. Specifically, the gene cluster boxes should also be included in the right panels, and the gene locations should be displayed on the left in a uniform format across all figures (e.g., formatting Figures 7 and 8 to match the style of Figures 5 and 6).

Figures 5 and 6 have a similar structure to each other because we were focusing on all of the genes in that chromosomal region. Figures 7 and 8 are different because we are focusing on how the region around a certain hotspot of interest changes.

It is also important to note that the genes plotted in Figures 8C and 8D are not the same. Concerning these two panels, it would be valuable to clarify whether the data presented pertains exclusively to monocytes. If so, information regarding the number of cells analyzed and the number of donors from which they were drawn would also be beneficial.

These figures are generated using scRNA-seq data. They represent all of the genes expressed in that region of the genome, in their chromosomal position. If a gene is not expressed in the scRNA-seq data, then it is not shown. I have debated with myself a lot on how to show gene expression in a region of the genome, but I think this is the clearest way to show this; including the genes that have no expression would make it more confusing. But yes, if you compare HC and AH, you see some differences in the list of genes. We have added more clarity to the figure legend for this figure.

References

(1) Kim, A., Bellar, A., McMullen, M. R., Li, X. & Nagy, L. E. Functionally Diverse Inflammatory Responses in Peripheral and Liver Monocytes in Alcohol-Associated Hepatitis. Hepatol Commun 4, 1459-1476 (2020). https://doi.org:10.1002/hep4.1563

(2) Stansfield, J. C., Cresswell, K. G. & Dozmorov, M. G. multiHiCcompare: joint normalization and comparative analysis of complex Hi-C experiments. Bioinformatics 35, 2916-2923 (2019). https://doi.org:10.1093/bioinformatics/btz048

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eLife Assessment

This manuscript presents a valuable antiviral approach using an engineered ACE2-Fc fusion protein that demonstrates broad-spectrum neutralization capacity against SARS-CoV-2 variants and achieves significant prophylactic protection in animal models through a novel Fc-mediated phagocytosis mechanism. The study provides convincing evidence for protective efficacy through rigorous in vivo validation in mice, mechanistic characterization via transcriptomic analysis and biodistribution studies, and demonstration of antibody-dependent cellular phagocytosis as the primary clearance mechanism mediated by the decoy. The work will be of interest to researchers working in vaccine development and associated immune responses.

Reviewer #1 (Public review):

Summary:

This manuscript by Wang et al. describes the development of an optimized soluble ACE2-Fc fusion protein, B5-D3, for intranasal prophylaxis against SARS-CoV-2. As shown, B5-D3 conferred protection not only by acting as a neutralizing decoy, but also by redirecting virus-decoy complexes to phagocytic cells for lysosomal degradation. The authors showed complete in vivo protection in K18-hACE2 mice and investigated the underlying mechanism by a combination of Fc-mutant controls, transcriptomics, biodistribution studies, and in vitro assays.

Strengths:

The major strength of this work is the identification of a novel antiviral approach with broad-spectrum and beyond simple neutralization. Mutant ACE2 enables broad and potent binding activity with the S proteins of SARS-CoV-2 variants, while the fused Fc part mediates phagocytosis to clear the viral particles. The conceptual advance of this ACE2-Fc combination is convincingly validated by in vivo protection data and by the completely abrogated protection of Fc LALA mutant.

Additionally:

The authors include a discussion (in Discussion part) about a previously reported ACE2 decamer (DOI: 10.1080/22221751.2023.2275598) and compared with the ACE2-Fc fusion protein developed in this study. The authors also tested the off-target activity and showed no evidence of toxicity in vivo.

Reviewer #2 (Public review):

Summary:

Wang et al. engineered an ACE2 mutant by introducing two mutations (T92Q and H374N), and fused this ACE2 mutant to human IgG1-Fc (B5-D3). Experimental results suggest that B5-D3 exhibits broad-spectrum neutralization capacity and confers effective protection upon intranasal administration in SARS-CoV-2-infected K18-hACE2 mice. Transcriptomic analysis suggests that B5-D3 induces early immune activation in lung tissues of infected mice. Fluorescence-based bio-distribution assay further indicates rapid accumulation of B5-D3 in the respiratory tract, particularly in airway macrophages. Further investigation shows that B5-D3 promotes viral phagocytic clearance by macrophages via an Fc-mediated effector function, namely antibody-dependent cellular phagocytosis (ADCP), while simultaneously blocking ACE2-mediated viral infection in epithelial cells. These results provide some insights into improving decoy treatments against SARS-CoV-2 and other potential respiratory viruses.

Strengths:

The protective effect of this ACE2-Fc fusion protein against SARS-CoV-2 infection has been evaluated in a reasonable way.

Weaknesses:

(1) Some of the mice experiments suffer from insufficient sample numbers, which affect the statistical power and reliability of the results. The author acknowledged this weakness, noting that the supply of aged mice was limited, while arguing that, although the sample size is small, the data from these mice are consistent.

(2) Compared to 6 hours, intranasal administration of B5-D3 at 24 hours before viral infection results in reduced protective efficacy. However, only survival and body weight data are provided, with no supporting evidence from virological assays such as viral titer measurement. The author acknowledged that such data would be more comprehensive and attributed the limitation to constraints in animal services.

(3) The efficacy of the B5-D3-LALA group was not as good as that of the B5-D3 group. The author suggested that there might be a certain degree of viral variation, and viral infection in the lungs may be uneven in the B5-D3-LALA group.

Reviewer #3 (Public review):

Strengths:

The core strength of this study lies in its innovative demonstration that an engineered sACE2-Fc fusion redirects virus-decoy complexes to Fc-mediated phagocytosis and lysosomal clearance in macrophages, revealing a distinct antiviral mechanism beyond traditional neutralization. Its complete prophylactic protection in animal models and precise targeting of airway phagocytes establish a novel therapeutic paradigm against SARS-CoV-2 variants and future respiratory viruses.

Weaknesses:

The study attributes the complete antiviral protection to Fc-mediated phagocytic clearance, a central claim that requires more rigorous experimental validation. The observation that abrogating Fc functions compromises protection could be confounded by potential alterations in the protein's stability, half-life, or overall structure. To firmly establish this mechanism, it is crucial to include a control molecule with a mutated Fc region that lacks FcγR binding while preserving the Fc structure itself. Without this critical control, the conclusion that phagocytic clearance is the primary mechanism remains inadequately supported. The strategy of deliberately targeting virus-decoy complexes to phagocytes via Fc receptors inherently raises the question of Antibody-Dependent Enhancement (ADE) of disease. While the authors demonstrate a lack of productive infection in macrophages, this only addresses one facet of ADE. The risk of Fc-mediated exacerbation of inflammation (ADE) remains a critical concern. The manuscript would be significantly strengthened by a direct discussion of this risk and by including data, such as cytokine profiling from treated macrophages, to more comprehensively address the safety profile of this approach. The exclusive use of the K18-hACE2 mouse model, which exhibits severe disease, limits the generalizability of the findings. The "complete protection" observed may not translate to models with more robust and naturalistic immune responses or to human physiology. Furthermore, the lack of data against circulating SARS-CoV-2 variants of concern. The concept of sACE2-Fc fusion proteins as decoy receptors is not novel, and numerous similar constructs have been previously reported. The manuscript would benefit from a clearer demonstration of how the optimized B5-D3 mutant represents a significant advance over existing sACE2-Fc designs. A direct comparative analysis with previously published benchmarks, particularly in terms of neutralizing potency, Fc effector function strength, and in vivo efficacy, is necessary to establish the incremental value and novelty of this specific agent.

Comments on revised version:

The author has successfully addressed the raised issue.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript by Wang et al. describes the development of an optimized soluble ACE2-Fc fusion protein, B5-D3, for intranasal prophylaxis against SARS-CoV-2. As shown, B5-D3 conferred protection not only by acting as a neutralizing decoy, but also by redirecting virus-decoy complexes to phagocytic cells for lysosomal degradation. The authors showed complete in vivo protection in K18-hACE2 mice and investigated the underlying mechanism by a combination of Fc-mutant controls, transcriptomics, biodistribution studies, and in vitro assays.

Strengths:

The major strength of this work is the identification of a novel antiviral approach with broad-spectrum and beyond simple neutralization. Mutant ACE2 enables broad and potent binding activity with the S proteins of SARS-CoV-2 variants, while the fused Fc part mediates phagocytosis to clear the viral particles. The conceptual advance of this ACE2-Fc combination is convincingly validated by in vivo protection data and by the completely abrogated protection of Fc LALA mutant.

We thank the reviewer for his recognition and positive comments on our study.

Weaknesses:

Some aspects could be further modified.

(1) A previously reported ACE2 decamer (DOI: 10.1080/22221751.2023.2275598) needs to be mentioned and compared in the Discussion part.

We thank the reviewer for pointing out this weakness.

Indeed, previous studies reported that the ACE2-IgM decamer, taking advantage of the decameric structure of IgM, exhibited higher avidity to spikes and greater potency for viral neutralization [1-3]. In particular, the study by Guo et al. has demonstrated a broad-spectrum neutralization ability of the ACE2-IgM decamer against multiple SARS-CoV-2 variants and reported the efficacy of intranasal prophylaxis in preventing lethal SARS-CoV-2 challenge in K18-hACE2 mice.

We agree with the reviewer that it is promising that our B5-D3 design would benefit from switching to the IgM isotype. However, the distinct biological features imposed by IgM Fc, including short serum half-life and restricted tissue penetration [4], may complicate the study design and diverge our focus.

In our current study, we would focus on the IgG1 Fc-based decoy design, while inactivating the enzyme activity of ACE2 to avoid disturbing the renin angiotensin system. This design allowed us to compare diverse administration routes and regimens and to gain useful insights into the potential of sACE2-Fc decoy in combating SARS-CoV-2 in vivo.

We appreciated the reviewer‘s insightful suggestion. In the revised manuscript, we have included additional discussion regarding ACE2-IgM decamer, addressing the relevant concern on page 17 lines 409–414.

(2) Limitations of this study, such as off-target binding and potential immunogenicity, should also be discussed.

We thank the reviewer for his insightful comments and agree that off-target activity is a major concern for designing the ACE2 decoy.

(1) In our study, the representative sACE2-Fc decoy candidate B5-D3 contains H374N mutation (D3) that is designed to inactivate ACE2 enzyme activity by causing dyscoordination of Zn2+. Our in vitro enzymatic activity assay has demonstrated that the H374N mutation (D3), as well as other three single mutations D1, D4 and D5, in either WT sACE2-Fc or B5 mutant, could effectively abolish the hACE2 enzyme activity (Supplementary Fig. 2e, h).

(2) To further address the concern on off-target activity, we performed AAV-based overexpression experiments in K18-hACE2 mice and examined serum levels of RAS hormones, using ELISA methods that specifically detect serum renin, Angiotensin II (Ang II), and Ang (1-7). While our data from WT sACE2-Fc overexpression revealed significantly elevated serum renin and Ang II, indicating a disruption of the RAS (Supplementary Fig. 4d, e); the results from examined double mutants, including B5-D3, showed negligible change in any of these metabolite levels, demonstrating no off-target effect and minimal disturbance to the RAS activity in K18-hACE2 mice (Supplementary Fig. 4d–f).

(3) Moreover, in this experiment, after the prolonged overexpression of all these molecules in K18hACE2 mice, histological examination of multiple organs showed no evidence of immune cell infiltration and tissue damage and no difference was observed between the mice receiving WT sACE2-Fc or B5-D3(Supplementary Fig. 4g).

In the revised manuscript, we have included the results from the AAV-delivered in vivo overexpression of WT sACE2-Fc and three most promising double mutants (B5-D3, B5-D4 and B5-D5) on page 5 lines 118–122 and on page 6 lines 123–135 in the main text. The relevant data were presented in the new Supplementary Fig. 4.

Reviewer #2 (Public review):

Summary:

Wang et al. engineered an optimized ACE2 mutant by introducing two mutations (T92Q and H374N) and fused this ACE2 mutant to human IgG1-Fc (B5-D3). Experimental results suggest that B5-D3 exhibits broad-spectrum neutralization capacity and confers effective protection upon intranasal administration in SARS-CoV-2-infected K18-hACE2 mice. Transcriptomic analysis suggests that B5D3 induces early immune activation in lung tissues of infected mice. Fluorescence-based biodistribution assay further indicates rapid accumulation of B5-D3 in the respiratory tract, particularly in airway macrophages. Further investigation shows that B5-D3 promotes viral phagocytic clearance by macrophages via an Fc-mediated effector function, namely antibody-dependent cellular phagocytosis (ADCP), while simultaneously blocking ACE2-mediated viral infection in epithelial cells. These results provide insights into improving decoy treatments against SARS-CoV-2 and other potential respiratory viruses.

Strengths:

The protective effect of this ACE2-Fc fusion protein against SARS-CoV-2 infection has been evaluated in a quite comprehensive way.

We thank the reviewer for his recognition and positive comments on our study.

Weaknesses:

(1) The paper lacks an explanation regarding the reason for the combination of mutations listed in Supplementary Figure 2b. For example, for the mutations that enhance spike protein binding, B2-B6 does not fully align with the mutations listed in Table S1 of Reference 4, yet no specific criteria are provided.

We thank the reviewer for pointing out this negligence.

We constructed the B2-B6 mutants based on the study by Chan et al. [5] (Reference 4 in the previous version), mainly referencing to their Fig. 1A rather than to their Table S1. In Chan’s study, each of the proposed mutations were discovered as single mutations in monomeric sACE2 molecules based on the enrichment in target cell-binding. T92 was a notable hot spot for enriched mutations in their Fig. 1A.

Since monomeric and dimeric forms of sACE2 showed dramatically different kinetics for ACE2-RBD interaction, we selected five proposed mutations and further examined their affinity and activity in dimeric sACE2-Fc in our study. We chose not only the combinations of mutations, such as B3, B4, and B6 proposed in their Table S1, but also explored less-complicated mutation(s) like B2 (T27Y/L79T) and B5 (T92Q) in their Fig. 1A, which were in silico predicted to enhance ACE2-RBD binding but not tested in sACE2-Fc in Chan’s study.

Interestingly, although our results confirmed enhanced viral neutralization by all these mutations, the activity increase compared to WT ACE2-Fc was rather limited. Hence, we chose not to explore other mutations but to focus on B2–B6 to construct an enhanced ACE2-Fc decoy as a representative, to investigate the potential of ACE2-Fc decoys in combating SARS-CoV-2 infections.

In the revised manuscript, we have further amended the writing on page 4 lines 84–87 to enhance the readability. Whereas for conciseness of the manuscript, we did not describe in too much detail how we selected the mutations to be tested.

Second, for the mutations that abolished enzymatic activity, while D1 and D2, D3, D4, and D5 are cited from References 12, 11, and 33, respectively, the reason for combining D3 and D4 into A2, and D1 and D2 into A3 remains unexplained. It is also unclear whether some of these other possible combinations have been tested. Furthermore, for the B5-derived mutations, only double-mutant combinations with D1-D5 are tested, with no attempt made to evaluate triple mutations involving A2 or A3.

We thank the reviewer for pointing out this negligence.

A2 and A3 mutations were originally proposed as double mutations [6,7]. A2 (H374N/H378N) was first reported by Guy et al. [6] (Reference 11 in the previous version), while A3 (R273G/T445G) was originally proposed in Payandeh et al.’s study [7] (Reference 33 in the previous version).

In this study, we further split the two mutations in A2 and A3, to generate the single enzymedeactivating mutations, D1 and D2 from A3, and D3 and D4 from A2. Among these single mutations, D2 failed to inactivate ACE2 enzymatic activity (Supplementary Fig. 2e), and it was excluded in subsequent analyses.

D5 (H345L) was a single mutation directly adopted from the report by Glasgow et al. [8] (Reference 12 in the previous version).

After combining the B5 with the enzyme-deactivating mutations (A2, A3, D1, D3, D4, D5), our neuralization assay results showed that, the simpler compound mutants with only two mutations, like B5-D1, B5-D3, B5-D4 and B5-D5, exhibited stronger neutralization capacity than B5-A2 and B5-A3 with triple mutations. Moreover, since fewer mutations were more favorable to reduce risks in causing protein structure alteration and evoking host immunity, we then focused on the sACE2-Fc double mutants B5-D3, B5-D4 and B5-D5 in the subsequent neutralization and overexpression assays (Supplementary Fig. 3 and 4), and examined B5-D3 as a representative candidate in the in vivo infection tests and follow-up analysis (Figure 2–6, and Supplementary Figures 5–18).

We agree that the lack of explanation for splitting A2 and A3 into D1 to D4 single mutations made the rationale unclear. In the revised manuscript, we have included our previous test results on B5-A2 and B5-A3, cited Lei et al.’s study using A2 in ACE2 decoy [9], and explained the rationale for splitting A2 and A3 into D1 to D4 mutations. Relevant revision was made on page 4 lines 94–97 in the main text, while the design and data for B5-A2 and B5-A3 were included in the revised Figure 1b and Supplementary Figure 2b, f–h.

(2) Figures 1b, 1d, and 1e lack statistical analyses, making it difficult to determine whether B5 and D3 exhibit significant advantages. For Wuhan-Hu-1 strain, B2 and B5 are similar, and for D614G strain, B2, B3, B4, B5, and B6 display comparable results. However, only the glycosylation-related single mutant B5 is chosen for further combinatorial constructs. Moreover, for VOC/VOI strains, B5 is superior to B5-D3; for the Alpha strain, B5-D4 and B5-D5 are superior to B5-D3; and for the Delta and Lambda strains, B5-D5 is superior to B5-D3. These observations further highlight the need for a clearer explanation of the selection strategy.

We agree with the reviewer’s insightful observations.

Indeed, although our results confirmed enhanced viral neutralization by these reported mutations, the activity increases compared to WT ACE2-Fc were generally limited. Importantly, these observations were largely consistent with other reports (including the study by Chan et al. [5]), suggesting limited potential of mutagenesis in enhancing the ACE2-RBD/Spike interaction. Therefore, we chose to selectively examine B2-B6 to construct an enhanced ACE2-Fc decoy with reasonable performance, as a representative candidate to study the application potential of ACE2-Fc decoy.

The IC₅₀ values in Figures 1b, 1d, and 1e were calculated from neutralization curves, measuring infection reduction at multiple concentrations in duplicates, which therefore were presented with statistical support. Based on the multiple neutralization assays, B5-D3 consistently showed a high performance among other top-performers (Figure 1, Supplementary Fig. 2f,g, and Supplementary Fig. 3).

We agree that B2 and B5 performed comparably well in neutralization assays, but B2 contains two mutations (T27Y/T92Q) while B5 carries a single mutation (T92Q). Hence, we decided to focus on B5 due to its lowest mutational burden and least potential risk.

We agree that for VOC/VOI strains, B5 was superior to B5-D3 in pseudovirus-neutralization assays. However, B3-D3 was enzymatically inactive, which is essential for generating safe ACE2 decoy and, therefore, justifies our usage of B5-D3 over B5.

We agree with the reviewer that, altogether, the B5-D3 did not show significant advantages than other top performers like B5-D4 and B5-D5. Here, B5-D3 was selected as a representative, which performed equally well rather than being the most outstanding candidate, for subsequent examination of efficacy, safety, and mechanistic insights.

We thank the reviewer for his valuable feedback. In the revised manuscript, we have further amended our description of B5-D3, as a “representative” candidate, to improve the readability. Relevant changes can be found on page 4 line 84, page 5 line 109, page 14 line 333 and page 15 line 360.

(3) Figure 1e does not specify the construct form of the control hIgG1, namely whether it is an hIgG1 Fc fragment or a full-length hIgG1 protein. If the full-length form is used, the design of its Fab region should be clarified to ensure the accuracy and comparability of the experimental control.

We thank the reviewer for pointing out this negligence.

In this study, we used the in vivo grade recombinant human IgG1 isotype control antibody in its full length (Syd labs, #PA007125) as the negative control. It is the 4F17 clone, which is widely used and showed low or no specific binding to any human samples [10] (Human IgG1 Isotype Control Antibody | Recombinant, in vivo Grade - Syd Labs). We have added the relevant information in the MATERIALS AND METHODS on page 23 lines 548–549.

(4) In Figure 2a, all three PBS control mice died, whereas in Figure 2f, three out of five PBS control mice died, with the remaining showing gradual weight recovery. This discrepancy may reflect individual immune variations within the control groups, and it is necessary to clarify whether potential autoimmune factors could have affected the comparability of the results. Also, the mouse experiments suffer from insufficient sample sizes, which affects the statistical power and reliability of the results. In Figure 2a, each group contains only 4 replicates, one of which was used for lung tissue sampling. As a result, body weight monitoring data is derived from only 3 mice per group (the figure legend indicating n=4 should be corrected to n=3). Such a small sample size limits the robustness of the conclusions. Similarly, in Figure 2f, although each group has 5 replicates, body weight data are presented for only 4 mice, with no explanation provided for the exclusion of the fifth mouse. Furthermore, the lung tissue experiments in Figure 3a include only 3 replicates, which is also inadequate.

We thank the reviewer for his valuable feedback.

Figure 2a was the first in vivo infection experiment of this study, and we performed the test in aged female K18-hACE2 mice at 10–12 months old. Whereas for the subsequent experiments in Figure 2f and Figure 3, we changed to young female K18-hACE2 mice at 2–3 months old, because the limited supply of old mice. While in Figure 2a, four aged mice (not three) in the PBS control group all died within 7 dpi, results of Figure 2f and Figure 3 consistently showed heterogeneous responses among young mice in the PBS control groups. Since increased susceptibility to SARS-CoV-2 infection has been broadly observed among aged human populations and it was also supported by mouse study [11], here we would attribute the observed discrepancy to the age difference between the two cohorts in Figure 2a and 2f. In the revised manuscript, we have further elucidated this observation in results (on page 7 lines 163–167) and included a new reference for better clarification (page 7 line 167).

Furthermore, because the PBS control mice in both Figure 2a and 2f died within 7 dpi, which was too soon for autoimmune factors to take place. Moreover, we have performed AAV-based prolonged overexpression experiments in K18-hACE2 mice (new Supplementary Fig. 4), which showed no tissue damage in either WT sACE2-Fc or B5-D3 treated mice, suggesting low immunogenicity. Collectively, the autoimmune factors are unlikely the reason leading to the different survival between PBS controls in Figure 2a and 2f.

We thank the reviewer for pointing out the weakness regarding small sample sizes in our study.

(1) In Figure 2a–c, the experiment was performed in an aged cohort at 10–12 months old, starting with 5 mice in each virus-inoculated group and 4 mice in the mock control group. At 4 dpi, we sacrificed one mouse from each group for tissue analysis. Therefore, in the survival analysis, there were 4 mice in each virus-inoculated group and 3 mice in the mock control group, whose survival and body weight changes were presented in Figure 2b, c.

Despite the relatively small sample sizes in Figure 2b, c, all 4 PBS control mice died, while all 4 mice in 6-hour B5-D3 IN prophylaxis group survived, demonstrating 100% survival and no sign of body weight loss. The survival and body weight data were highly consistent, strongly supporting that B5-D3 intranasal prophylaxis could protect the mice from lethal SARS-CoV-2 infection.

To enhance clarity, in the revised manuscript, we have added the sample size information in chart legends in Figure 2a–c.

(2) In Figure 2f–h, the experiment was performed in a young cohort at 2–3 months old and the body weight and survival data were presented for 5 mice in each group (not for 4 mice). Notably, although 2 out of 5 young mice in the PBS control group eventually survived from the viral infection, they had suffered significant weight loss during 4–7 dpi, similarly to the died. Whereas all 5 mice in the – 6hr B5-D3 IN prophylaxis group showed no sign of weight loss. Hence, these data were highly consistent with Figure 2b, c, supporting the efficiency of B5-D3 IN prophylaxis in protection against SARS-CoV-2 infection.

We noticed that some data points in Figure 2g, h were very close to each other, making it difficult to distinguish the data line for individual mice. To enhance clarity, in the revised manuscript, we have added sample-size information in chart legends in Figure 2g and 2h.

(3) In Figure 3a, we aimed to examine the lung tissues at early time points. For each treatment, we have 3 mice sacrificed at a single selected time point. Hence, total 9 mice were examined in the PBS control group and B5-D3 IN group, yielding results at 1 dpi, 2 dpi and 4 dpi that consistently supported each other. Moreover, the viral titers, S, and N protein expression analysis all showed significant difference among different groups. Therefore, our experiments have enough discrepancy between different treatment groups to draw the conclusion.

(5) Compared to 6 hours, intranasal administration of B5-D3 at 24 hours before viral infection results in reduced protective efficacy. However, only survival and body weight data are provided, with no supporting evidence from virological assays such as viral titer measurement. Therefore, the long-term effectiveness lacks sufficient experimental validation.

In Figure 2f–h, we aimed to compare the efficacies of IN administration of B5-D3 at different timepoints, mainly focusing on the body weight change and survival data along the infection and recovery time. As indicated by early data in Figure 2d, viruses were largely cleared by 4 dpi in mice treated with B5-D3 prophylaxis. Therefore, in this test, we did not examine virus titers in the recovered animals by the end of observation at 14 dpi. Instead, we examined plasma levels of virus-neutralizing antibodies in the survivors at the endpoint, which indeed supported that the 6-hours and 24-hours IN B5-D3 prophylaxis provided effective protection against the SARS-CoV-2 infection and resulted in minimal levels of neutralizing antibodies in plasma, as shown in Figure 2i.

Collectively, the body weight, survival, and antibody data all supported that 6-hour IN B5-D3 prophylaxis achieved the best efficacy. Hence, we performed comprehensive viral titer and profiling analysis at early time points like 1 dpi, 2 dpi, and 4 dpi, focusing only on the 6-hour IN B5-D3 prophylaxis. This works also included B5-D3-LALA control to examine viral titers, host immune responses, and underlying mechanisms (Figure 3,4).

We agree with the reviewer that it would be more comprehensive if our experiments could include indepth analysis of the 24-hours IN B5-D3 prophylaxis group. However, due to limited capacity of animal service, we chose to focus on the best-performing group as a representative treatment to study the underlying mechanisms.

(6) In Figures 3b and 3c, viral spike (S) and nucleocapsid (N) RNA relative expression levels are quantified by qPCR. The results show significant individual variation within the B5-D3-LALA treatment group: one mouse exhibits high S and N expression, while the other two show low expression. Viral load levels are also inconsistent: two mice have high viral loads, and one has a low viral load. Due to this variability, the available data are insufficient to robustly support the conclusion.

We understand the reviewer’s concern on the variability within the B5-D3-LALA group. However, we have some reservations about the importance of further increasing the sample sizes in this test.

First, since viral gene transcription and viral particle levels represented different phases in viral life, they may follow different kinetics during infection progression and lead to variability. Second, we used different parts of the lung tissues from each mouse for extracting RNA and tissue homogenates, which were then used for detection of S/N expression and viral load levels, respectively. The uneven viral infection in the lung might also contribute to the variability. Furthermore, in this test, both our qPCR and viral load analysis data consistently demonstrated that the B5-D3-LALA was less effective than B5-D3, indicating that Fc function played an important role in supporting full protection by B5-D3 against lethal SAS-CoV-2 infections. This observation is also supported by other studies [12].

We appreciate the valuable feedback from the reviewer. In the revised manuscript, we have further clarified these observations on page 8, lines 192–194, and included alveolar thickening data on page 9, lines 202–204.

(7) Figure 3e: "H&E staining indicated alveolar thickening in all groups," including the Mock group. Since the Mock group did not receive virus or active drug treatment, this observed change may result from local tissue reaction induced by the intranasal inoculation procedure itself, rather than specific immune activation. A control group (no manipulation) should be set to rule out potential confounding effects of the experimental procedure on tissue morphology, thereby allowing a more accurate assessment of the drug's effects.

We thank the reviewer for his insightful comments and suggestions.

We have further examined our H&E staining and quantified alveolar thickening in different treatment groups. Indeed, the data suggested a transient alveolar thickening in the mock group at 1 dpi, which was improved at 2 dpi. This observation supports that the intranasal procedure itself indeed caused a transient alveolar thickening, that was evident at 1 dpi but disappeared at 2 dpi.

Notably, moderate alveolar thickening was found to be persistent in the B5-D3-treated mice till the end point at 4 dpi. Whereas the PBS groups with intensive SARS-CoV-2 infection progressively developed severe structural damage and showed much stronger alveolar thickening than B5-D3 or mock groups at 4 dpi. Consistent with the partial protection by B5-D3-LALA, histological analysis of lung samples in this group revealed severer yet heterogenous alveolar thickening. These observations suggested that -6h IN B5-D3 treatment prevented tissue damage brought by infection with minimal yet efficient immune activation.

In the revised manuscript, we have included the quantitation results of alveolar thickening on page 9, lines 200–204 and presented the data in new Supplementary Fig. 7.

(8) In Supplementary Figure 11b, a considerable number of alveolar macrophages (AMs) are observed in both the PBS and B5-D3 groups. This makes it difficult to determine whether the observed accumulation is specifically induced by B5-D3.

We thank the reviewer for pointing out this issue.

In this experiment, the cell populations examined in previous Supplementary Fig. 11b and Fig. 5h are different, though graphs appear similar.

Supplementary Fig. 11b (new Supplementary Fig. 12b) showed the analysis among CD45+ immune cells, regardless of B5-D3-AF750 signal. The dominance of AMs among immune cell populations is a normal physiological feature of BALF cells. To make this clear, we have added new data of BALF cells from untreated mice in the revised manuscript and new Supplementary Fig. 12b.

Fig. 5h displayed for cell type analysis among the CD45+ B5-D3-AF750+ cells —only CD45+ immune cells that took up the AF750-labeled B5-D3.

To enhance clarity, in the revised manuscript, we have amended the labels as CD45+ B5-D3-AF750+ in Figure 5h (and similarly in revised Supplementary Fig. 13), to differentiate the data from that in CD45+ cells shown in the revised Supplementary Fig. 12b.

(9) In the flow cytometry experiment shown in Figure 5, the PBS control group is not labeled with AF750, which necessarily results in a value of zero for "B5-D3+ cells" on the y-axis. An appropriate control (e.g., hIgG1-Fc labeled with AF750) should be included.

We thank the reviewer for his valuable question.

In this experiment, we intended to analyze all immune cells with positive AF750 signals, to identify the major immune cell types that took up AF750-B5-D3 as the candidate cells responsible for the observed activation of innate immunity. Hence, here we deliberately set PBS vehicle treatment without AF750 signal as the control group for gating.

This analysis aimed to provide an overall picture of immune cell types that actively take up ACE2 decoy, likely via Fc receptor-mediated binding. Control IgG1 labeled with AF750, with an Fc region, may show similar profile and biodistribution among BALF immune cells, which, therefore, was not examined as control for gating.

Instead, in the revised manuscript, we have added new analysis results comparing the efficiencies of B5-D3 and IgG1 in mediating pseudovirus uptake in THP-1-derived macrophages. IgG1 isotype control was examined to address ACE2-specific effect. Indeed, we observed no pseudovirus uptake based on p24 signal, in the IgG1 treated samples, indicating that the presence of B5-D3 is crucial for efficient pseudovirus uptake in macrophages due to the sACE2-spike affinity. These results have been added on page 13 lines 310–316 in the main text, and the relevant data was presented in new Supplementary Fig. 17.

(10) The Methods section: a more detailed description of the experimental procedures involving HIV p24 and SARS-CoV-2 should be included.

We thank the reviewer for pointing out this weakness.

In the revised manuscript, we have provided further details of the relevant experimental procedures in the Materials and Methods part, on page 21, lines 507–517.

Reviewer #3 (Public review):

Strengths:

The core strength of this study lies in its innovative demonstration that an engineered sACE2-Fc fusion redirects virus-decoy complexes to Fc-mediated phagocytosis and lysosomal clearance in macrophages, revealing a distinct antiviral mechanism beyond traditional neutralization. Its complete prophylactic protection in animal models and precise targeting of airway phagocytes establish a novel therapeutic paradigm against SARS-CoV-2 variants and future respiratory viruses.

We thank the reviewer for his recognition and positive comments on our study.

Weaknesses:

The study attributes complete antiviral protection to Fc-mediated phagocytic clearance, a central claim that requires more rigorous experimental validation. The observation that abrogating Fc functions compromises protection could be confounded by potential alterations in the protein's stability, half-life, or overall structure. To firmly establish this mechanism, it is crucial to include a control molecule with a mutated Fc region that lacks FcγR binding while preserving the Fc structure itself. Without this critical control, the conclusion that phagocytic clearance is the primary mechanism remains inadequately supported.

We thank the reviewer for his insightful comments and suggestions.

The L234A/L235A mutations in human IgG1 Fc region are most widely used to abolish its FcγR binding and Fc effector functions [13]. In this study, we have used B5-D3-LALA in the in vivo infection experiments in K18-hACE2 mice, as the control molecule that lacks FcγR binding while preserving the Fc structure (Figure 3, 4).

To address the reviewer’s concern, we further performed new analysis comparing the efficiencies of different versions of B5-D3 in mediating pseudovirus uptake in THP-1-derived macrophages. In this test, B5-D3-LALA and B5-D3 were examined side-by-side to address the role of Fc effector functions in the phagocytosis process. Meanwhile, IgG1 isotype control was examined to address ACE2-specific effect. Indeed, we detected significant reduction of pseudovirus uptake based on p24 signal, in the B5D3-LALA treated samples compared to those receiving B5-D3. This decreased pseudoviral uptake correlated with the loss of Fc-mediated effector functions in B5-D3-LALA, indicating the involvement of Fc functions in efficient macrophage uptake of B5-D3-virus complex.

In the revised manuscript, we have included these results on page 13 lines 310–316 in the main text and presented relevant data in Supplementary Fig. 17.

The strategy of deliberately targeting virus-decoy complexes to phagocytes via Fc receptors inherently raises the question of Antibody-Dependent Enhancement (ADE) of disease. While the authors demonstrate a lack of productive infection in macrophages, this only addresses one facet of ADE. The risk of Fc-mediated exacerbation of inflammation (ADE) remains a critical concern. The manuscript would be significantly strengthened by a direct discussion of this risk and by including data, such as cytokine profiling from treated macrophages, to more comprehensively address the safety profile of this approach.

(1) We thank the reviewer for his insightful comments and suggestions regarding the ADE issue.

Indeed, Antibody-Dependent Enhancement (ADE) of viral infection is a critical concern when developing the ACE2 decoy strategy. In this study, we have carefully examined the relevant risk based on our data from various in vitro and in vivo assays.

In our in vivo infection experiments, all B5-D3 prophylaxis and treatment groups, regardless of the administration times and routes, showed improved outcomes like less body-weight loss and better survival, compared to the PBS control groups (Figure 2). None of these treatment groups demonstrated worsened infections, indicating that ADE phenomenon was not occurring or did not play a major role during the B5-D3 treatments. Instead, moderate immune activation was observed in the lung of B5-D3 treated mice, which occurred much earlier but was milder compared to that in the PBS groups, and may reflect responses that lead to the efficient early clearance of viruses without observable symptoms (Figure 3 and 4).

In our in vitro assays shown in Figure 6, B5-D3 treatments in epithelial or non-immune cell models (hACE2-Galu-3 and hACE2-293T) significantly blocked the entry of pseudovirus into cells and yielded much reduced luciferase signals (Figure 6d–g). Whereas in the THP-1-derived macrophages, although the presence of B5-D3 largely enhanced the entry of SARS-CoV-2 pseudovirus into cells (Figure 6a,b), it did not result in active infection and produced no luciferase signal (Figure 6g). These results were robustly reproducible, indicating that pseudoviruses did not successfully release its genome RNA and viral proteins (like RTase and integrases) after entering macrophages. Instead, colocalization analysis of p24 (pseudoviruses), sACE2-Fc (B5-D3), and LAMP1 (lysosome) signals suggested probability of pseudovirus degradation in endosomes/lysosomes after cell entry (Figure 6a,c). Consistently, examination of the macrophages that had taken up pseudovirus showed that the Spike (S) proteins from the pseudovirus particles were not cleaved to release S2’ fragment at a distinct smaller size (Figure 6h). As the cleavage of S protein in host cells is critical for effective membrane fusion, it is essential and regarded as hallmark for successful viral entry and escape from endosome. Collectively, these data consistently indicated that the SARS-CoV-2 pseudoviruses were degraded directly in lysosomes after entering macrophages, showing no sign of ADE.

(2) We thank the reviewer for his valuable suggestion and have performed RNA-seq analysis to profile immune responses in the treated macrophages.

We performed RNA-Seq analysis to investigate major transcriptional changes in THP-1-derived macrophages after the pseudovirus infection, with or without B5-D3 treatments. Although no individual genes fulfilled the cutoff threshold of significant up-/down-regulation, we observed antiviral responses in the pseodovirus-B5-D3 treated samples by GSEA (new Supplementary Fig. 18). This observation indicated that the B5-D3 treatment and subsequent cell-entry of pseudovirusB5-D3 complexes into macrophages induced immune activation at moderate levels, but not evoking strong immune responses that can be harmful to the host.

In the revised manuscript, we have included the new RNA-seq analysis results on macrophage infection tests on page 13 lines 317–322 and page 14 lines 323–325 in the main text and presented the relevant data in the new Supplementary Fig. 18. Furthermore, we agree that ADE is a critical issue and have further enriched our discussion on page 17 lines 415–417, to emphasize that the risk for ADE should be thoroughly evaluated to further develop the decoy strategy for human use.

The exclusive use of the K18-hACE2 mouse model, which exhibits severe disease, limits the generalizability of the findings. The "complete protection" observed may not translate to models with more robust and naturalistic immune responses or to human physiology.

We thank the reviewer for pointing out the limitation of the mouse model used.

(1) Given that wild type mice are not susceptible to SARS and SARS-CoV-2 infection, transgenic mice have been generated to express hACE2, through various designs and strategies, serving as models for viral infection and drug development. However, many of these hACE2 transgenic mouse models exhibit mild infections due to moderate hACE2 levels, failing to develop the severity observed in SARS and COVID patients [14].

(2) The K18-hACE2 transgenic mouse line (B6. Cg-Tg(K18-ACE2)2Prlmn/J, Jackson Laboratory) used in our study carries multiple copies of K18-hACE2 transgene cassette [15]. Compared to other hACE2 transgenic mouse models, this K18-hACE2 line shows higher expression of hACE2 in airway and other epithelia and supports severer infections by both SARS and SARS-CoV2 viruses, successfully causing lethality [16]. Hence, K18-hACE2 mice is a widely used model to study SARS and SARS-CoV2 virus infections and drug developments.

(3) We agree that K18-hACE2 mice is a relatively weak transgenic line with poor productivity. However, it demonstrates best susceptibility to SARS-CoV-2 infection among established mouse models. In this study, we observed robust responses to SARS-CoV-2 infection in both aged and young cohorts, with all infected mice consistently demonstrating significant body weight loss during 4 dpi to 7 dpi (the PBS groups in Figure 2b, g)

We agree with the reviewer that it would be more convincing to assess the efficacy of B5-D3 using additional animal models. However, we have some reservations about the importance of these additional tests. First, the generality of ACE2-Fc decoy concept and its efficacy have been reported in other studies using various models [17,18]. Moreover, different transgenic mice or animal models exhibit distinct kinetics in the pathogenesis process and immune responses to SAS-CoV-2 infections, which differ from that in human patients at varied aspects. Hence, given the limited capacity of animal facility, we chose to focus on the K18-hACE2 mice that have demonstrated most robust and convincing infection data, to investigate the potential of B5-D3 administered through various strategies as well as the underlying mechanisms for the full protection observed in IN prophylaxis.

In the revised manuscript, we have further enriched our discussion regarding this limitation, on page 17 lines 417–422.

Furthermore, the lack of data on circulating SARS-CoV-2 variants is a concern

We thank the reviewer for his valuable comment.

In this study, we have demonstrated the viral neutralization capacity of B5-D3, as a representative of the enhanced sACE2 decoy, using multiple pseudoviruses and authentic SARS-CoV-2, which collectively covered eleven variants (up to Omicron strains). Our results from both in vitro neutralization and PRNT experiments confirmed the robust resilience of B5-D3 against viral evolution (Figure 1c–g). This observation aligns well with other studies and is broadly supported by various investigations, as was pointed out below by the reviewer.

Furthermore, studies on viral evolution have observed a robust trend that later-emerging SARS-CoV-2 variants exhibit a higher affinity for the ACE2 receptor, enhancing their infectivity and transmissibility [19]. Therefore, it is unlikely for a newly emerged SARS-CoV-2 variant to escape from B5-D3mediated neutralization.

Collectively, all evidence consistently supports the principle of decoy design, B5-D3 (or other effective ACE2 decoys) possess the intrinsic ability to neutralize new circulating SARS-CoV-2 variants, as long as the virus variants rely on ACE2 receptor for cell entry. Hence, although further tests on circulating viral variants would add strengths to our study, the significance of this additional data may be limited.

In the revised manuscript, we have further addressed this concern in the discussion, on page 16 lines 394–397.

The concept of sACE2-Fc fusion proteins as decoy receptors is not novel, and numerous similar constructs have been previously reported. The manuscript would benefit from a clearer demonstration of how the optimized B5-D3 mutant represents a significant advance over existing sACE2-Fc designs.

We thank the reviewer for his valuable comments.

Indeed, previous research has reported multiple ACE2 mutations to enhance its binding to spike proteins and neutralization against SARS-CoV-2. However, combining ACE2 mutations based on in silico predictions to both enhance spike binding and eliminate the ACE2 enzymatic activity resulted in accumulated burdens. For instance, ACE2 decoy candidates with up to five mutations like K31F/N33D/H34S/E35Q/H345L [8] and L79F/M82Y/Q325Y/H374A/H378A [12] have demonstrated excellent potency to neutralize SARS-CoV-2 in both in vitro and in vivo assays. However, the extensive mutations could be associated with structural instability and reduced production efficiency [8,12]. Furthermore, the high mutation loads increase risks for immunogenicity, which is a critical issue in future clinical applications. Corroboratively, Urano et al. detected in vitro T cell stimulation elicited by the L79F mutation, whereas the T92Q mutation (included in our decoy design) showed much lower immunogenicity and enhanced spike binding affinity [20].

In our ACE2 decoy design, we incorporated only two mutations (like T92Q and H374N in B5-D3) to enhance neutralization potency while eliminating enzymatic activity, resulting in simplest ACE2 mutants desired for engineering enhanced decoy. B5-D3, as one representative, not only exhibited minimal mutation-related risks (Supplementary Fig. 2i) but also top-level neutralization potencies among all candidate mutants tested (Figure 1, Supplementary Fig. 2f,g and Supplementary Fig. 3). To further address the safety of B5-D3 for in vivo use, we have performed prolonged in vivo overexpression of B5-D3 ACE2 decoy through AAV delivery in immune-competent K18-hACE2 mice, which indeed showed no sign of RAS disturbance or immune infiltration causing tissue damage. (In the revise manuscript, we have included these new results on page 5 lines 118–122 and page 6 lines 123–135 in the main text and presented the data in new Supplementary Fig. 4).

Therefore, instead of demonstrating advantage over existing sACE2-Fc designs, our study used the optimized B5-D3 as a representative ACE2 decoy of top performers, to systematically examined various administration strategies as well as the underlying mechanisms for the full protection observed in IN prophylaxis. Aligned with this effort, our study identified 6-hours IN prophylaxis as the most effective regimen to confer complete protection against SARS-CoV-2 infection in K18-hACE2 mice. Further investigation through transcriptomics, bio-distribution, and phagocytosis analysis revealed that IN-delivered B5-D3 not only neutralizes viruses but also engaged airway phagocytes to promote early viral clearance and host immune activation, uncovering a distinct antiviral mechanism for the universal “decoy strategy” to combat unknown air-borne respiratory virus in the future.

In the revised manuscript, we have further clarified our focus on using B5-D3 as a “representative” of ACE2 decoy on page 4 line 84, page 5 line 109, page 14 line 333, and page 15 line 360.

A direct comparative analysis with previously published benchmarks, particularly in terms of neutralizing potency, Fc effector function strength, and in vivo efficacy, is necessary to establish the incremental value and novelty of this specific agent.

We thank the reviewer for his valuable comments.

Indeed, our study has aimed to address this concern and made partial progress through in vitro neutralization assays (Figure 1b and Supplementary Fig. 2c,d,f,g). Our results from the limited yet meaningful comparisons with the sACE2 lacking Fc domain and selected sACE2-Fc mutants published/proposed previously clearly demonstrated “substantial enhancement through Fc-fusion” (Supplementary Fig. 1d) and modest improvement from protein mutagenesis at ACE2-Spike interaction interface” (Figure 1b and Supplementary Fig. 2c,d,f,g).

Based on the results from our various neutralization assays, we chose B5-D3 as a representative of enhanced decoy for in vivo infection, which identified 6-hours IN prophylaxis to confer complete protection against infection, demonstrating significant impact of administration strategies on in vivo efficacy of B5-D3 (Figure 2). Subsequent analysis further uncovered intriguing phenomena regarding the cellular distribution of IN-administered B5-D3 and the early immune activation triggered in the lung, which underlies the full protection by IN prophylaxis and represents an important novelty of this study.

We agree with the reviewer that further analysis with additional benchmark versions would enhance the value of this study, but we have reservation regarding the importance. To enhance clarity, in the revised manuscript, we have further emphasized our study focus on using B5-D3 as a representative ACE2 decoy throughout the text and enriched the discussion on page 15 line 348–365.

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(15) McCray PB, Pewe L, Wohlford-Lenane C, Hickey M, Manzel L, Shi L, Netland J, Jia HP, Halabi C, Sigmund CD, Meyerholz DK, Kirby P, Look DC & Perlman S. Lethal Infection of K18hACE2 Mice Infected with Severe Acute Respiratory Syndrome Coronavirus. Journal of Virology 81, 813-821 (2007).

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(17) Urano E, Itoh Y, Suzuki T, Sasaki T, Kishikawa JI, Akamatsu K, Higuchi Y, Sakai Y, Okamura T, Mitoma S, Sugihara F, Takada A, Kimura M, Nakao S, Hirose M, Sasaki T, Koketsu R, Tsuji S, Yanagida S, Shioda T, Hara E, Matoba S, Matsuura Y, Kanda Y, Arase H, Okada M, Takagi J, Kato T, Hoshino A, Yasutomi Y, Saito A & Okamoto T. An inhaled ACE2 decoy confers protection against SARS-CoV-2 infection in preclinical models. Sci Transl Med 15, eadi2623 (2023).

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eLife Assessment

This study integrates large-scale behavioral, genetic, and molecular analyses in animal models to investigate morphine response. Utilizing high-quality, time-series Quantitative Trait Loci (QTL) mapping, the work provides compelling evidential support for novel, time-dependent genetic interactions (epistasis). A fundamental result of this rigorous analysis is the discovery of a novel Oprm1-Fgf12-MAPK signaling pathway, which offers new insights into the mechanisms of opioid sensitivity.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have appropriately addressed the comments raised in the previous round of review.]

Summary:

The study by Lemen et al. represents a comprehensive and unique analysis of gene networks in rat models of opioid use disorder, using multiple strains and both sexes. It provides a time-series analysis of Quantitative Trait Loci (QTLs) in response to morphine exposure.

Strengths:

A key finding is the identification of a previously unknown morphine-sensitive pathway involving Oprm1 and Fgf12, which activates a cascade through MAPK kinases in D1 medium spiny neurons (MSNs). Strengths include the large-scale, multi-strain, sex-inclusive design, the time-series QTL mapping provides dynamic insights, and the discovery of an Oprm1-Fgf12-MAPK signaling pathway in D1 MSNs, which is novel and relevant.

Reviewer #2 (Public review):

Summary:

This highly novel and significant manuscript re-analyzes behavioral QTL data derived from morphine locomotor activity in the BXD recombinant inbred panel. The combination of interacting behavioral-pharmacology (morphine and naltrexone) time course data, high-resolution mouse genetic analyses, genetic analysis of gene expression (eQTLs), cross-species analysis with human gene expression and genetic data, and molecular modeling approaches with Bayesian network analysis produces new information on loci modulating morphine locomotor activity.

Furthermore, the identification of time-wise epistatic interactions between the Oprm1 and Fgf12 loci is highly novel and points to methodological approaches for identifying other epistatic interactions using animal model genetic studies.

Strengths:

(1) Use of state-of-the art genetic tools for mapping behavioral phenotypes in mouse models.

(2) Adequately powered analysis incorporating both sexes and time course analyses.

(3) Detection of time and sex-dependent interactions of two QTL loci modulating morphine locomotor activity.

(4) Identification of putative candidate genes by combined expression and behavioral genetic analyses.

(5) Use of Bayesian analysis to model causal interactions between multiple genes and behavioral time points.

Appraisal:

The authors largely succeeded in reaching goals with novel findings and methodology.

Significance of Findings:

This study will likely spur future direct experimental studies to test hypotheses generated by this complex analysis. Additionally, the broad methodological approach incorporating time course genetic analyses may encourage other studies to identify epistatic interactions in mouse genetic studies.

Reviewer #3 (Public review):

Summary:

This is a clearly written paper that describes the reanalysis of data from a BXD study of the locomotor response to morphine and naloxone. The authors detect significant loci and an epistatic interaction between two of those loci. Single-cell data from outbred rats is used to investigate the interaction. The authors also use network methods and incorporate human data into their analysis.

Strengths:

One major strength of this work is the use of granular time-series data, enabling the identification of time-point-specific QTL. This allowed for the identification of an additional, distinct QTL (the Fgf12 locus) in this work compared to previously published analysis of these data, as well as the identification of an epistatic effect between Oprm1 (driving early stages of locomotor activation) and Fgf12 (driving later stages).

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The study by Lemen et al. represents a comprehensive and unique analysis of gene networks in rat models of opioid use disorder, using multiple strains and both sexes. It provides a time-series analysis of Quantitative Trait Loci (QTLs) in response to morphine exposure.

Strengths:

A key finding is the identification of a previously unknown morphine-sensitive pathway involving Oprm1 and Fgf12, which activates a cascade through MAPK kinases in D1 medium spiny neurons (MSNs). Strengths include the large-scale, multi-strain, sex-inclusive design, the time-series QTL mapping provides dynamic insights, and the discovery of an Oprm1-Fgf12-MAPK signaling pathway in D1 MSNs, which is novel and relevant.

Weaknesses:

(1) The proposed involvement of Nav1.2 (SCN2A) as a downstream target of the Oprm1-Fgf12 pathway requires further analysis/evidence. Is Nav1.2 (SCN2A) expressed in D1 neurons?

The authors mentioned that SCN8A (Nav1.6) was tested as a candidate mediator of Oprm1-Fgf12 loci and variation in locomotor activity. However, the proposed model supports SCN2A as a target rather than SCN8A. This is somewhat unexpected since SCN8A is highly abundant in MSN.

Can the authors provide expression data for SCN2A, Oprm1, and Fgf12 in D1 vs. D2 MSNs?

Author response image 1.

We generated Author response image 1 to show both Scn2a and Scn8a are ubiquitously expressed in MSN and GABAergic neurons.

(2) The authors should consider adding a reference to FGF12 in Schizophrenia (PMC8027596) in the Introduction.

This is a relevant reference. We have cited it in the discussion section instead of introduction because we felt that is more relevant.

(3) There is recent evidence supporting the druggability of other intracellular FGFs, such as FGF14 (PMC11696184) and FGF13 (PMC12259270), through their interactions with Nav channels. What are the implications of these findings for drug discovery in the context of the present study? Could FGF12 be considered a potential druggable therapeutic target for opioid use disorder (OUD)?

The recent success in targeting FGF14 and FGF13 protein-protein interactions with sodium channels suggests that FGF12 could indeed be a druggable target for OUD. We have added a section to the Discussion exploring the potential for developing small-molecule modulators of the FGF12-Nav interface as a novel therapeutic strategy.

Reviewer #2 (Public review):

Summary:

This highly novel and significant manuscript re-analyzes behavioral QTL data derived from morphine locomotor activity in the BXD recombinant inbred panel. The combination of interacting behavioral-pharmacology (morphine and naltrexone) time course data, high-resolution mouse genetic analyses, genetic analysis of gene expression (eQTLs), cross-species analysis with human gene expression and genetic data, and molecular modeling approaches with Bayesian network analysis produces new information on loci modulating morphine locomotor activity.

Furthermore, the identification of time-wise epistatic interactions between the Oprm1 and Fgf12 loci is highly novel and points to methodological approaches for identifying other epistatic interactions using animal model genetic studies.

Strengths:

(1) Use of state-of-the art genetic tools for mapping behavioral phenotypes in mouse models.

(2) Adequately powered analysis incorporating both sexes and time course analyses.

(3) Detection of time and sex-dependent interactions of two QTL loci modulating morphine locomotor activity.

(4) Identification of putative candidate genes by combined expression and behavioral genetic analyses.

(5) Use of Bayesian analysis to model causal interactions between multiple genes and behavioral time points.

Weaknesses:

(1) There is a need for careful editing of the text and figures to eliminate multiple typographical and other compositional errors.

We have performed a thorough review of the manuscript and corrected typographical errors, including "ddactivates" and other compositional issues.

(2) There are multiple examples of overstating the possible significance of results that should be corrected or at least directly pointed out as weaknesses in the Discussion. These include:

(a) Assumption that the Oprm1 gene is the causal candidate gene for the major morphine locomotor Chr10 QTL at the early time epochs. Oprm1 is 400,000 bp away from the support interval of the Mor10a QTL locus, and there is no mention as to whether the Oprm1 mRNA eQTL overlaps with Mor10a.

We have clarified this in the text. While Oprm1 is located proximal to the peak, its massive size and the presence of a strong mRNA cis-eQTL in the NAc and hippocampus that precisely overlaps with the Mor10a QTL support interval provide robust evidence for its candidacy. We have added this detail to the Results section.

(b) Although the Bayesian analysis of possible complex interactions between Oprm1, Fgf12, other interacting genes, and behaviors is very innovative and produces testable hypotheses, a more straightforward mediation analysis of causal relationships between genotype, gene expression, and phenotype would have added strength to the arguments for the causal role of these individual genes.

We agree that mediation analysis would be a valuable addition. We revised the Results section to acknowledge that while the Bayesian network provides a comprehensive causal hypothesis, future studies employing formal mediation analysis could further strengthen these individual gene-to-behavior links.

(c) The GWAS data analysis for Oprm1 and Fgf12 is incomplete in not mentioning actual significance levels for Oprm1 and perhaps overstating the nominal significance findings for Fgf12.

We have updated the manuscript to include the specific significance levels for the human GWAS findings related to Oprm1 and Fgf12. We have clarified that the OPRM1 variant rs1799971 reached genome-wide significance (OR = 1.046, p = 4.92 × 10^-9). Furthermore, we have ensured that the findings for FGF12 are described as nominally significant to avoid any overstatement of the results. For example, we now specify that the top FGF12 SNP rs1553460 achieved nominal significance (OR = 1.015, p = 0.021). The Results and Discussion sections have been revised to reflect these precise statistical values.

Appraisal:

The authors largely succeeded in reaching goals with novel findings and methodology.

Significance of Findings:

This study will likely spur future direct experimental studies to test hypotheses generated by this complex analysis. Additionally, the broad methodological approach incorporating time course genetic analyses may encourage other studies to identify epistatic interactions in mouse genetic studies.

Reviewer #3 (Public review):

Summary:

This is a clearly written paper that describes the reanalysis of data from a BXD study of the locomotor response to morphine and naloxone. The authors detect significant loci and an epistatic interaction between two of those loci. Single-cell data from outbred rats is used to investigate the interaction. The authors also use network methods and incorporate human data into their analysis.

Strengths:

One major strength of this work is the use of granular time-series data, enabling the identification of time-point-specific QTL. This allowed for the identification of an additional, distinct QTL (the Fgf12 locus) in this work compared to previously published analysis of these data, as well as the identification of an epistatic effect between Oprm1 (driving early stages of locomotor activation) and Fgf12 (driving later stages).

Weaknesses:

(1) What criteria were used to determine whether the epistatic interaction was significant? How many possible interactions were explored?

By design we only tested for epistasis between the Oprm1 and the Fgf12 loci—a single test of a non-linear interaction. As such there is no correction for multiple tests and no need for permutation. In other words the “nominal” P value in this case is the only relevant P value. We have added this clarification in the Results and Methods.

(2) Results are presented for males and females separately, but the decision to examine the two sexes separately was never explained or justified. Since it is not standard to perform GWAS broken down by sex, some initial explanation of this decision is needed. Perhaps the discussion could also discuss what (if anything) was learned as a result of the sex-specific analysis. In the end, was it useful?

We chose to analyze sexes separately AND jointly due to significant sex differences and sex by strain interactions in locomotion data. This rationale has been added to the results section. We also discussed sex-specific results in the revision.

(3) The confidence intervals for the results were not well described, although I do see them in one of the tables. The authors used a 1.5 support interval, but didn't offer any justification for this decision. Is that a 95% confidence interval? If not, should more consideration have been given to genes outside that interval? For some of the QTLs that are not the focus of this paper, the confidence intervals were very large (>10 Mb). Is that typical for BXDs?

The 1.5 LOD support interval is a standard metric for most QTL mapping studies, and does correspond approximately to a 95% confidence or support interval. Large intervals are common in BXD studies when effect sizes are moderate or recombination density is lower in specific regions. We have clarified the use of the 1.5 LOD interval in the Results section.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

In the vast majority of the figures, the text is too small to read.

We have adjusted the font size in most of the figures.

Reviewer #2 (Recommendations for the authors):

(1) There is a need for careful editing of the text and figures to eliminate multiple typographical and other compositional errors. Examples of these include:

(a) Figure 2E&F lacks identification of Oprm1 as the gene for cis-eQTL studies.

(b) Figure 2H is fairly uninterpretable given the small font sizes. It should be excluded, put as a supplemental figure, or reconfigured to highlight the most important findings in a more legible manner.

(c) Figure 4b: columns in the table need to be identified by a header row.

We thank the reviewer for these comments and have addressed them in the revised version.

Oprm1 is now labeled in Figure 2E and 2F, Figure 2G and 2H is now moved to the Supplementary material. And a header row is added to the table in Figure 4b.

Reviewer #3 (Recommendations for the authors):

Abstract

(1) For the abstract, it might be simpler to name the alleles as "the C57BL/6J allele", etc., since B allele will confuse people unfamiliar with mouse nomenclature.

It is critical to not confound the organism known as C57BL/6J with the genotype, allele, or haplotype that a mouse happens to inherit. Diverse types of mice inherit reference alleles but they may be only very distantly related the C57BL/6J strain. And even the C57BL/6J strain is a moving target that accumulates mutations that are not even consider reference. For example the mutation in Gabra2 of C57BL/6J is a de novo mutation that is not carried by many of the BXD strains since this mutation happened in JAX foundation stock after the BXDs were first established by Dr. Ben Taylor in the 1970s.

The convention is to refer to mouse strains by one string and RRID, the abbreviation of that strain by a common code (often B6), and the abbreviation of the allele, genotype, or haplotype by the italic letter B. This has been the recommendation of the Mouse Nomenclature Committee (on which one of the authors has been a member) for well over 50 years.

(2) I wondered if "also associated with a high B allele" could be reworded somehow; I had to re-read that sentence several times.

This sentence has been reworded for clarity.

(3) Parts of the abstract are written in the present tense, but then it switches to past ("we generated" but then "a Bayesian network analysis supports...").

We have thoroughly revised the abstract. Following standard scientific writing conventions, we now utilize the past tense to describe the specific experimental actions and results of this study. We have maintained the present tense for established biological facts and the broader significance of the findings.

(4) While the -log(p) values are all impressive, the abstract should indicate what threshold is used for genome-wide significance and how that threshold was obtained.

We have added the significance threshold to the Abstract.

(5) Do the details of the MAP kinase cascade need to be explained in the abstract? It feels like a lot of detail for an abstract and represents one of the most speculative aspects of the paper. Maybe just say you identified a possible network, but save the details for the main paper.

This is a valid suggestion. We removed the specific MAP kinase from the abstract.

Introduction

(1) You could add a sentence explaining why using an LMM (GEMMA) was an improvement over the prior analysis.

We have added a sentence explaining that GEMMA improves mapping power and better controls for population structure compared to previous methods.

(2) When mentioning Philips 2010, you could indicate that it identified Oprm1. This might be easier than "In addition to Oprm1" which confused me at first because it had not been mentioned before, so 'in addition' was jarring.

We have revised the text to state that Philip et al. (2010) originally identified the Oprm1 locus.

Results

(1) There are additional instances of the tense switching between past and present in the results section.

We have standardized the tenses in the Results section.

(2) "Ostn, Uts2d, Ccdc50, Gm10823, Fgf12, and Mb21d2" - before giving arguments for fgf12, can you clarify if there are coding variants or eQTLs for any of these genes?

We have added a statement clarifying the coding variants for other genes in this interval and highlighting their eQTL status.

(3) "a total number of 4,495 high-quality nuclei transcriptomes". Consider removing the word "number".

Removed.

(4) "approximately 6 males and 6 females" - could you point the reader to a supplementary table that has the exact number of individuals at the end of this sentence?

The exact number of mice used in each of the BXD strains is not recorded in the original publication by Philip et al., with only mean and max was given. We have clarified that 6 is the average.

(5) "computed using a subset" - please explain how you selected this subset (I assumed LD pruning, but why not be explicit. How many SNPs/markers were there originally, and how many are retained?

We have specified that the subset of markers was selected via LD pruning to represent the genetic diversity of the BXDs.

(6) A few words about how the significant threshold was obtained (permutation?) are needed.

We have clarified that the significance threshold was obtained through 1,000 permutations.

(7) Some of the GWAS results are presented for males and females separately (as well as combined). This is not typical, and so maybe a sentence explaining why the authors thought there might be sex specific GWAS results would be warranted.

The rationale for sex-specific analysis is provided in the results section (significant sex difference and sex by strain interaction)

(8) The correlation between the sexes of 0.68 could be evidence that there are sex-specific genetic effects, but could it also just be due to increased noise as you reduce sample size? What is the confidence interval for that number? Does it include 1? Or 0? If you randomly split the dataset, rather than splitting on the basis of sex, would you obtain higher correlations? The idea of sex differences is interesting, but a bit more work is needed to clarify these concerns.

The correlation of 0.68 (95% CI: 0.52–0.79) significantly excludes both 0 and 1. The drop from r = ~0.86 at earlier intervals suggests a biological shift rather than noise due to sample size, as n remains constant (n = ~ 6 /sex/strain) across all time points. This divergence is driven by sex-specific genetic modifiers, such as the Fgf12 locus, which is more than twice as strong in females (LOD 10.6) as in males (LOD 4.3). We have addressed this in the revision.

(9) Maybe I missed it, but how did you determine the threshold for significance for the epistatic interaction? Could you also clearly indicate how many possible cases of epistasis were examined/considered, since that dictates the correction for multiple testing.

We only tested the interaction between the Fgf12 and the Oprm loci.

(10) "To further examine whether Oprm1 and Fgf12 were co-expressed in the same cells of the NAc," can you first give an indication as to why you looked in NAc versus other brain areas you might have considered?

We have added a sentence explaining that the NAc was chosen due to its central role in opioid reward and the observed strain differences in dopamine release in this region.

(11) "...from every cell type conveyed a weak but significant positive correlation (r = 0.08, p = 1.8e-8) between the expression of Oprm1 and Fgf12 (Figure 7e). When we performed Pearson's correlation analysis within each individual cell cluster, only D1-MSN-3 had a significant positive correlation (r = 0.35, p = 6.1e-8, Figure 7f). In contrast, D1-MSN-2 had a significantly weak negative correlation (r = -0.12, p = 0.02, Figure 7g)." Can you explain why these correlations are relevant? What hypothesis are you testing?

We have clarified that these correlations were used to test the hypothesis that Oprm1 and Fgf12 are co-expressed and potentially co-regulated within the same neuronal subtype to support their epistatic interaction.

(12) "After the morphine locomotion tests were complete," can you give a specific timepoint? Like, was it exactly 180 minutes after the morphine injection?

We have specified that naloxone was injected exactly 180 minutes after the morphine injection.

(13) I appreciate the desire to relate the results of this paper to human GWAS results; however, I don't feel there is much worth discussing beyond the Oprm1 finding. Therefore, I would suggest removing this from the results section and instead just making it a discussion topic. The results presented are clearly the weakest part of this paper, and I personally think it is a shame to end the results section with something that is not very informative. But I suspect the authors may wish to retain this section, and I leave that decision to them and the editor.

We have retained this section but moved some of the more speculative human data discussion to the Discussion section as suggested.

Discussion

(1) Typo "deactivates".

Corrected to "activates".

(2) The last sentence in the first paragraph again discusses the comparison to humans; I would remove this.

That sentence is condensed.

(3) "These data indicate that Oprm1 is a strong candidate gene for the Chr 10 locus associated with morphine-induced locomotion response." I would remind them of the eQTL for Oprm1 since this is a key piece of evidence supporting this gene as a candidate.

We have added a reminder of the overlapping mRNA cis-eQTL for Oprm1.

(4) "It is likely that differences in morphine-induced dopamine release are involved in the highly variable locomotor responses to morphine across the BXD family." I agree this might be true, but since you have no evidence to support this claim, is it worth mentioning at all?

We have rephrased this as a hypothesis or cited relevant literature supporting this link in parental strains.

(5) Could you include a sentence or two about why Philip 2010 didn't find Fgf12? Lack of markers? The difference between an LM and an LMM?

We have added an explanation that the use of a high-density WGS-based marker set and the LMM (GEMMA) allowed for the detection of this novel locus that was previously missed.

(6) Section titled "Cell-type specific gene expression in NAc". While this is interesting, you might also want to remind the reader that epistatic interactions do not necessarily require the genes to be expressed in the same cell or for their gene products to physically interact.

We have added this caveat to the Discussion.

(7) I think the Bayesian network section is not very strong. For example, they did not compare the results for their two chosen genes to the results they might have obtained if they had chosen other genes from their QTL intervals. My guess is that those other genes might have also produced results that were equally convincing. I'm not asking them to do that, but it reflects the risk of false positive results when taking an approach like this. Nevertheless, I am guessing the authors would prefer to include this section.

We appreciate the reviewer pointing out this possibility and agree with this concern. We have added a statement acknowledging the risk of false positives in Bayesian modeling in this context and noting that these findings are intended as testable hypotheses

Methods

(1) How were the 2 HS rats selected? I had the impression that Dr. Telese's lab had access to snRNA-seq data from more than 2 HS rats.

We have clarified that these rats were selected based on their addiction-like behavior phenotypes from a larger cohort.

(2) I didn't look back, but did the main paper point out that the rats are treated with oxycodone rather than morphine?

We have clarified this distinction in the Methods section.

This article shows how greenwashing claims in consumer products function as misleading language that lacks any real transparency. Ethically, this creates a strong feeling of betrayal among consumers because brands are exploiting environmental trust for profit while continuing to generate massive waste. This deceptive behavior goes beyond bad marketing, it highlights critical legal risks because failing to provide honest product data increasingly triggers strict regulatory penalties and sanctions from consumer protection authorities.

eLife Assessment

This important study investigates how the nervous system adapts to changes in the mechanics of the body, which are altered through a tendon transfer surgery affecting finger extensor and flexor muscles. By measuring task performance, joint kinematics, and muscle activity for several weeks post surgery, the authors provide convincing evidence that monkeys undergo a two-phase adaptation process. First, they adopt a maladaptive strategy to overcome the functional challenges imposed by the surgery, and then revert to a strategy that uses the same patterns of muscle coactivation observed pre-tendon transfer.

Reviewer #1 (Public review):

Summary:

Many studies have investigated adaptation to altered sensorimotor mappings or to an altered mechanical environment. This paper asks a different but also important question in motor control and neurorehabilitation: how does the brain adapt to changes in the controlled plant? The authors addressed this question by performing a tendon transfer surgery in two monkeys during which the swapped tendons flexing and extending the digits. They then monitored changes in task performance, muscle activation and kinematics post-recovery over several months, to assess changes in putative neural strategies.

Strengths:

(1) The authors performed complicated tendon transfer experiments to address their question of how the nervous system adapts to changes in the organisation of the neuromusculoskeletal system, and present very interesting data characterising neural (and in one monkey, also behavioural) changes post tendon transfer over several months.

(2) The fact that the authors had to employ to two slightly different tasks -one more artificial, the other more naturalistic- in the two monkeys and yet found qualitatively similar changes across them makes the findings more compelling. After all these are very challenging experiments!

(3) The paper is well written, the analyses are sound, and the authors interpret the data appropriately, acknowledging the key limitations.

Weaknesses:

None of note.

Reviewer #3 (Public review):

Summary:

In this study, Philipp et al. investigate how a monkey learns to compensate for a large, chronic biomechanical perturbation--a tendon transfer surgery, swapping the actions of two muscles that flex and extend the fingers. After performing the surgery and confirming that the muscle actions are swapped, the authors follow the monkeys' performance on grasping tasks over several months. There are several main findings:

- There is an initial stage of learning (around 60 days), where monkeys simply swap the activation timing of their flexors and extensors during the grasp task to compensate for the two swapped muscles.

- This is (seemingly paradoxically) followed by a stage where muscle activation timing returns almost to what it was pre-surgery, suggesting that monkeys suddenly swap to a new strategy that is better than the simple swap.

- Muscle synergies seem remarkably stable through the entire learning course, indicating that monkeys do not fractionate their muscle control to swap the activations of only the two transferred muscles.

- Muscle synergy activation shows a similar learning course, where the flexion synergy and extension synergy activations are temporarily swapped in the first learning stage and then revert to pre-surgery timing in the second learning stage.

- The second phase of learning seems to arise from making new, compensatory movements (supported by other muscle synergies) that get around the problem of swapped tendons.

Strengths:

This study is quite remarkable in scope, studying two monkeys over a period of months after a difficult tendon-transfer surgery. As the authors point out, this kind of perturbation is an excellent testbed for the kind of long-term learning that one might observe in a patient after stroke or injury, and provides unique benefits over more temporary perturbations like visuomotor transformations and over studying learning through development. Moreover, while the two-stage learning course makes sense, I found the details to be genuinely surprising--specifically the fact that: 1) muscle synergies continue to be stable for months after the surgery, despite being maladaptive; and 2) muscle activation timing reverts to pre-surgery levels by the end of the learning course. These two facts together initially make it seem like the monkey simply ignores the new biomechanics by the end of the learning course, but the authors do well to explain that this is mainly because the monkeys develop a new kind of movement to circumvent the surgical manipulation.

I found these results fascinating, especially in comparison to some recent work in motor cortex, showing that a monkey may be able to break correlations between the activities of motor cortical neurons, but only after several of coaching and training (Oby et al. PNAS 2019). Even then, it seemed like the monkey was not fully breaking correlations but rather pushing existing correlations harder to get succeed at the virtual task (a brain-computer interface with perturbed control).

Weaknesses:

I found the analysis to be reasonably well considered and relatively thorough. The authors have also suitably addressed my comments on the previous version. One minor weakness that remains (understandably so) is that the two animals in the study performed different tasks, and the results of the secondary synergy analysis seem to be quite different (Figure 10). That said, I don't think this weakness reduces the impact of the study, and though multiple replications of the same results would provide more convincing evidence, I don't think it's necessary to make the points that the authors are making.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

(1) I think this is an important paper, but I’m puzzled about a tension in the results. On the one hand, it looks like the behavioural gains post-TT happen rather smoothly over time (Figure 5). On the other hand, muscle synergy activations change abruptly at specific days (around day ~65 for Monkey A and around day ~45 for Monkey B; e.g., Figure 6). How do the authors reconcile this tension? In other words, how do they think that this drastic behavioural transition can arise from what appears to be step-by-step, continuous changes in muscle coordination? Is it “just” subtle changes in movements/posture exploiting the mechanical coupling between wrist and finger movements, combined with subtle changes in synergies, and they just happen to all kick in at the same time? This feels to me to be the core of the paper and should be addressed more directly.

We thank the reviewer for this insightful comment, as it touches upon the central finding of our study. The apparent tension between the smooth behavioral recovery and the abrupt shift in neural strategy is indeed a key feature of the adaptation process. We propose that this reflects the interaction of two distinct, parallel processes operating on different timescales:

A slow, gradual skill-learning process, where the monkeys incrementally developed and refined a compensatory motor strategy (i.e., the tenodesis effect). This slow refinement is responsible for the smooth improvement seen in the behavioral metrics over many weeks.

A fast, switch-like adaptive process, which governs the activation of the primary muscle synergies. The initial ‘swap’ strategy, while simple, was biomechanically conflicting and inefficient. The CNS only abandoned this flawed strategy abruptly once the slow learning process had rendered the new compensatory strategy “good enough” to be a viable alternative.

Therefore, the abrupt neural shift does not cause the behavioral improvement but is rather enabled by the gradual, underlying development of a better motor solution. To address this important point more directly within the manuscript, we added a new subheading to the Discussion section. This section is dedicated to explicitly framing our findings within this multi-timescale learning model, ensuring the link between the gradual behavioral recovery and the abrupt neural shift is clearly articulated.

(2) The muscle synergy analyses, which are an important part of the paper, could be improved. In particular:

(a) When measuring the cross-correlation between the activation of synergies, the authors should include error bars and should also look at the lag between the signals.

We thank the reviewer for these excellent suggestions to improve our analysis.

Error Bars: We agree that showing trial-to-trial variability is important. In our revision, we have added a shaded envelope (representing the SD across trials) to the cross-correlation plots in Figures 6, 9 and 10.

Time Lag: We have performed the cross-correlation analysis allowing for variable time lags and extracted the lag yielding the maximum correlation coefficient (max CC) for each session, in addition to the zero-lag correlation presented in the main figures. As hypothesized, allowing variable lags often resulted in high max CC values throughout the adaptation period, potentially obscuring the clear swap-and-revert pattern visible in the zerolag analysis. This is likely because the primary adaptation involved changes in synergy timing rather than fundamental shape. However, the analysis of the lag itself proved informative. We observed significant fluctuations in the optimal lag during the early and mid-adaptation phases, particularly around the time of the ‘switch-back’, before the lag stabilized closer to zero in the late phase.

We have added a description of this analysis to the Methods section. The results of the lag analysis are now presented in a new Supplementary Figure S6 and S7, and a sentence summarizing this finding has been added to the Results section.

(b) Figure 7C and related figures, the authors state that the activation of muscle synergies reverts to pre-TT patterns toward the end of the experiments. However, there are noticeable differences for both monkeys (at the end of the “task range” for synergy B for monkey A, and around 50% task range for synergy B for monkey B). The authors should measure this, e.g., by quantifying the per-sample correlation between pre-TT and post-TT activation amplitudes. Same for Figures 8I, J, etc.

We thank the reviewer for this detailed and insightful suggestion. We agree that our use of the term ‘reversion’ should be nuanced, as the recovery of the synergy activation patterns is substantial but not perfect.

To formally quantify these remaining differences, we performed a rigorous quantitative comparison between the pre-surgery and final-day post-surgery activation profiles. We calculated the Cosine Similarity to assess the recovery of the temporal shape, and used a Permutation Test (n=10,000) to test for statistical distinctness between the pre- and post-surgery trajectories.

Results: We found that while the temporal shapes were highly similar (Cosine Correlation > 0.90 for all synergies), the Permutation Test confirmed that the profiles remained statistically distinct (p < 0.0001) in both animals.

We have added this quantification to the text (Results). This confirms our nuanced interpretation: while the primary temporal features of the synergies reverted, the recovered motor program represents a novel, ‘good enough’ solution that is robust and functional, rather than a mathematically perfect restoration of the original baseline.

(c) In Figures 9 and 10, the authors show the cross-correlation of the activation coefficients of different synergies; the authors should also look at the correlation between activation profiles because it provides additional information.

We thank the reviewer for this comment and the opportunity to clarify our terminology. We agree that analyzing the correlation between the full activation profiles is the most informative approach. In our manuscript, the terms ‘activation coefficients’ and ‘activation profiles’ both refer to the complete, time-varying activation patterns of the muscle synergies. Therefore, the crosscorrelation analysis presented in Figures 9 and 10 is indeed the correlation between these full activation profiles. To prevent any potential ambiguity for future readers, we have revised the manuscript to use the term ‘activation profiles’ exclusively and consistently when referring to these time-varying synergy activations.

(d) The muscle synergy analysis for Monkey B is hindered by the fact that the authors lost the ability to record from the (very) functionally relevant FDS muscle. I’d repeat the synergy analyses without this muscle to understand to what extent the observed changes with respect to baseline are driven by the lack of this data.

We thank the reviewer for raising this important methodological point. We agree that controlling for changes in the recorded muscle set is crucial for a valid comparison between pre- and post-surgical synergy structures. The reviewer’s concern is based on the premise that the FDS muscle was included in the pre-surgical analysis for Monkey B but absent from the postsurgical analysis.

We would like to clarify that this is not the case. Due to the loss of the FDS signal post-surgery, we made the deliberate decision to exclude the FDS muscle from ALL synergy analyses for Monkey B, including the pre-surgical baseline period. This was done for the precise reason the reviewer identifies: to ensure a direct and unbiased “apples-to-apples” comparison and to avoid introducing the lack of this muscle as a confound. Therefore, the changes in synergy structure that we report for Monkey B can be confidently attributed to genuine physiological adaptation rather than an artifact of a changing input dataset.

(e) Figure 11: The authors talk about a key difference in how Synergy B (the extensor finger) evolved between monkeys post-TT. However, to me this figure feels more like a difference in quantity - the time course than quality, since for both monkeys the aaEMG levels pretty much go back to close to baseline levels - even if there’s a statistically significant difference only for Monkey B. What am I missing?

We thank the reviewer for this insightful question, as it has prompted us to refine our interpretation of this key finding. The reviewer correctly notes that the recovery trajectories of Synergy B appear different, and we agree that our original explanation can be improved.

A more parsimonious interpretation, and one that we believe aligns better with the data, is that both monkeys likely underwent a similar ‘arms race’, but we captured different phases of this process. In Monkey A, our recordings (starting Day 29) captured the escalating phase of this neuromuscular conflict. In contrast, for Monkey B, recordings began on Day 20, by which time this rapid escalation had likely already occurred and peaked. This difference in the timing of the ‘arms race’ is consistent with our behavioral observations; Monkey A struggled for a longer period before performing the task proficiently, suggesting a more protracted overall adaptation process. Thus, the apparent difference in the figures is likely a reflection of the observational window and the individual adaptation rate of each animal, rather than a fundamental qualitative difference in their adaptive strategy. We have revised the text to present this more unified and coherent interpretation.

(f) Lines 408-09 and above: The authors claim that “The development of a compensatory strategy, primarily involving the wrist flexor synergy (Synergy C), appears crucial for enabling the final phase of adaptation”, which feels true intuitively and also based on the analysis in Figure 8, but Figure 11 suggests this is only true for Monkey B. How can these statements be reconciled?

We believe the reviewer may be referring to Monkey A in their comment, as the strong compensatory effect is indeed seen in this animal. The core of this issue, which we have clarified in our revision, is that both monkeys developed a compensatory tenodesis grasp but used different neural strategies to achieve it.

For Monkey A, strong evidence for this strategy is provided by a clear temporal shift in the activation of its dedicated wrist flexor synergy (Synergy C). As we have now clarified in the manuscript, the peak of this synergy’s activation moved from occurring just after object contact to just before it, a re-timing well-suited to enable a tenodesis grasp.

For Monkey B, the strategy was one of subtle re-timing rather than scaling. While the total aggregated activation of its primary flexor synergy (Synergy A) did not significantly increase, its temporal profile shifted. Specifically, activation prior to object contact increased, providing the necessary wrist flexion for its assistive tenodesis grasp, which was kinematically confirmed in Figure 12. This was achieved by reallocating activation from the post-contact phase, resulting in an earlier activation peak for the synergy overall. Crucially, a finer-grained analysis reveals a precise temporal sequence within this synergy’s activation: the wrist flexor component (PL) consistently peaked just before object contact to enable hand opening, while the finger flexor component (FDP) peaked just after contact to secure the grasp.

This timing resolves the apparent biomechanical conflict. It also reveals that while both monkeys converged on the same biomechanical solution (a tenodesis grasp), the observable neural implementation appeared different. However, we must be cautious in directly comparing the computed synergy structures themselves, as the analysis for Monkey B was performed without the FDS muscle. The apparent “multi-functional synergy” in Monkey B is most likely a consequence of this missing data. What is clear and robust, however, is that both monkeys converged on a remarkably similar temporal solution: they both learned to re-time the activation of their key wrist flexor muscles to the pre-grasp phase.

In Monkey A, this was observed in the temporal shift of its dedicated wrist flexor synergy (Synergy C). In Monkey B, this was observed in the temporal shift of the Palmaris Longus (PL) muscle itself (which, in our computed synergies, was grouped into Synergy A). This convergence on an identical temporal adaptation, regardless of the computed modular organization, is the key finding. We have revised the manuscript to articulate this more precise and defensible interpretation.

(3) Experimental design: at least for the monkey who was trained on the “artificial task” (Monkey A), it would have been good if the authors had also tested him on naturalistic grasping, like the second monkey, to see to what extent the neural changes generalise across behaviours or are task-specific. Do the authors have some data that could be used to assess this even if less systematically?

We thank the reviewer for raising this important point regarding the generalizability of our findings across different behaviors. We fully agree that a direct comparison of both tasks in the same animal would have been a valuable experiment. Unfortunately, we do not have systematic data on naturalistic grasping for Monkey A that would allow for such a direct comparison. We therefore view the two tasks as providing complementary evidence. Monkey A’s data shows the adaptation process during a highly stereotyped behavior, while Monkey B’s data demonstrates that a similar two-phase adaptive process occurs during a more naturalistic, unconstrained task. The convergence of these findings strengthens our overall conclusion that this multi-timescale adaptation is a robust principle of motor learning. Nonetheless, the reviewer raises a fascinating question about the task-specific tuning of motor synergies, which remains an excellent direction for future studies.

(4) Monkey B’s behaviour pre-tendon transfer seems more variable than that of Monkey A (e.g., the larger error bars in Figure 5 compared to monkey A, the fluctuating crosscorrelation between FDS pre and EDC post in Figure 6Q). This should be quantified to better ground the results since it also shows more variability post-TT.

We thank the reviewer for this excellent suggestion to formally quantify the presurgery behavioral variability. We have performed the suggested analysis on the "Grip Formation Time" metric (Fig. 5A), which was the comparable metric between the two tasks. Our calculation of the Coefficient of Variation (CV) confirms the reviewer’s observation. Monkey B’s pre-surgery performance was substantially more variable (CV = 81.93%) than Monkey A’s (CV = 46.62%). Furthermore, a non-parametric test for equal variances (Ansari-Bradley test) confirmed that this difference is highly statistically significant (p < 0.0001). We have added a description of this analysis to the Methods and reported this finding in the Results section to provide a clearer context for the baseline differences between the subjects.

(5) Minor: Figure 12 is interesting and supports the idea that monkeys may exploit the biomechanical coupling between wrist and fingers as part of their functional recovery. It would be interesting to measure whether there is a change in such coupling (tenodesis) over time, e.g., by plotting the change in wrist angle vs change in MCP angle as a scatter plot (one dot per trial), and in the same plot show all the days, colour coded by day. Would the relationship remain largely constant or fluctuate slightly early on? I feel this analysis could also help address my point (1) above.

We thank the reviewer for this excellent and insightful suggestion. We have performed the suggested analysis for Monkey B, plotting the trial-by-trial relationship between wrist and MCP angles for all recording days (New Figure 13).

The results clearly show the gradual refinement of the tenodesis coupling. Pre-surgery, there was no correlation (R²=0.00). Immediately post-surgery (Day 22), the relationship was weak and variable (R²=0.16), reflecting an exploratory phase. Over the following weeks, the coupling became progressively stronger and more consistent, with the R² value peaking at 0.58 around Day 56, indicating a robust exploitation of the new strategy. The relationship then stabilized at a moderate level (R² ~0.2-0.3) in the final days. This analysis provides direct kinematic evidence for the slow, gradual skill-learning component of our two-state model. It beautifully complements our response to the reviewer’s first point by visualizing the underlying refinement process that occurred concurrently with the more abrupt neural shifts. We have added this new figure and a description of these results to the manuscript.

Reviewer #2 (Public review):

Weaknesses:

The most notable weakness of the study is the incompleteness of the data. [...] As a result, it is difficult to make general conclusions from the study, and it awaits further analysis or the addition of another subject.

We thank the reviewer for this critical and accurate assessment of the study’s limitations. The reviewer is correct that the datasets for the two monkeys are incomplete in different ways and that the tasks were not identical. We fully acknowledge these limitations throughout the manuscript. Rather than viewing these differences as a weakness that prevents generalization, we propose that they offer a unique strength in the form of complementary evidence. We consider the two animals not as a direct replication, but as two distinct case studies that test the same underlying hypothesis under different conditions.

Monkey A, with its high-quality EMG and highly stereotyped task, provides a detailed, quantitative view of the neural adaptation process, allowing us to precisely characterize phenomena like the ‘neuromuscular arms race’.

Monkey B, with its kinematic data and more naturalistic task, provides crucial evidence that the same fundamental principles, a two-phase adaptation and the eventual development of a compensatory strategy, generalize to a less constrained, more behaviorally relevant context. We believe the key finding is the convergence of the results. Despite the differences in individual strategy, task demands, and available data, both animals demonstrated the same core "swapand-revert" adaptive process. We propose that this convergence from heterogeneous sources lends support to the generalizability of our conclusions, suggesting that the multi-timescale adaptation we describe may be a general feature of motor learning following such perturbations. We agree that future studies with more subjects are needed to fully establish this principle. Nonetheless, we feel that the convergent evidence from these two complementary cases provides a valuable foundation for the model we present.

A second weakness is the insufficient analysis of the movements themselves, particularly for Monkey A. [...] Since the authors have video data for both monkeys, it is surprising that it was not used to extract landmarks for kinematic analysis, or at least hand/endpoint trajectory, and how it is adjusted over time. Adding more behavior data and aligning it with the EMG data would be very helpful for characterizing motor recovery and is needed to support conclusions about underlying neural control strategies for functional improvement.

We thank the reviewer for this important suggestion. The reviewer’s comment prompted us to re-examine our behavioral data, and we have now performed additional analyses that we agree provide a much clearer link between the neural changes and functional recovery.

For Monkey A, we have quantified the ‘pull times’ on a day-by-day basis. This analysis reveals a clear, gradual learning curve: pull times were initially long and variable post-surgery but steadily decreased and stabilized over the recovery period. This provides a direct, quantitative measure of motor performance recovery for this animal.

For Monkey B, we have performed a detailed analysis of the ‘grasp aperture’ prior to object contact. This kinematic analysis is particularly revealing, as it shows the development of the compensatory strategy in real-time. The grasp aperture was initially very small post-surgery, reflecting the monkey’s inability to open its hand. It then steadily increased over the next ~40 days as the monkey learned and refined the compensatory tenodesis grasp, before stabilizing at a new, functional baseline.

We believe these new analyses directly address the reviewer’s concern by providing a more detailed picture of motor recovery. The grasp aperture data, in particular, offers a clear kinematic correlate for the slow, skill-learning process that we propose runs in parallel to the more abrupt neural reorganization. We have added these results as a new figure in the main text of our revised manuscript.

Considering specific conclusions, the statement that the monkeys learned to use “tenodesis” over time by increasing activation of a wrist flexor muscle synergy does not seem to be fully supported by the data. [...] Given these issues, it is not clear how to align the EMG and kinematic data and interpret these findings.

We thank the reviewer for this detailed and critical analysis. They raise an excellent point and have correctly observed that the adaptation is not a simple, uniform increase in wrist flexor synergy amplitude. Our interpretation, which we have clarified in the manuscript, is that the monkeys learned a more sophisticated strategy: a precise re-timing of the wrist flexor activation to occur earlier in the movement, specifically to pre-shape the hand for the grasp.

For Monkey A: The reviewer correctly notes that the peak amplitude of Synergy C (the wrist flexor synergy) around the moment of grasp (0% task range) is lower in the final phase compared to baseline. However, the crucial change is temporal: the peak of this synergy’s activation shifts from occurring just after the grasp (~+1%) to occurring just before it (~-2%). This re-timing is perfectly suited to enable finger extension via the tenodesis effect immediately prior to object contact. The subsequent lower amplitude may reflect a more efficient, less forceful movement once this new skill was refined.

For Monkey B: The reviewer is right that this monkey does not have a dedicated wrist flexor synergy and that the overall amplitude of the PL muscle does not increase dramatically. However, a closer look at its activity profile (Fig. S2-AN) reveals a clear and consistent increase in activation specifically in the pre-contact phase (~7% task range). This is the precise neural signature of the assistive tenodesis grasp that is kinematically confirmed in Figure 12. The monkey is not simply scaling up the synergy; it is strategically activating it earlier to prepare for the grasp.

In summary, the key evidence linking the EMG to the tenodesis strategy is in the temporal domain. The learned re-timing of the wrist flexor activation to the pre-grasp phase is the crucial link that aligns the neural and kinematic data. We have revised the manuscript to make this distinction between amplitude scaling and temporal shifting clearer.

A more minor point regarding conclusions: statements about poor task performance and high energy expenditure being the costs that drive exploration for a new strategy are speculative and should be presented as such. Although the monkeys did take longer to complete the tasks after the surgery, they were still able to perform it successfully and in less than a second and no measurements of energy expenditure were taken.

We thank the reviewer for this important point regarding the precision of our language. We agree that statements regarding ‘high energy expenditure’ and the specific drivers for exploring a new strategy are interpretations of the data, not direct measurements, and should be framed as such.

Our speculation about energetic cost is based on the significant increase in muscle co-activation we observed (e.g., Fig. 11), a phenomenon widely understood to be metabolically expensive. Similarly, while the monkeys were still successful, their prolonged movement times and inefficient motor patterns represent a clear performance deficit compared to their highly optimized presurgical baseline, which we propose acted as a driver for further adaptation. In our full revision, we have carefully revised the manuscript to soften these claims. We have used more speculative language, such as “we hypothesize that...”, “the likely cost of...”, or “may have provided the impetus for...” to ensure that our interpretations are clearly distinguished from our direct empirical findings.

A small concern is whether the tendon transfer effect may fail over time, either due to scar tissue formation or tendon tearing, and it would be ideal if the integrity of the intervention were re-assessed at the end of the study.

We thank the reviewer for raising this important point regarding the long-term integrity of the tendon transfer. We agree that a terminal anatomical re-assessment would be an ideal control. While a terminal assessment was not performed as part of this study’s protocol, we were able to monitor the transfer’s integrity throughout the study. We are confident the transfer remained functionally intact for two key reasons:

(1) Physical Monitoring: We periodically used ultrasound imaging to non-invasively visualize the tendon repair, which allowed us to confirm its continued physical integrity.

(2) Functional Evidence: This physical confirmation was corroborated by the functional data. Both animals achieved stable, proficient task performance that was maintained for months. Furthermore, the late-phase neuromuscular control strategies became highly consistent. A significant failure, such as a tendon tear or prohibitive mechanical scarring, would be incompatible with this sustained behavioral and neural stability.

Nevertheless, we agree that a terminal assessment is an excellent methodological suggestion that should be incorporated into the design of future long-term studies of this nature.

Reviewer #3 (Public review):

(1) First, I find myself wondering about the physical healing process from the tendon transfer surgery and how it might contribute to the learning. Specifically, how long does it take for the tendons to heal and bear forces? If this itself takes a few months, it would be nice to see some discussion of this.

We thank the reviewer for this insightful question about the potential contribution of the physical healing process to the adaptation timeline. Our surgical protocol was specifically designed to ensure the tendon transfer was biomechanically robust from the outset, minimizing the role of healing as a rate-limiting factor.

We used a Pulvertaft weave technique, which is known to achieve mechanical strength equivalent to that of a native tendon shortly after the procedure (Graham et al., 2023). The repair involved more than two weaves and utilized high-strength suture material to maximize its initial forcebearing capacity. While full fibrous integration around the suture site typically occurs within approximately six weeks, the repair itself was strong enough to bear physiological forces immediately post-surgery. Therefore, the prolonged, complex, two-phase multi-month behavioral recovery and the neural reorganization we observed cannot be attributed to a slow physical healing process. Instead, this supports our conclusion that the observed timeline reflects the challenges and constraints of a purely neural adaptation and skill-learning process. To make this crucial point clear to all readers, we have added these details about the surgical method to the Methods section and included a brief discussion of its implications in the Discussion.

(2) Second, I see that there are some changes in the muscle loadings for each synergy over the days, though they are relatively small. The authors mention that the cosine distances are very small for the conserved synergies compared to distances across synergies, but it would be good to get a sense for how variable this measure is within synergy. For example, what is the cosine similarity for a conserved synergy across different pre-surgery days? This might help inform whether the changes post-surgery are within a normal variation or whether they reflect important changes in how the muscles are being used over time.

We thank the reviewer for this excellent and insightful suggestion. Establishing a baseline for normal day-to-day variability is an important control for our synergy analysis.

We have performed this analysis in full. Specifically, to quantify baseline stability, we calculated the cosine similarity between the spatial synergy weights (W) of each individual recording day and the pre-surgery average. This provides a rigorous measure of day-to-day variability relative to the stable baseline structure. We have added these data to Figure 7 (Panel I), which plots the pre-surgery similarity (blue traces) alongside the post-surgery adaptation (red traces).

We found that baseline stability was remarkably high, with cosine similarity consistently exceeding 0.99 (e.g., Monkey A: 0.99 ± 0.001). This quantification allows the reader to formally assess that the changes observed post-surgery (e.g., drops to ~0.80 or ~0.60 in Monkey B) are well outside the range of normal physiological fluctuation, representing subtle but genuine structural adaptation.

(3) Last, and maybe most difficult (and possibly out of scope for this work): I would have ideally liked to see some theoretical modeling of the biomechanics so I could more easily understand what the tendon transfer did or how specific synergies affect hand kinematics before and after the surgery. Especially given that the synergies remained consistent, such an analysis could be highly instructive for a reader or to suggest future perturbations to further probe the effects of tendon transfer on long-term learning.

We thank the reviewer for this excellent and forward-thinking suggestion. We completely agree that a detailed biomechanical model of the tendon transfer would be a powerful tool for understanding the mechanical consequences of the surgery and for interpreting the function of the recorded muscle synergies. However, creating a subject-specific musculoskeletal model with the fidelity required to accurately simulate synergy-to-kinematic transformations is a highly complex project that we feel is well beyond the scope of the current manuscript. Such an endeavor would constitute a major research project in its own right.

Our study’s primary focus was to provide a detailed, longitudinal characterization of the in-vivo neural adaptation following this perturbation, a dataset that is itself rare and valuable. We aimed to document the physiological learning process as it unfolded over many months. Nonetheless, the reviewer’s point is exceptionally well-taken. Currently, we are constructing a monkey musculoskeletal model and performing tendon transfer on this model to investigate what kind of characteristics in the learning process reproduce the synergy changes observed in the experiments. Although this project is still in progress, to date, we have demonstrated that the robustness of synergies themselves is necessary for changes in muscle activity at the synergy level (Nakajima N, Wang S, Ogihara N, Oya T, Seki K, Funato T, Upper Limb Musculoskeletal Model of Macaque Monkey for Approaching Adaptation Mechanism to Tendon Transfer, Society for Neuroscience 2023, Washington DC, USA, 2023).

The rich dataset we have collected in the present research could serve as an excellent foundation for developing and validating such a model in the future. We believe that combining these two approaches is a critical and exciting next step for the field, and we have highlighted this as a key future direction in our discussion.

Recommendations for the authors:

Reviewing Editor Comments:

When revising the manuscript for resubmission, please try to improve the visual presentation of the data, which is a point highlighted by all three reviewers during the discussion, including making the presentation of monkey-specific results more consistent across subjects.

We have comprehensively revised the figures to ensure a consistent and clear visual presentation, as requested. Specifically, we standardized the layout across all main and supplementary figures (placing Monkey A consistently in the top rows or left columns and Monkey B in the bottom rows or right columns) and applied unified color schemes throughout the manuscript. Furthermore, we harmonized the presentation of the analytical results, such as the specific cross-correlation pairings in Figures 9 and 10, to ensure that the data for both subjects are presented with identical logic, facilitating direct comparison.

Reviewer #1 (Recommendations for the authors):

(1) Please revise the writing; some words are missing (line 90), and some sentences could be clarified slightly, even if the paper is well written (lines 317-320). The paragraph including the idea of tenodesis could also be further clarified, I think.

Thank you for pointing these out. We have corrected the missing word (osteoarthritis) on line 90. We have also revised lines 317-320 to remove ambiguity. Furthermore, the section describing the tenodesis effect (now section "Distinct neural implementations...") has been substantially rewritten for improved clarity, incorporating a more detailed explanation of the biomechanics.

(2) In the Introduction, the authors cite Hunter and Eckstein 2009 and Mercuri and Muntoni 2013 without describing the pathological conditions; this will not be clear for not nonspecialists.

Thank you. We have added brief descriptions ("osteoarthritis, a degenerative joint disease," and "muscular dystrophy, which involves progressive muscle weakness,") directly into the Introduction sentence where these references appear.

(3) Data presentation: I often thought that the data could be presented more clearly:

(a) For example, Figure 3D and 4D should show error bars around the mean to have a sense of the consistency of pre-lesion behaviour. Same for other figures like Figure 6.

We appreciate the reviewer's suggestion to visualize data consistency. (a) Figures 3D, 4D, and 6 (EMG Profiles): For these figures, we opted to display mean traces and peak markers to clearly illustrate the temporal shifts and relationships between muscles. Overlaying multiple standard deviation envelopes in these comparative plots would significantly reduce legibility. However, to fully address the reviewer's request to see the consistency of pre-lesion behavior, we direct attention to Supplementary Figure S1, which presents the complete EMG profiles with full error tubes (Mean ± SD) for every recorded muscle. (b) Quantitative Analysis Figures: We ensured that variability is explicitly visualized in all statistical analyses. The crosscorrelation time-courses in Figures 6 (G-Q), 9, and 10 are plotted with shaded error tubes to show variance. Similarly, the aggregated EMG analysis in Figure 11 utilizes bar plots with explicit error bars to quantify the statistical consistency of the changes.

(b) The autocorrelation analysis in Figure 6 should also include measures of lag if it’s not at zero lag. If it’s the latter, please specify it in the Methods.

We thank the reviewer for this question regarding the cross-correlation analysis presented in Figure 6 (Panels G-J, P-Q). We confirm that this analysis was performed at zero time lag. To clarify this, we have added a sentence to the Methods section (Subsection "Crosscorrelation analysis") explicitly stating that the EMG cross-correlations shown in Figure 6 were calculated at zero lag. We have also added a clarifying note ("at zero time lag") to the description of these panels within the Figure 6 caption.

(c) Seeing EMG patterns similar to those presented in Figures 3D and 4D at different times post-lesion (e.g., as a Supplementary figure) would also give readers a better intuition of the neural changes.

We thank the reviewer for this suggestion to provide more intuitive examples of the neural changes. We realize we did not sufficiently highlight this in the main text, but this complete data is already available in the manuscript. Supplementary Figures S1 and S2 provide a comprehensive overview of the EMG patterns for all recorded muscles in Monkey A and Monkey B, respectively. These figures show the pre-surgery and post-surgery average profiles for all recording sessions as well as the average profiles from five different post-surgery landmark days, covering the entire adaptation period. We have added explicit cross-references to these figures in the main text.

(d) I couldn’t fully understand the analysis in Figure 4E; clarify.

We thank the reviewer for noticing this oversight. The reviewer is correct that Figure 4E was not referenced in the main text. This panel was intended to show the baseline kinematic profiles (MCP and wrist angles) for Monkey B's control session, corresponding to the average EMGs shown in panel 4D. Given that our more comprehensive kinematic analyses are now presented in Figure 12 and the new Figure 13, we believe panel 4E is largely redundant. To improve the clarity and focus of Figure 4, we have removed panel 4E and its description from the revised manuscript.

(e) Some figures showing neural changes (e.g., Figures 6G-J, 6P,Q, Figures 9 and 10, and even Figure 11 for different reasons) would become more understandable if they were accompanied by the behavioural changes (e.g., something like Figure 5A on top of them).

We agree that visualizing the temporal link between neural reorganization and behavioral recovery is essential for interpreting the data. We have implemented this suggestion by overlaying behavioral metrics onto the right y-axes of Figures 6 (G-Q), 9, 10, and 11. However, regarding the specific behavioral metric, we opted to overlay the maladaptive behavior/aberrant reaching metric (from Figure 5B) rather than the grip formation time (Figure 5A). We found that the maladaptive behavior profile provided a clearer and more direct correlate to the neural data, as its peak coincides precisely with the ‘swapped’ synergy phase, thereby effectively illustrating the functional cost of that specific neural state.

(f) Some figure captions could be improved by adding more detail (e.g., for Figure 6).

We agree. We have substantially expanded and improved the captions for Figure 6 and Figure 7 to make them more self-contained and guide the reader more effectively through the key findings presented in the panels. We have also reviewed other captions for clarity.

(g) I’d show the cosine distance between synergies across days as a main figure, e.g., as part of Figure 7, because this is an important result.

We agree that the longitudinal stability of the synergy structures is a crucial result that deserves prominence. We have implemented this suggestion by adding a new panel, Figure 7 (I, K) for primary synergies and Figure 8 (K, L) for secondary synergies, which plots the cosine similarity of the spatial synergy weights across the entire experimental timeline. This figure explicitly visualizes the high stability of the pre-surgery baseline (blue traces, similarity > 0.99) and contrasts it with the dynamic structural tuning observed during the post-surgery adaptation (red traces), providing a clear, day-by-day account of synergy evolution as requested.

(h) In Figure 7C, D and G, H, it’d be interesting to also see in the background the EMG for the transferred muscle that belongs to each synergy, to appreciate their relationship.

We thank the reviewer for this suggestion. To illustrate the close relationship between the primary synergies and their key constituent muscles, while avoiding visual clutter in the complex post-surgery plots, we have modified the pre-surgery panels of Figure 7 (C, D, G, H). In these panels, we have now overlaid the average pre-surgery EMG profile of the primary transferred muscle belonging to that synergy (e.g., FDS for Synergy A, EDC for Synergy B) as a thin, gray, dashed line. This visually confirms the tight correlation between the synergy profile and the muscle’s activity at baseline.

(i) In page 10, the authors report as maladaptive behaviour the duration of the aberrant reaching component from day 29 (monkey A) and day 20 (monkey B). What was happening before those recording dates? Were the monkeys recovering?

Thank you for this question. We have added two sentences to the start of the Results section (“Functional Recovery Follows...”) clarifying that the period between surgery and formal recordings included approximately one week of home cage recovery followed by several weeks of assisted task practice. Formal recordings began once the monkeys could perform the task consistently without assistance.

(j) In the Methods (EMG Analysis), the authors state that they resumed their recordings post-TT “once they (the monkeys) were able to perform the task on their own”. It would be good if the authors made this more precise (e.g., based on success rate or another metric).

We thank the reviewer for this suggestion to increase precision. We have revised the Methods section to include the specific criteria used for resuming post-surgical recordings. Recordings were restarted once the monkeys were able to perform the task independently (i.e., without assistance from the experimenter) and consistently achieved a successful trial count of at least 100 trials within a single experimental session.

(k) Line 266- reads “Alternation of EMG activity in non-transferred muscle suggests one possibility: TT might alter the control strategy of coordinated muscle activity for hand movement by modifying the transferred muscles and their agonists as a cohesive unit”, however, some “muscles showed patterns that were incompatible with a simple swap” (Lines 255-256). Doesn’t this observation suggest that what happens is not a simple change in muscle synergies?

We thank the reviewer for this insightful question regarding the interpretation of muscles with adaptive patterns incompatible with the primary ‘swap-and-revert’. We agree that these observations require careful consideration within the modular framework. Our interpretation is that these muscles do not represent evidence against modular control, but rather reflect the involvement of multiple modules adapting concurrently. Specifically, muscles like FCR and PL, which showed distinct patterns, are primary members of Synergy C (the wrist flexor synergy) in Monkey A. Their adaptive profile is therefore consistent with the task-specific recruitment and retiming of Synergy C as part of the compensatory tenodesis strategy, rather than being a deviation from the swap observed in Synergies A and B. Synergies represent the dominant, shared variance in muscle activity. While they capture the overall strategy, some degree of individual muscle variation or the influence of secondary synergies is expected. We have added a sentence to the Results section to clarify that these diverse patterns likely reflect the differential involvement of muscles in multiple adapting synergies. We believe the overall evidence still strongly supports the modulation of stable synergies as the primary mechanism of adaptation in this paradigm.

(l) You may want to call synergy A and synergy B, synergy F and synergy E to make recall easier? (Same for synergy C and D, which could be F2 and E2).

We thank the reviewer for this helpful suggestion aimed at improving clarity. We considered renaming the synergies based on function (e.g., F/E). However, given the number of figures and the complexity of a global change, and the fact that the functional roles of Synergies C and D differed between animals, we decided to retain the original A/B/C/D labels for consistency. To ensure clarity for the reader, we have carefully checked the manuscript to ensure that we consistently define the primary functional role of each synergy (e.g., "Synergy A, the primary finger flexor synergy") when it is discussed.

(m) Lines 315-317 - “These pattens of changes in synergy 3 and 4, both contributed minimally to the EMG of transferred muscles” -> This statement puts the causality as synergies cause muscles to activate according to certain patterns, which is supported by work by several groups -including the authors- however, they could also reflect biomechanical and task constraints as other have argued; perhaps this tone would be better for the discussion?

We thank the reviewer for this nuanced point regarding the interpretation of synergy contributions. We agree that the causal relationship between computed synergies and muscle activity is complex and can reflect both neural commands and task constraints. To address this, we have revised the sentence in question in the Results section. Instead of stating that the synergies "contributed minimally," we now state that the changes in these synergies "were associated with minimal EMG activity in the transferred muscles." This phrasing is more descriptive of the observation and less implicitly causal, while retaining the key point within the flow of the results. The subsequent sentences, which offer interpretation, are already framed speculatively ("This suggests...", "may have served...").

(n) Line 403 How do the authors conclude from the synergy patterns in Figure 11 that the early post-TT is characterised by “an unstable and inefficient neural control strategy”? To me, this is shown clearly in the behaviour, not in these plots, unless I’m missing something?

We thank the reviewer for this comment, which highlights the need to clearly connect our neural findings to the behavioral outcome. The reviewer is absolutely correct that the behavioral data (Fig. 5) provides the most direct evidence of instability and inefficiency during the early adaptation phase. Our intention was to argue that the neural patterns observed in Figure 11 provide a physiological correlate for this behavioral inefficiency. Specifically, the escalating aggregated EMG activity observed in the conflicted extensor synergy (Synergy B), which we term the ‘arms race’, represents significant muscle co-activation. Such co-activation is widely understood to be energetically costly and reflects a suboptimal control strategy where the CNS is essentially "fighting itself" against the altered mechanics. To make this link clearer, we have revised the concluding sentence of the relevant paragraph in the Discussion ("The early adaptation phase...") to explicitly state that this escalating co-activation is a known marker of inefficient recruitment and that it occurred concurrently with the period of poor behavioral performance shown in Figure 5.

(o) Lines 469-471. The authors suggest that muscle synergies may be preserved post-TT because a modular approach (to motor control) may be computationally easy and metabolically cheap. To me, recent data suggest that the most parsimonious explanation is what they later say: that the nervous system may not be plastic enough to change this (e.g., see Makin and Krakauer, “Against reorganisation” also in eLife).

We thank the reviewer for raising this important theoretical point and for referencing the relevant literature on constraints on cortical reorganization. We agree that the preservation of muscle synergies in the face of such a profound perturbation is a key finding that warrants careful interpretation. In our revised Discussion (section "The CNS Defaults to a Modular Strategy..."), we have now explicitly incorporated the perspective that synergy stability may reflect inherent constraints on neural plasticity, citing Makin and Krakauer (2023), alongside our original hypothesis regarding computational and metabolic efficiency. We present these ideas not as mutually exclusive, but as potentially complementary factors that both contribute to the CNS’s apparent preference for modulating existing modules rather than fundamentally restructuring them.

(p) Lines 501-503. Also on interpretation. Would the metabolic cost indeed be much higher? Couldn’t the observed change in strategy be explained purely based on performance metrics?

This is an important point. We agree that statements regarding high energy expenditure are interpretations, not direct measurements. We have carefully revised the manuscript (Abstract, Results, and Discussion) to soften these claims, using more speculative language (e.g., "likely costly," "what we propose was...") to clearly distinguish our interpretations from direct empirical findings.

(q) Lines 538-. The authors link the initial adaptation phase to the fast process reported in adaptation studies and say that this leads to poor retention. However, it seems from their data that the behaviour is stable across (early) days, so doesn’t this rule out such an interpretation?

We thank the reviewer for this insightful question regarding the interpretation of the early adaptive phase within the two-state model framework. The reviewer correctly notes that the early post-surgical behavior, while maladaptive, appeared relatively stable across days and did not show the rapid decay sometimes associated with the "poor retention" characteristic of the fast system. We agree that this apparent stability requires careful interpretation. In our revised Discussion (section "A Multi-Timescale Model..."), we now propose that the fast system is primarily responsible for the initial, rapid adoption of the ‘swap’ strategy in response to the large error signal. The subsequent persistence of this flawed but stable state for several weeks is likely not due to strong retention by the fast system itself, but rather reflects the time required for the parallel slow system to gradually develop a more effective compensatory strategy (i.e., the tenodesis grasp). Once this alternative strategy became viable, it enabled the abrupt "switchback," which we also attribute to the fast system recalibrating away from the highly costly swap strategy. Therefore, we believe our data is consistent with the involvement of a fast system driving rapid strategic shifts, even if the typical "poor retention" phenotype is masked by the lack of a viable alternative strategy during the early phase.

Reviewer #2 (Recommendations for the authors):

(1) The discussion would benefit greatly from a more careful comparison with prior work characterizing the response to experimental or clinical tendon or nerve transfer in different models.

We thank the reviewer for suggesting these important references and for the recommendation to compare our findings more carefully with prior work. This is an excellent point, and we agree it will significantly strengthen the discussion. In our full revision, we have added a new paragraph to the Discussion section dedicated to this comparison. We discuss how our findings relate to classic work showing primate adaptive capacity beyond simple maladaptive responses (Sperry, 1947), EMG evidence for the persistence of original neural patterns alongside new ones in human patients (Illert et al., 1986), the critical role of altered peripheral biomechanics and myofascial force transmission in complicating adaptation (Maas & Huijing, 2012), and how our observation of synergy stability aligns with evidence for modular adaptation strategies (Berger et al., 2013). This comparison helps situate our unique findings of a multi-timescale process and synergy timing modulation within the broader context of motor relearning after musculoskeletal rearrangement.

(2) Line 90 - Which disease or condition is studied in Hunter and Eckstein (2009)?

Thank you. We have clarified this in the Introduction; the reference pertains to osteoarthritis.

(3) Line 280 for clarity in text and as a reminder to the readers, please state which muscles are involved in each synergy grouping.

We have updated the text (Results, 'Adaptation occurs through modulating...') to explicitly list the main contributing muscles for each synergy grouping (e.g., Synergy A: FDS and FCU for Monkey A). This provides the requested clarity regarding the functional identity of each synergy while maintaining readability. For the complete, quantitative muscle weight composition including minor contributors, we referred the reader to Figure 7 and Supplementary Table 1.

(4) Line 180 There are differences in the time course for measurements between the behavioral metrics and EMGs. If not recorded at fixed time intervals, the differences in the time courses for the two monkeys should be explained.

We thank the reviewer for this question regarding the time courses of our measurements. We interpret this comment in two ways, both of which we have addressed in the revised manuscript.

First, if the reviewer is asking about the overall recording schedule, they are correct that sessions were not performed at fixed daily intervals, and the specific days sampled differed between monkeys. This non-uniform sampling was due to the practical constraints of longterm behavioral experiments (e.g., animal cooperation, scheduling, weekends) and the aim to capture data during key phases of adaptation. However, within any given session, behavioral (video) and EMG data were always collected concurrently.

Second, if the reviewer is asking whether the set of days included differs between the behavioral plots (e.g., Fig 5) and the EMG/synergy plots (e.g., Figs 6, 9-11), this is a possibility depending on data quality criteria. Our criterion for including a session in the behavioral analysis was a minimum of 20 successful trials. However, for the more demanding synergy analysis, we required a higher minimum of 100 successful trials to ensure robust factorization. It is possible that a few sessions met the behavioral criterion but not the synergy criterion and were thus excluded from the latter analysis, leading to slight differences in the days presented across figures. To ensure full clarity, we have added text to the Methods section explicitly stating: (A) the rationale for the non-uniform daily sampling schedule, and (B) the specific minimum trial count criteria used for including data in the behavioral versus the synergy analyses, noting if this resulted in different sets of days being analyzed for different figures.

(5) General figure comments - The figures are informative, but they could be better presented, designed, and formatted to explain the important results in the paper. The figures should be able to explain most of the key results without entirely referring to the text to find some of the details. I had a bit of trouble understanding Figure 9 & 10. I would also like to suggest that bringing raw data into some figures (e.g., EMG of different muscle groups), such as showing stability between the synergies, could improve the results and allow the story to flow with more clarity. Likewise, clearly showing the differences between baseline EMG measurements and post-surgery measurements could improve some of the result figures.

We thank the reviewer for these important general comments on data presentation. We agree that the figures are the key to our story and are implementing several revisions based on this and other reviewer feedback to improve their clarity.

General Presentation: We have conducted a thorough review of all figures to improve layout, consistency, and font legibility (addressing R3, 1 and the Reviewing Editor's comments). This includes adjusting the layouts of Figures 3, 4, and 6 for better alignment and clarity.

Figures 9 & 10 (Cross-correlation): The reviewer mentioned having trouble understanding these figures. In our revision, we have substantially rewritten the captions for Figures 9 and 10 to be much more descriptive. We explicitly walk the reader through how to interpret the plots (e.g., "The ‘swap’ is evidenced by the drop in self-correlation... and a concurrent rise in antagonist-correlation...").

Including "Raw Data" (EMG): We thank the reviewer for this suggestion to provide more intuitive examples of the neural changes. We realize we did not sufficiently highlight this in the main text, but this complete data is already available in the manuscript. Supplementary Figures S1 and S2 provide a comprehensive overview of the EMG patterns for all recorded muscles in Monkey A and Monkey B, respectively. These figures show the pre-surgery and post-surgery average profiles for all recording sessions as well as the average profiles from five different post-surgery landmark days, covering the entire adaptation period. These figures directly visualize the swap-and-revert pattern in the transferred muscles and their agonists (e.g., EDC, ED23), as well as the diverse and complex adaptations in other nontransferred muscles (e.g., FCR, PL), as requested. To make this clearer, we have added explicit cross-references to Supplementary Figures S1 and S2 within the main Results section to ensure readers are directed to this detailed data.

Showing Differences (Pre vs. Post): To "clearly show the differences between baseline... and post-surgery measurements," we implemented the point-by-point statistical comparison of pre- vs. final-day synergy profiles (as suggested in R1, 2b). This has resulted in a new Supplementary Figure visually highlighting the precise periods in the task where the final profiles still differ significantly from baseline (Fig. S9).

We believe these additions (new figures and improved captions) will make the results much clearer and more self-explanatory, as the reviewer suggested.

(6) Figure 1 A table with all the acronyms would help with identifying all the muscles and their respective synergies (supplemental), especially when describing the muscles in the result of the discussion section.

This is an excellent suggestion. We have created a comprehensive table (Supplementary Table 1) listing all muscle abbreviations, full names, primary functional groups, and assigned synergies for both monkeys. We have added a reference to this table in the Figure 1 caption and the Methods section.

(7) Figure 2 - is this mainly from Monkey A? If so, it should be stated.

We thank the reviewer for pointing out this omission. We have updated the caption for Figure 2 to clarify that the example data shown (ultrasound, trajectories, and quantitative plots) are from Monkey A.

(8) Figure 3 & Figure 4 seems unbalanced because of the descriptive need to explain Monkey B’s tasks? The figure alignments could be better.

We thank the reviewer for this comment on the visual presentation of Figures 3 and 4. The reviewer’s observation that the figures appeared ‘unbalanced’ was correct. This was a direct consequence of two issues: (1) the different tasks required slightly different schematics (the "descriptive need" the reviewer mentioned), and (2) the original Figure 4 contained an additional kinematic panel (formerly 4E) that was unique to Monkey B, which broke the parallel structure with Figure 3.

To address this and significantly improve the alignment, we have now moved the unique kinematic panel (formerly 4E) to a new Supplementary Figure (Supplementary Figure S8). This change has allowed us to re-arrange the panels in Figures 3 and 4 so that they now follow the exact same order. We have also adjusted the layout to ensure that corresponding panels are of a consistent size. We agree that this creates a much better visual balance and makes the comparison between the two monkeys far more direct and clear, as the reviewer suggested.

(9) Figure 5. It seems like the animals can still perform the task post-surgery, but with high variability. Maybe emphasize the differences in variability between baseline and postsurgery?

We thank the reviewer for this suggestion to emphasize the changes in variability. We have now quantified this using the Coefficient of Variation (CV) for key behavioral metrics across different phases (Pre-surgery, Early, Mid, Late post-surgery). The results confirm the reviewer’s observation of high variability post-surgery, particularly in the early phase. For instance, Monkey A’s grip formation time CV spiked dramatically (Pre: 47% vs Early: 133%), while Monkey B’s remained high (Pre: 82% vs Early: 76%). Interestingly, while Monkey A’s variability returned close to baseline levels in the late phase (Late: 55%), Monkey B’s variability increased further (Late: 97%), suggesting persistent inconsistency despite functional recovery.

We also observed metric-specific changes. Monkey A’s pull time became less variable than baseline later on (Pre: 65% vs Late: 43%), suggesting refinement of that action. Conversely, Monkey B’s grasp aperture remained consistently low throughout (Pre: 26% vs Late: 19%), indicating relatively precise kinematic control was maintained or quickly regained. We have added a summary of these findings to the Results section to provide a more complete picture of how behavioral variability evolved relative to baseline during the adaptation process.

(10) Figure 6 quite a confusing figure. This figure needs to be better presented. The figure legends are hard to see for Monkey A vs Monkey B. At first, I thought Monkey B’s figure legend also represented Monkey A. I would suggest reorganizing the figures for clarity and coherence.

We agree that the original presentation of Figure 6 was dense and potentially confusing. We have completely reorganized the figure to improve clarity and coherence.

(1) Clear Separation: The figure is now structured with a strict separation between Monkey A (Left Panels, A-J) and Monkey B (Right Panels, K-Q), with prominent headers for each subject to prevent ambiguity.

(2) Improved Legends: We have redesigned the legends to be larger and placed them explicitly within their respective subject’s section to ensure it is immediately clear which data they describe.

(3) Visual Consistency: We have standardized the color schemes and axis layouts across this and all other figures to reduce cognitive load and facilitate easier comparison between subjects.

(11) Figure 12 - This figure is incomplete without Monkey A’s results. The videos in the supplemental sections seem clear enough for some kinematic analysis. The story could be more supported with more thorough measurements of the kinematics from both animals to show how they differ over time and by highlighting the two phases. As a minor note, it would be helpful to present the kinematic data together with a schematic of when during the task the data are drawn from, using the % task range scale, since that is the standard throughout the paper.

We thank the reviewer for their suggestions regarding the kinematic analysis. We agree that a parallel kinematic analysis for Monkey A, similar to that in Figure 12, would be ideal. We did attempt this. Unfortunately, while the supplemental videos for Monkey A are sufficient for observing the overall movement trajectory, they are not suitable for the detailed joint angle analysis the reviewer suggests. The videos for Monkey A were recorded at an insufficient frame rate that did not allow to reliably extract the rapid joint angle positions of the wrist and fingers during the grasping movement. This is the reason why this detailed kinematic analysis was limited to Monkey B, for which we had high-speed video recorded at 240 fps, allowing for a robust analysis of these fast movements.

We have, however, expanded our kinematic analysis for Monkey B to show the refinement of the tenodesis strategy over the full time course (New Figure 13), which does help to highlight the different adaptive phases for that animal. We have also clarified in the manuscript (e.g., in the caption for Figure 12) that the lack of Monkey A data for this specific analysis was due to the lowresolution and low-frame-rate video available.

We agree that defining the precise timing of the kinematic snapshot relative to our normalized task range is critical for accurate interpretation. In response, we have added a new panel (Figure 12C) that explicitly maps the kinematic snapshot to our standardized task timeline. This schematic clarifies that the joint angle analysis captures the hand configuration during the pre-shaping phase, specifically at 83 ms prior to object contact (which corresponds to -0.02% of the normalized task range). This ensures the kinematic data can be directly interpreted within the same temporal context as the EMG and synergy results presented throughout the paper.

Reviewer #3 (Recommendations for the authors):

First and most major: I found many of the figures much too small and incredibly difficult to read. Possibly the most difficult was Figure 7, where I had to zoom in a great deal to read what muscles corresponded to which bars. I don’t have specific suggestions here other than to make sure that figures are legible.

We thank the reviewer for highlighting this important issue. We have comprehensively revised the figures to ensure they are legible at standard publication sizes. Specific improvements include:

(1) Figure 7: We have significantly increased the font size of the x-axis muscle labels and optimized the bar chart spacing to ensure the muscle identities are readable without excessive zooming.

(2) Global Updates: Across all figures, we have increased font sizes for axis labels and titles, removed unnecessary whitespace to maximize the data-to-ink ratio, and exported all final figures in high-resolution vector formats to ensure clarity.

Second and more minor: I liked the setup of the manuscript, where the authors explained the unique benefits of their experimental methods and the question they were going after (“When confronted with structural changes to the musculoskeletal system, does the CNS adapt by modulating existing synergies, or by shifting toward more fractionated control strategies?”). However, the evolution of the paper made the answer to this question seem very confusing to me as I read it. The results show that monkeys initially modulated existing synergies in phase 1, but then reverted to the original modulation. This, in addition to the way the question was set up initially, made me think the conclusion was going to be that the synergies themselves changed in the second phase, but this paradoxically was not the case--synergies were stable throughout. I was left confused for the back half of the results section, until the discussion on tenodesis and developing compensatory movement strategies. So the answer is that the monkey learns by modulating existing synergies, but using different strategies in different learning phases. I’m not entirely sure how to avoid this confusion, but I wonder if there’s a way to foreshadow this finding earlier on.

We thank the reviewer for this valuable feedback on the manuscript’s narrative structure. We understand how the initial framing (modulation vs. fractionation) followed by the reversion of the initial modulation could lead to confusion before the compensatory strategy is fully introduced. To address this, we have made two key adjustments in the revised manuscript:

(1) In the Introduction, after posing the central question, we have added a sentence to subtly foreshadow that the adaptive process might be complex and multi-phasic, requiring analysis over extended timescales.

(2) In the Results section, at the transition point between describing the reversion of the primary synergy timings and introducing the compensatory tenodesis strategy, we have added a short paragraph to explicitly signal that the reversion was not the complete solution and that a distinct compensatory strategy emerged concurrently.

We believe these changes improve the narrative flow, provide better signposting for the reader, and mitigate the potential for confusion identified by the reviewer, making it clearer that the ultimate solution involved modulating existing synergies but via different strategies across distinct learning phases. We appreciate the reviewer’s help in identifying this area for improvement.

also appropriate for small sample sizes

https://arxiv.org/pdf/2412.16980

Patrick - Mixed quality has the lowest (best) churn rate. LTV is also much higher than "Robust + fresh". In fact, "Robust + fresh" has the third lowest LTV. Why do you think that losing robust + fresh is rarer and more expensive?

Patrick - Churn in what time?

Patrick - Check with Claudia if there understanding is the same.

Patrick- We have 1M Paid Marketing cloud users. Do you mean by paid audience, audience of paid 1M users?

eLife Assessment

This useful study uses a chemoinformatics pipeline to identify a list of candidate mosquito repellants that may be pleasant to smell and safe for humans. The strength of evidence and in particular the computational methodology are incomplete because it is insufficiently benchmarked against other leading models. At the high concentrations tested, there may also be off-target effects of the repellents on the mosquitoes that are not considered.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors set up a pipeline to predict insect repellents that are pleasant and safe to humans. This is done by daisy chaining a new classification model based predicting repellents with a published model on predicting human perception. Models use a feature-engineered selection of chemical features to make their predictions. The predicted molecules are then validated against a proxy humanoid (heated brick) and its safety is tested by molecular assays of human cells. The humanistic approach to modeling these authors have taken (which consider cosmetic/aesthetic appeal and safety) is novel and a necessary step for consumer usage. However, the importance of pleasantness over effectiveness is still up for debate (DEET is unpleasant but still used often) and the generalization of safety tests is unknown and assumed. The effectiveness of the prediction models is also still warranted. They pass the authors own behavioral tests, but their contribution to the field is unknown as both models (new and published) have not been rigorously bench-marked to previous models. Moreover, the author's breadth of literature in this field is sparse, ignoring directly related studies.

Strengths:

Humanistic approach to modeling consider pleasantness and safety. Chaining models can help limit the candidate odorants from the vastness of odor space.

Weaknesses:

The current models need to be bench-marked against leading models predicting similar outcomes. Similarly, many of these papers need to be addressed and discussed in the introduction. The authors might even consider their data sources for model training to increase performance and lexical categorization for interoperability. For instance, the Dravnikes data lexicon, currently used in the human perception lexicon, has been highly criticized for its overlapping and hard to interpret descriptive terms ("FRAGRANT", "AROMATIC").

Human Perception<br /> Khan, R. M., Luk, C. H., Flinker, A., Aggarwal, A., Lapid, H., Haddad, R., & Sobel, N. (2007). Predicting odor pleasantness from odorant structure: pleasantness as a reflection of the physical world. Journal of Neuroscience, 27(37), 10015-10023.

Keller, A., Gerkin, R. C., Guan, Y., Dhurandhar, A., Turu, G., Szalai, B., ... & Meyer, P. (2017). Predicting human olfactory perception from chemical features of odor molecules. Science, 355(6327), 820-826.

Gutiérrez, E. D., Dhurandhar, A., Keller, A., Meyer, P., & Cecchi, G. A. (2018). Predicting natural language descriptions of mono-molecular odorants. Nature communications, 9(1), 4979.

Lee, B. K., Mayhew, E. J., Sanchez-Lengeling, B., Wei, J. N., Qian, W. W., Little, K. A., ... & Wiltschko, A. B. (2023). A principal odor map unifies diverse tasks in olfactory perception. Science, 381(6661), 999-1006.<br /> Related cleaned data: https://github.com/BioMachineLearning/openpom

Insect Repellents:<br /> Wright, R. H. (1956). Physical basis of insect repellency. Nature, 178(4534), 638-638.

Katritzky, A. R., Wang, Z., Slavov, S., Tsikolia, M., Dobchev, D., Akhmedov, N. G., ... & Linthicum, K. J. (2008). Synthesis and bioassay of improved mosquito repellents predicted from chemical structure. Proceedings of the National Academy of Sciences, 105(21), 7359-7364.

Bernier, U. R., & Tsikolia, M. (2011). Development of Novel Repellents Using Structure− Activity Modeling of Compounds in the USDA Archival Database. In Recent Developments in Invertebrate Repellents (pp. 21-46). American Chemical Society.

Wei, J. N., Vlot, M., Sanchez-Lengeling, B., Lee, B. K., Berning, L., Vos, M. W., ... & Dechering, K. J. (2022). A deep learning and digital archaeology approach for mosquito repellent discovery. bioRxiv, 2022-09.

The current study assumes that insect repellents repel via its odor valence to the insect, but this is not accurate. Insect repellents also mask the body odor of humans making them hard to locate. The authors need to consult the literature to understand the localization and landing mechanisms of insects to their hosts. Here, they will understand that heat alone is not the attractant as their behavioral assay would have you believe. I suggest the authors test other behaviors assays to show more convincing evidence of effectiveness. See the following studies:

De Obaldia, M. E., Morita, T., Dedmon, L. C., Boehmler, D. J., Jiang, C. S., Zeledon, E. V., ... & Vosshall, L. B. (2022). Differential mosquito attraction to humans is associated with skin-derived carboxylic acid levels. Cell, 185(22), 4099-4116.

McBride, C. S., Baier, F., Omondi, A. B., Spitzer, S. A., Lutomiah, J., Sang, R., ... & Vosshall, L. B. (2014). Evolution of mosquito preference for humans linked to an odorant receptor. Nature, 515(7526), 222-227.

Wei, J. N., Vlot, M., Sanchez-Lengeling, B., Lee, B. K., Berning, L., Vos, M. W., ... & Dechering, K. J. (2022). A deep learning and digital archaeology approach for mosquito repellent discovery. bioRxiv, 2022-09.

Comments on revisions:

The revisions made to the manuscript do not fully address the concerns raised in the previous round of review. The authors are encouraged to consider the following points to strengthen the work.

The benchmarking of the human perception models against Keller et al. (2017) and Gutiérrez et al. (2018) is insufficient, as the field has progressed considerably in the last five years with newer approaches using larger data sources. Benchmarking against more recent models would better situate the contribution of this work.

The exclusion of human repellency data from preprint Boyle et al. (2016) is worth reconsidering. For a study that takes an explicitly human-centric modeling approach, human behavioral data on repellency, pleasantness, and usage intent would directly support the central claims of the manuscript.

The key claims regarding repellency and consumer acceptability would be considerably strengthened by the addition of these data.

Reviewer #2 (Public review):

Summary:

This is an interesting study that seeks to identify novel mosquito repellents that smell attractive to humans. This is the second time I have reviewed, and the authors have not done anything to address the weaknesses. Although the subject matter may provide important new information for the development of new repellents, its current breadth is limited without additional assays. Arm-in-cage assays, testing the longevity of the new repellents, other ML analyses and confusion matrices, would strengthen the manuscript and demonstrate innovation. The lack of cohesion and new experimental results weakens the manuscript.

Strengths:

The combination of standard machine learning methods with mosquito behavioral tests is a strength.

Weaknesses:

The study would be strengthened by describing how other modern ML approaches (RF, decision trees) would classify and identify other potential repellents.

A comparison of the repellent activity between DEET and the top ten hits identified in this new study indicates little change in repellent activity (~3%), suggesting that DEET remains the gold standard. Without additional toxicity tests and longevity tests, the study is arguably incremental. The study's novelty should be better clarified.

The Methods in the repellency tests are sparse, and more information would be useful. Testing the top repellents at low doses (<<1%) and for long periods (2-12 h) would strengthen the manuscript. Without this information, the manuscript is lacking in depth.

Testing human subjects on their olfactory percept of the repellents would also increase the depth and utility of the manuscript. Without additional experiments, the authors' conclusions lack support and have limited impact on the state-of-the-art.

This manuscript is a mix of different approaches, which makes it lack cohesion. There is the ML method for classifying new repellents that smell good, but no testing of the repellents on human volunteers. The repellents are not tested at realistic concentrations and durations. And the calcium mobilization test is strange, and makes little sense in the context of the other experiments and framing of the manuscript.

Comments on revisions:

The authors have a potentially strong manuscript. However, I would urge the authors to address the reviewer comments in a substantive manner.

Author response:

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

Mosquito-transmitted diseases cause nearly a million deaths every year and significant worldwide morbidity. Moreover, the geographical range of mosquito vectors is rapidly expanding due to climate change and mosquito-borne disease risks are emerging in new parts of the world.

Innovation in finding new repellents has been slow due to limitations in current research approaches and high costs for EPA registration (especially for synthetic compounds). Since DEET was discovered in the 1940s only a handful of additional actives have been approved by the EPA for repellent products. In the 20+ years since discovery of insect odorant receptors from genomes, not a single novel repellent compound has been identified that was registered by the EPA. Thus, there is a both a strong need for new approaches to find insect repellents and need for new active ingredients that are safe and strategically effective.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors set up a pipeline to predict insect repellents that are pleasant and safe for humans. This is done by daisy-chaining a new classification model based on predicting repellents with a published model on predicting human perception. Models use a feature-engineered selection of chemical features to make their predictions. The predicted molecules are then validated against a proxy humanoid (heated brick) and its safety is tested by molecular assays of human cells. The humanistic approach to modeling these authors have taken (which considers cosmetic/aesthetic appeal and safety) is novel and a necessary step for consumer usage. However, the importance of pleasantness over effectiveness is still up for debate (DEET is unpleasant but still used often) and the generalization of safety tests is unknown and assumed. The effectiveness of the prediction models is also still warranted. They pass the authors' own behavioral tests, but their contribution to the field is unknown as both models (new and published) have not been rigorously benchmarked to previous models. Moreover, the author's breadth of literature in this field is sparse, ignoring directly related studies.

Strengths:

Humanistic approach to modeling considers pleasantness and safety. Chaining models can help limit the candidate odorants from the vastness of odor space.

Weaknesses:

The current models need to be bench-marked against leading models predicting similar outcomes. Similarly, many of these papers need to be addressed and discussed in the introduction. The authors might even consider their data sources for model training to increase performance and lexical categorization for interoperability. For instance, the Dravnikes data lexicon, currently used in the human perception lexicon, has been highly criticized for its overlapping and hard-to-interpret descriptive terms ("FRAGRANT", "AROMATIC"). 

Human Perception:

Khan, R. M., Luk, C. H., Flinker, A., Aggarwal, A., Lapid, H., Haddad, R., & Sobel, N. (2007). Predicting odor pleasantness from odorant structure: pleasantness as a reflection of the physical world. Journal of Neuroscience, 27(37), 10015-10023.

Keller, A., Gerkin, R. C., Guan, Y., Dhurandhar, A., Turu, G., Szalai, B., ... & Meyer, P. (2017). Predicting human olfactory perception from chemical features of odor molecules. Science, 355(6327), 820-826.

Gutiérrez, E. D., Dhurandhar, A., Keller, A., Meyer, P., & Cecchi, G. A. (2018). Predicting natural language descriptions of mono-molecular odorants. Nature communications, 9(1), 4979.

Lee, B. K., Mayhew, E. J., Sanchez-Lengeling, B., Wei, J. N., Qian, W. W., Little, K. A., ... & Wiltschko, A. B. (2023). A principal odor map unifies diverse tasks in olfactory perception. Science, 381(6661), 999-1006.

The human perception predictions were performed using models that we had reported in two earlier publications which we have now indicated clearly in the results and methods sections of the VOR: Kowalewski & Ray, iScience (2020b) and Kowalewski, Huynh & Ray, Chem. Senses (2021). Three of the four references pointed out by the referee were cited in these prior studies, which involved computational validation by predicting on a test set of the data which was left out of training (as typically done), and also predicting across different human studies with a high degree of success. A rigorous benchmarking of the odor perception models was done in Kowalewski, Huynh & Ray, Chem. Senses (2021) and a mini-review published in the same issue of the journal by Gerkin, Chem. Senses, (2021). This included a favorable comparison with the two references indicated by the referee: Keller et al. Science (2017) as well as the Gutiérrez et al. Nat. Communication (2018).

The 4th reference, Lee et al, Science (2023) describes a neural network approach and was published well after our mosquito behavior studies were completed. Although using an advanced Neural network model Lee et al. worked with 2-D structures of compounds in contrast to our 3-D approach. They also did not report cross-study validations or comparisons with Keller et al, 2017 or benchmark to past studies, so it is difficult to compare advances if any. We have added this reference in the VOR.

The intent of the current study was to move beyond testing approaches, of which there are many, and instead work on a practical use case. As we see it, it is not necessarily the prediction of fragrance character or quality alone that matters but overlap with other predicted bioactivities. From the perspective of human use, a molecule with a pleasing scent that also repels insects is likely to be far more useful than one with an unappealing scent. Accordingly, our task in this study was to select molecules that fit into specific use categories: display strong insect repellency, have pleasing scent profiles, are natural in origin and are potentially repurposed from flavors and fragrances.

Insect Repellents:

Wright, R. H. (1956). Physical basis of insect repellency. Nature, 178(4534), 638-638.

Katritzky, A. R., Wang, Z., Slavov, S., Tsikolia, M., Dobchev, D., Akhmedov, N. G., ... & Linthicum, K. J. (2008). Synthesis and bioassay of improved mosquito repellents predicted from chemical structure. Proceedings of the National Academy of Sciences, 105(21), 7359-7364.

Bernier, U. R., & Tsikolia, M. (2011). Development of Novel Repellents Using Structure− Activity Modeling of Compounds in the USDA Archival Database. In Recent Developments in Invertebrate Repellents (pp. 21-46). American Chemical Society.

The Katritzky et al. PNAS (2008) paper is cited in our study, and we have indicated that the chemical analogs reported therein are part of the training data set in our study. We thank the reviewer for pointing us to the book chapter by Bernier & Tsikolia (2011), which reviews the QSAR approaches taken for repellent discovery and in large measure focuses on the Katritzky et al. PNAS (2008) paper. We did cite two relevant studies by Uli Bernier.

The current study assumes that insect repellents repel via their odor valence to the insect, but this is not accurate. Insect repellents also mask the body odor of humans making them hard to locate. The authors need to consult the literature to understand the localization and landing mechanisms of insects to their hosts. Here, they will understand that heat alone is not the attractant as their behavioral assay would have you believe. I suggest the authors test other behaviour assays to show more convincing evidence of effectiveness. See the following studies:

De Obaldia, M. E., Morita, T., Dedmon, L. C., Boehmler, D. J., Jiang, C. S., Zeledon, E. V., ... & Vosshall, L. B. (2022). Differential mosquito attraction to humans is associated with skin-derived carboxylic acid levels. Cell, 185(22), 4099-4116.

McBride, C. S., Baier, F., Omondi, A. B., Spitzer, S. A., Lutomiah, J., Sang, R., ... & Vosshall, L. B. (2014). Evolution of mosquito preference for humans linked to an odorant receptor. Nature, 515(7526), 222-227.

Wei, J. N., Vlot, M., Sanchez-Lengeling, B., Lee, B. K., Berning, L., Vos, M. W., ... & Dechering, K. J. (2022). A deep learning and digital archaeology approach for mosquito repellent discovery. bioRxiv, 2022-09.

In this study we took an unbiased approach to compile the training data set, including several known insect repellents of varying chemical structures and volatility, for most of which there is no information on how they are sensed by insects. Not surprisingly, the repellents we identified are varied in structure and in functional groups, and are likely detected in more than one way by the mosquitoes, using olfactory and/or gustatory systems. We did not consider “masking” of skin attraction as a factor in the training data set in this study, which precluded the need to discuss the papers pointed out by the referee. In fact there is an extremely vast and rich body of literature regarding human skin odor, CO₂ and breath emanations, which includes our own contributions of research, and review articles that are not discussed in the current paper.

We did in fact conduct human arm-in-cage experiments with a few of the compounds reported in this study using female Aedes aegypti mosquitoes; a preprint describes the smaller scale analysis, the results of which show very strong repellency, in Boyle et al. bioRxiv (2016) https://doi.org/10.1101/060178 (Figure 4). That line of experimentation falls outside the scope of this current study and are being pursued in a separate form. We have added the citation for this preprint in the results section of the VOR.

However, heat with CO₂ as used in this study offers a practical proxy for evaluating prospective repellents in a high-throughput manner. It would certainly be desirable to further evaluate additional candidates from the heat attraction assay with human subjects in the future.

We thank the reviewer for pointing out the preprint by Wei, et al. bioRxiv (2022). Our approaches differ in that Wei et al do not consider properties such as fragrance and toxicity. We also cannot assume that their newer neural network model is superior because although the model uses a large training dataset, it does not use 3D chemical structures that are extremely relevant for biological activity. While very little information is available for the actives reported in Wei et al., we independently evaluated their top compounds similar or better than DEET (CAS#3731-16-6, 4282-32-0, 2040-04-2, 32940-15-1 and 3446-90-0) and could not find information about toxicity, smell, or natural source. In contrast, the top repellents that we identify here as similar or better than DEET (N=8) are all classified as GRAS (Generally Regarded as Safe) compounds by the Flavor and Extract Manufacturers (FEMA), are all naturally occurring (plum, jasmine, mushroom, grapes, etc), and have pleasant smells. The Dermal toxicity values in rabbits are known for six of our compounds and are at the best possible levels (≥5000mg/kg).

Reviewer #2 (Public Review):

Summary:

This is an interesting study that seeks to identify novel mosquito repellents that smell attractive to humans.

Strengths:

The combination of standard machine learning methods with mosquito behavioral tests is a strength.

Weaknesses:

The study would be strengthened by describing how other modern ML approaches (RF, decision trees) would classify and identify other potential repellents.

The current approach already shows a success rate >85% for repellency coefficient >0.5 and identifies eight naturally occurring GRAS compounds with repellency as strong as or greater than DEET. This substantially expands the repertoire of strong natural repellents. Since the 1950s only six active ingredients have been registered by US EPA for use in topical repellents, of which only two are natural in origin (Oil of lemon eucalyptus and catmint oil) and they typically do not protect as well as DEET does. That being said, we have since explored other predictive algorithms, for instance Neural Networks. The experimental evaluation of these newer pipelines will take significant resources and time and will be the focus of future grants.

A comparison in the repellent activity between DEET and the top ten hits identified in this new study indicates little change in repellent activity (~3%), suggesting that DEET remains the gold standard. Without additional toxicity tests, the study is arguably incremental. The study's novelty should be better clarified.

There is an urgent need to find new insect repellents that have better chances of being adopted by people who avoid DEET, such as in Africa and Asia. Having more natural actives that are effective, expands the tools against disease transmitting mosquitoes. As mentioned above, the top repellents that we identified as similar to or better than DEET (N=8) are all classified as GRAS (Generally Regarded as Safe) compounds by the Flavor and Extract Manufacturers (FEMA), are all naturally occurring (plum, jasmin, mushroom, grapes), and have pleasant smells. The Dermal toxicity values in rabbits are known for six and they are of the best possible levels (≥5000mg/kg).

The Methods in the repellency tests are sparse, and more information would be useful. Testing the top repellents at low doses (<<1%) and for long periods (2-12 h) would strengthen the manuscript. Without this information, the manuscript is lacking in depth.

The US Environmental Protection Agency (EPA) regulates mosquito repellents, and DEET-based commercial products are typically assigned protection times that vary with concentration (10% ~2 hrs, 30% ~5hrs, 100% ~8hrs). These would be the relevant concentrations for testing protection times on human volunteers, not lower as suggested. Such studies fall within the realm of EPA registration efforts, involving extensive GLP-testing for safety, physical chemistry, and Human Subjects Board approvals. This is outside the scope of the current study and is typically accomplished during development efforts.

Testing human subjects on their olfactory perceptions of the repellents would also increase the depth and utility of the manuscript. Without additional experiments, the authors' conclusions lack support and have limited impact on the state-of-the-art.

This manuscript is a mix of different approaches, which makes it lack cohesion. There is the ML method for classifying new repellents that smell good, but no testing of the repellents on human volunteers. The repellents are not tested at realistic concentrations and durations. And the calcium mobilization test is strange and makes little sense in the context of the other experiments and framing of the manuscript.

The human olfaction validation that we present in this paper is consistent with most current publications in the field (for example, Keller et al, Gutiérrez et al.). More systematic validation of the human odor character prediction pipelines used was presented in two previous papers Kowalewski & Ray, iScience (2020b) and Kowalewski, Huynh & Ray, Chem. Senses (2021) and a mini-review published in the same issue of the journal by Gerkin, Chem. Senses, (2021).

Reviewer #3 (Public Review):

While I am not a specialist in this field, I do have some knowledge of the subject matter and the computational aspects involved. The authors employ simple machine learning techniques (such as SVM) for the following purposes:

(a) Prediction of aversive valence.

(b) Predicting anti-repellent chemicals.

(c) Predicting calcium mobilization.

The approach is commonplace in chemoinformatics literature.

Weaknesses:

All the above models are presented discretely, making it difficult to discern experiment design principles and connectedness.

The ML work is rudimentary, lacking adequate details. Chemoinformatics has reached great heights, and SVM does not seem contemporary.

There is significant existing research on finding repellents.

In the current study, we aimed to showcase how computational research may be combined with basic science to create scalable pipelines that address real world problems, rather than to demonstrate methodological novelty of chemoinformatics approaches. Specifically we wanted to use different predictive models to identify compounds that display strong insect repellency, have pleasing scent profiles, are natural in origin and are potentially repurposed from flavors and fragrances. Unfortunately, there is very little existing research on insect repellents that have these types of properties, which would make them better candidates for EPA registration. Most tested compounds are synthetic, and are often analogs of known repellents like DEET, and necessitate substantial time and resources to register. Moreover the identities of chemosensory receptors that are responsible for repellency to DEET and other compounds, and that are conserved across Anopheles, Aedes and Culex mosquitoes are not known.

It is true that the field of cheminformatics has experimented with a variety of newer approaches, based in part on neural networks (e.g., Graph Neural Networks and graph embeddings to encode chemical structure rather than a more conventional Extended Connectivity Fingerprint (ECFP)). Importantly, however, novelty does not imply usefulness. The mosquito behavior experiments that we present show a very high success rate (>85%), validating our approach and identifying several excellent candidates already.

Strengths:

Authors attempt to make a case for calcium mobilization in the context of repellency. This aspect sounds interesting but is not surprising.

Behavioral profiling of repellents could be useful.

We thank the referee for this comment. We have indeed done behavioral profiling for several repellents that evoke calcium mobilization, but we do not see any clear correlation thus far.

Version 4 of this preprint has been peer-reviewed and recommended by Peer Community in Genomics.<br /> See the peer reviews and the recommendation.

Results in large effect sizes

The file-drawer problem is a bias in scientific research where studies with non-significant results (results that fail to reach statistical significance, usually p>.05) are much less likely to be published. Instead, they often stay hidden in researchers’ “file drawers.”

This section places text directly on top of a detailed background image, which reduces contrast and makes the writing harder to read. Module 2 explains that colour should never be the only cue for communicating information, and text needs strong, consistent contrast from its background to stay "Perceivable." Because the images behind the text have many colours and textures, the contrast changes and the words become less clear for users with low vision, colour-vision deficiencies, or anyone viewing the page in bright light.

eLife Assessment

This manuscript proposes a valuable idea on how cortical networks may learn a helpful representation of sensory stimuli. The model implementing this idea is tested in multiple experimental paradigms. However, the evidence remains incomplete as to whether the method supports both invariance and equivariance and whether it can estimate the dynamics of the moving object.

Reviewer #1 (Public review):

Summary:

The paper describes a biologically plausible version of JEPA using recurrent neural networks called RPL for recurrent predictive learning. Given an embedding z_t, a recurrent neural network processes these inputs with the form: c_t+1 = RNN(c_t, z_t). Then the predictive network f is predicting the future inputs with the format: min || f(c_t) - stop_grad(z_t+delta t) ||^2. I understand that a prediction error is defined as: e = z_t+delta t - f(c_t) to model cortical measurements in the oddball task.

The RPL model is also shown to build an internal world model, with "real-world" data like the movement of moving animals or speech signals. The representation is then compared to V1 data and expected prediction error signals in an oddball setting. In a stacked hierarchy of RNN learning with RPL, the higher layers appear to learn high-level latent variables, although gradients are not propagated downward to the lower layers.

Strengths:

(1) The paper tackles an open question: Self-supervised learning is thought to be a fundamental principle to explain how computation is structured in the brain. Cortical data suggest qualitatively that prediction error is a core principle of representation learning in the brain, but the field is still looking for a simple yet expressive model that would explain how the cortex learns its representations. RPL contributes in that direction by making a useful link between cortical representation learning in RNN models and the JEPA learning algorithm that was demonstrated to scale to large world model learning from video data by Lecun's group. It is very useful to connect this popular deep learning algorithm to cortical data.

(2) The model formalism is relatively elegant and simple: Simple next input prediction objectives are conceptually simple but not necessarily trivial to build at scale. There is a clear benefit in comparison with contrastive or IL methods because they are free from dataset-specific data augmentation and negative samples. Thereby moving the comp neuro field towards conceptually simpler models of representation in the cortex. Yet predictive only models (and in particular predictive models in latent space instead of pixel space) are not easy to build in a stable fashion. JEPA family is basically intended to solve this question; it is very nice and timely to bring this to comp neuro.

(3) The methodology combining comp neuro and deep learning makes sense: The conceptual and qualitative analogy with cortical prediction errors is relevant and consistent with what is expected as a model of self-supervised learning in cortical models. The methodology to compare RPL with IL and CL is methodologically meaningful and grounded: showing, for instance, how some of the models fail to represent some latent structure in some toy datasets is interesting.

(4) h-RPL: The h-RPL is perhaps the most creative departure from the JEPA model family. It would be interesting to say more about what was particularly difficult to see in the latent variables emerging in the hierarchical model. I often find it magical that layer-wise learning rules of this type are not learning redundant representations. Any insights why this is not the case here would be potentially insightful.

Weaknesses:

In general, I fully support the type of question and ideas that the paper is putting forward. It is, however, very hard in this research field to gain insight into specific conceptual contributions or specific bits of experimental data that the model puts forward. In pointing to the following weaknesses, I am encouraging the authors to lay out more clearly what the unique hypothesis is or the contribution of the RPL model that we should remember it for.

(1) The devil is in the details:

1a) Comparison with JEPA variants: JEPA variants are integrating different details into the learning algorithm. Integrating, for instance, "masking" of the latent encoder targets, or EMA in the style of BYOL or Siamese networks, for the predicted representations. It is great that RPL does not seem to need any of those (next input prediction is a natural implementation of masking, and EMA does not seem to be used). It is notoriously hard for the JEPA model to work without these features. Since some of these details are sometimes surprisingly crucial for a simulation to work, it would be good to report which of the other important details were key to live without EMA and masking. Is it the difference in learning rate, for instance? Or maybe the tasks considered are simply easy enough for any model to work; if so, it could be useful to acknowledge to what extent this is true.

1b) Comparison with IL and CL: On a high level, the comparison with IL and CL algorithms is written as conclusive. I suspect that the failure modes of IL and CL that are described are not due to the algorithms themselves, but rather to the construction of invariance statistics or the choice of negative sample sets (the sets of samples among which variance 1 is requested by VICreg). For instance, if variance (or negative sample set) is taken only across time, the variance object identity is expected to collapse. Similarly, if the variance is taken across the object identity, the variance across time can collapse. So I wonder if the failure of IL and CL is induced by the construction of the variance definition.

(2) Prediction error: When compared to the recording of cortical activity in Figure 7. It is not obvious from the figure which latent space we are talking about mathematically. Is the vector z, c or the prediction error e? This is rather important from a neuroscientific point of view, because the prediction error e is expected to explain the neuronal data. On the other hand, the prediction error e is only used in the learning algorithm to define the loss function, but it is not the communication medium between the RNN units c (or with the encoder z).

In the brain, since the measurements are recorded as neural activity, they are communication channels between specific units (z or c). It is probably c or z that would already explain the oddball prediction error. I believe that other models, like Forward-forward of Nejad et al., have tried quite hard to address this apparent tension. Whether or not this is resolved by RPL, it thinks it would be beneficial to state the problem and clarify how the algorithm addresses or ignores the issue.

(3) Successor representation without value? I believe the term successor representation is historically relevant in a reinforcement learning (RL) setting and has a precise mathematical definition. Without RL, I feel that learning successor representation is conceptually identical to learning a transition matrix (aka, a primitive world model). I therefore wonder if the pitch for high-level framing of the successor representation is appropriately described or trivial.

(4) Learning in RNN: Learning with recurrent networks appears to be a key in this model presented here (it is in the algorithm name). Yet, this aspect of the model and the literature on biologically plausible learning rules for RNN is not really discussed.

Reviewer #2 (Public review):

This is a very interesting manuscript, which proposes a novel idea on how cortical networks may learn useful representations of sensory stimuli. The model implementing this idea is thoroughly tested in multiple experimental paradigms. The manuscript is very clearly written. I feel it may have a significant impact on our understanding of cortical circuitry.

Reviewer #3 (Public review):

Summary:

This paper presents Recurrent Predictive Learning (RPL), a self-supervised model conceptually similar to Joint-Embedding Predictive Architecture (JEPA) models. RPL sequentially observes dynamic scenes to predict subsequent observations. A central claim of the work is that the model's trained representations are simultaneously invariant and equivariant to transformations, such as movement properties that emerge without explicit supervision. These representational qualities are demonstrated through three experiments utilizing two simulated datasets and one naturalistic dataset. Furthermore, the latent embeddings are qualitatively compared with neural data, showing that the model reproduces the successor representation observed in human V1 and the local/global oddball effect in the monkey Prefrontal Cortex.

Strengths:

(1) The paper addresses a fundamental question relevant to both computational neuroscience and machine vision: how the brain learns representations that are simultaneously invariant and equivariant to transformations. The manuscript is well-written, easy to follow, and supported by clear visualizations.

(2) While JEPA-style models have recently gained significant traction in the artificial intelligence community, this paper nicely bridges the gap to neuroscience. By framing these architectures as a theory for visual learning in the brain, the authors provide valuable insights into how predictive frameworks can explain cortical processing.

(3) The qualitative alignment with V1 and PFC data is a particularly strong contribution, as it offers a potential mechanistic explanation for observed neural phenomena through the lens of self-supervised learning.

Weaknesses:

(1) The central claim, that both invariance and equivariance emerge spontaneously, requires further scrutiny (see Ghaemi et al., NeurIPS, 2025; Garrido et al., arXive, 2024). In particular, the synthetic "moving animal" dataset used in this paper may be too simple to fully support this claim. In latent space prediction, a model must predict both the scene content and the dynamics of movement. Because movement (whether ego-motion or external) is often highly uncertain (or multi-modal), predictive models in naturalistic settings often "collapse" toward learning purely invariant representations, ignoring the hard-to-predict dynamics. In the provided simulations, the movements are extremely predictable. In more complex scenarios, the model would likely prioritize content (invariance) over dynamics (equivariance) unless aided by action-conditioning or explicit factor estimation (Zhang et al., ICLR, 2026). The authors' results in Figure 5 using naturalistic video seem to reflect this limitation, given the lower performance on the naturalistic videos compared to the synthetic datasets.

(2) The framing of the RPL model as an entirely new theory of representation learning is slightly overstated. The focus on prediction in representation space rather than input space is the defining characteristic of JEPA and various other Self-Supervised Learning (SSL) models, even sequential prediction. While this paper clarifies the connection between these AI frameworks and cortical circuits, the work would be strengthened by more explicitly positioning RPL within the context of existing JEPA-style models and prior SSL theories of the visual system.

(3) A significant challenge in latent-space SSL is avoiding "representational collapse" (where the model provides a trivial constant output). While the paper alludes to JEPA-like solutions, it lacks a detailed explanation (in both the text and the architectural schematics) of the specific technique used to prevent collapse. Consequently, it is difficult to evaluate the authors' claim of "biological plausibility," as the biological equivalents of common machine learning techniques (such as stop gradient) are not discussed.

(4) Recent work has shown that the capacity (size) of the predictor significantly influences the learned representations in a JEPA-type world model (Gorrido et al., 2024). In simpler scenarios, a large enough predictor can allow a model to "memorize" dynamics rather than learning generalized equivariant features. It would be beneficial to see how the ratio of predictor size to encoder size affects the emergence of these features.

Methodological Clarifications:

(1) The authors mention a contrastive learning comparison but provide few details. Since contrastive learning is primarily a technique to avoid collapse, it would be a more rigorous baseline if implemented within the same architecture as RPL to isolate the effect of the predictive objective.

(2) In the PFC data comparison (Figure 7f), there appears to be a discrepancy where the local and global conditions show nearly identical results in PFC, while different dynamics in the model. It is unclear if this is a visualization error or a genuine model deviation.

(3) The criteria for selecting specific model variables for comparison with V1 versus PFC are not explicitly defined. Clarification is needed on whether the same latent variables were used for both brain regions or if different layers were selected.

Author response:

Public Reviews:

Reviewer #1 (Public review):

The paper describes a biologically plausible version of JEPA using recurrent neural networks called RPL for recurrent predictive learning. Given an embedding z_t, a recurrent neural network processes these inputs with the form: c_t+1 = RNN(c_t,z_t). Then the predictive network f is predicting the future inputs with the format: min||f(c_t) − stop grad(z_t+∆_t)||². I understand that a prediction error is defined as: e = z_t+∆_t − f(c_t) to model cortical measurements in the oddball task.

The RPL model is also shown to build an internal world model, with ”real-world” data like the movement of moving animals or speech signals. The representation is then compared to V1 data and expected prediction error signals in an oddball setting. In a stacked hierarchy of RNN learning with RPL, the higher layers appear to learn high-level latent variables, although gradients are not propagated downward to the lower layers.

The paper tackles an open question: Self-supervised learning is thought to be a fundamental principle to explain how computation is structured in the brain. Cortical data suggest qualitatively that prediction error is a core principle of representation learning in the brain, but the field is still looking for a simple yet expressive model that would explain how the cortex learns its representations. RPL contributes in that direction by making a useful link between cortical representation learning in RNN models and the JEPA learning algorithm that was demonstrated to scale to large world model learning from video data by Lecun’s group. It is very useful to connect this popular deep learning algorithm to cortical data.

The model formalism is relatively elegant and simple: Simple next input prediction objectives are conceptually simple but not necessarily trivial to build at scale. There is a clear benefit in comparison with contrastive or IL methods because they are free from dataset-specific data augmentation and negative samples. Thereby moving the comp neuro field towards conceptually simpler models of representation in the cortex. Yet predictive only models (and in particular predictive models in latent space instead of pixel space) are not easy to build in a stable fashion. JEPA family is basically intended to solve this question; it is very nice and timely to bring this to comp neuro.

The methodology combining comp neuro and deep learning makes sense: The conceptual and qualitative analogy with cortical prediction errors is relevant and consistent with what is expected as a model of self-supervised learning in cortical models. The methodology to compare RPL with IL and CL is methodologically meaningful and grounded: showing, for instance, how some of the models fail to represent some latent structure in some toy datasets is interesting.

(1.1) h-RPL: The h-RPL is perhaps the most creative departure from the JEPA model family. It would be interesting to say more about what was particularly difficult to see in the latent variables emerging in the hierarchical model. I often find it magical that layer-wise learning rules of this type are not learning redundant representations. Any insights why this is not the case here would be potentially insightful.

We thank the reviewer for this comment. Regarding representational collapse in h-RPL: each local circuit independently applies the same collapse-preventing strategy as the single-level RPL model: namely, the asymmetric prediction architecture combined with the stop-grad operator. Since this mechanism operates locally within each circuit, it is sufficient to prevent collapse at every level of the hierarchy independently (see also our response to Point P1.3).

The more subtle question is why the circuits learn non-redundant rather than identical representations across the hierarchy. We believe two mechanisms are at play here: First, the hierarchical encoder is a stacked convolutional network, meaning that receptive field sizes grow with depth. This architectural inductive bias naturally encourages successive circuits to operate on increasingly spatially integrated features, creating a structural pressure toward learning complementary rather than redundant representations. Second, the growing expressivity of the network with depth means that higher circuits have access to richer, more abstract inputs from which they can extract higher-level latent structure that is not already captured by lower circuits. Together these factors: the local collapse-preventing mechanism and the depth-dependent growth in receptive field size and network expressivity presumably explain why h-RPL builds an increasingly refined and non-redundant representational hierarchy.

What we will do: We will expand our discussion on this point in the revised manuscript. We plan to expand our quantification on how abstractions emerge in h-RPL in future work in which we will also study variations with top-down connections.

(1.2) In general, I fully support the type of question and ideas that the paper is putting forward. It is, however, very hard in this research field to gain insight into specific conceptual contributions or specific bits of experimental data that the model puts forward. In pointing to the following weaknesses, I am encouraging the authors to lay out more clearly what the unique hypothesis is or the contribution of the RPL model that we should remember it for.

Thanks for the positive feedback along with the constructive criticism, and we agree that articulating the core contributions more crisply would strengthen the paper.

At its heart, we believe the paper makes two contributions we hope it will be remembered for. First, while prior work has established that invariant representations can be learned via local Hebbianlike learning rules, we show that learning equivariant representations alongside a latent dynamics model requires something qualitatively different: a local circuit; one with recurrent dynamics and an asymmetric predictive architecture. RPL provides a minimal concrete instantiation of this principle.

Second, and perhaps more broadly, the model makes a structural prediction about (cortical) neuronal circuit organization: since the encoder, integrator, and predictor each perform functionally distinct computations, the framework implies the existence of corresponding cell types and connectivity patterns one should look for in experimental data.

What we will do: We will sharpen these above messages in the revised manuscript to ensure these contributions are prominently highlighted throughout the paper.

(1.3) Comparison with JEPA variants: JEPA variants are integrating different details into the learning algorithm. Integrating, for instance, “masking” of the latent encoder targets, or EMA in the style of BYOL or Siamese networks, for the predicted representations. It is great that RPL does not seem to need any of those (next input prediction is a natural implementation of masking, and EMA does not seem to be used). It is notoriously hard for the JEPA model to work without these features. Since some of these details are sometimes surprisingly crucial for a simulation to work, it would be good to report which of the other important details were key to live without EMA and masking. Is it the difference in learning rate, for instance? Or maybe the tasks considered are simply easy enough for any model to work; if so, it could be useful to acknowledge to what extent this is true.

We thank the reviewer for raising this important point. There are two key mechanisms that ensure stable, non-trivial training in RPL. First, using a higher learning rate for the predictor relative to the encoder is crucial for stable training. This prevents the predictor from collapsing the encoder representations and was already noted empirically by Chen et al. (2021).

Second, and more fundamentally, predicting at the level of the memoryless encoder output, rather than at the level of the recurrent integrator, is essential to prevent a degenerate solution in which the RNN simply learns to generate an internally predictable time series unrelated to the input. By anchoring the prediction target to the encoder, the model is forced to ground its representations in the sensory input. Intuitively, otherwise the RNN can simply “make up” a predictable time series, which satisfies the learning objective, but would not yield useful internal representations.

Beyond these architectural points, previous work from our group (Srinath Halvagal et al., 2023) has shown mathematically that JEPAs without EMA avoid collapse via an implicit variance regularization mechanism, and we believe RPL benefits from the same principle. Indeed, we now have a more complete theoretical understanding of this, including identifiability proofs for the latent dynamical model under relatively mild assumptions (Mikulasch et al., 2026). This work has recently been accepted at ICML. Other than that, one has to ensure that representations are not already nearly collapsed at the beginning of training. In this paper, we used normalization layers (batchnorm) in the encoder to ensure this.

Finally like all SSL paradigms the augmentation strength is an important hyperparameter that impacts the quality of learned representations. In the temporal predictive setting, the augmentation strength is fixed by the world itself. The only knob we have to play with is the prediction horizon ∆. While we typically focused on next-time-step (∆ = 1) prediction, we saw a clear effect in the case of the speech dataset where ∆ = 8, but not ∆ = 1, yielded useful representations for the tasks (Fig. 5b).

What we will do: We will discuss the above points more prominently in the discussion to avoid them being overlooked in the methods. Additionally, we will include a plot on the empirical prediction horizon for the speech dataset in the supplementary material for reference.

(1.4) Comparison with IL and CL: On a high level, the comparison with IL and CL algorithms is written as conclusive. I suspect that the failure modes of IL and CL that are described are not due to the algorithms themselves, but rather to the construction of invariance statistics or the choice of negative sample sets (the sets of samples among which variance 1 is requested by VICreg). For instance, if variance (or negative sample set) is taken only across time, the variance object identity is expected to collapse. Similarly, if the variance is taken across the object identity, the variance across time can collapse. So I wonder if the failure of IL and CL is induced by the construction of the variance definition.

We thank the reviewer for this thoughtful point. Both RPL and CL implement an implicit variance regularizer by virtue of being JEPAs (Srinath Halvagal et al., 2023), whereas IL uses an explicit regularizer computed along both the batch and time dimensions to avoid representational and dimensional collapse. The failure modes of IL and CL therefore cannot be entirely attributed to the statistics of the input samples chosen for variance regularization, but are instead primarily determined by the choice of prediction and target representations.

What we will do: We will clarify this in the Methods section of the revised manuscript.

(1.5) Prediction error: When compared to the recording of cortical activity in Figure 7. It is not obvious from the figure which latent space we are talking about mathematically. Is the vector z, c or the prediction error e? This is rather important from a neuroscientific point of view, because the prediction error e is expected to explain the neuronal data. On the other hand, the prediction error e is only used in the learning algorithm to define the loss function, but it is not the communication medium between the RNN units c (or with the encoder z).

In the brain, since the measurements are recorded as neural activity, they are communication channels between specific units (z or c). It is probably c or z that would already explain the oddball prediction error. I believe that other models, like Forward-forward of Nejad et al., have tried quite hard to address this apparent tension. Whether or not this is resolved by RPL, it thinks it would be beneficial to state the problem and clarify how the algorithm addresses or ignores the issue.

Thanks for pointing out the issue with regards to clarity and for raising the important but subtle point about prediction error representation. To answer the immediate question asking which vector we use in Figure 7, it is the vector c corresponding to the integrator representations. We agree this should be stated explicitly and will update the manuscript accordingly.

On the more general point, we agree that the tension between recordable neural activity and the computational role of prediction errors is an important issue. We do already briefly engage with it in the Discussion (subsection “Relation to previous modeling work”), where we note that under RPL “inter-areal communication is dominated by representations rather than error signals”. However, we agree that this point should be surfaced more directly.

To elaborate, under classical predictive coding, prediction errors are the inter-areal communication channel and are therefore expected to be directly observable in neural recordings, e.g., as oddball responses. Under RPL, this is not the case: e is computed locally within a circuit and serves only as a learning signal for synaptic plasticity, not as a signal propagated between circuits or areas. What cortex primarily encodes and communicates in our framework are predictive representations, not reconstruction errors. Accordingly, what should map onto recorded population activity are the representations c (and z), while locally computed prediction errors could in principle remain observable as more circumscribed or transient mismatch-like signals within a circuit.

We would like to push this point further. The reviewer frames this as a tension that RPL needs to resolve, but growing neurophysiological evidence suggests that classical residual-difference prediction errors may not be a dominant mode of cortical encoding in the first place. Furutachi, Franklin, et al. (2024) showed that V1 responses to unexpected visual stimuli do not encode how input deviates from predictions, but instead selectively amplify the representation of the unexpected stimulus itself. Very recently, Furutachi and Hofer (2026) generalize this into a revised framework in which feedforward pathways transmit sensory representations modulated by prediction-error magnitude, rather than residual differences. Vasilevskaya et al. (2026) constrain the space of plausible cortical algorithms via functionalinfluence experiments, also concluding that no variant of standard predictive processing is consistent with the full pattern of layer 2/3 ↔ layer 5 interactions; they propose a JEPA-based model, citing RPL as a promising candidate. The model by Nejad et al. (2025) similarly shares with RPL the property that representations, rather than residual errors, propagate between circuit elements.

Taken together, the apparent tension may be less a problem RPL needs to resolve than one it is well positioned to explain, remaining consistent with the emerging picture of cortex as encoding amplified sensory features rather than transmitting residual errors across areas.

What we will do: We will add missing information to the main text and sharpen the Discussion with these arguments.

(1.6) Successor representation without value? I believe the term successor representation is historically relevant in a reinforcement learning (RL) setting and has a precise mathematical definition. Without RL, I feel that learning successor representation is conceptually identical to learning a transition matrix (aka, a primitive world model). I therefore wonder if the pitch for high-level framing of the successor representation is appropriately described or trivial.

The reviewer makes a valid point on the concept of successor representations. To answer the immediate question, it is not entirely trivial, as we not only observe the emergence of the transition structure (Fig. 6c), but also the encoding of decaying future (but not past) state occupancy (Fig 6d,e). We largely adapted the terminology “successor-like representations” from the study by (Ekman et al., 2023), but we will elaborate a bit further for why we stuck to it. As nicely pointed out by the reviewer, the term “successor representations” was introduced in the RL literature (Dayan, 1993), but further adopted in neuroscience to describe the idea that a neuronal population encodes a predictive representation that reflects the expected future occupancy of future states under a given policy. Ekman et al. (2023) use the term “successor-like representations” to explain the phenomena where the neural activity in V1 (and hippocampus) represent both current and (discounted) future, but not past, state occupancies in a sequence learning task with no explicitly defined policy or value training. In other words, successor-like representations are simply predictive representations.

What we will do: To deal with this dichotomy, we will replace “successor-like representations” with the term “predictive representations” in the abstract and clarify this distinction in the Results section of the revised manuscript.

(1.7) Learning in RNN: Learning with recurrent networks appears to be a key in this model presented here (it is in the algorithm name). Yet, this aspect of the model and the literature on biologically plausible learning rules for RNN is not really discussed.

We thank the reviewer for raising this concern. While h-RPL is one step toward more biologically plausible and spatially local learning rules, exploring it further in terms of temporal credit assignment is beyond the scope of the present study and would require a more systematic and in-depth analysis. However, moving toward more biologically plausible learning rules is an interesting research direction that we plan to explore, as we also mentioned in the Discussion (“Limitations and future research directions”).

We think a viable strategy could be to combine a slim spatial credit assignment strategy such as feedback alignment (Nøkland, 2016; Lillicrap et al., 2016) with an online learning rule using eligibility traces for temporal credit assignment such as SuperSpike (Zenke et al., 2018) or e-prop (Bellec et al., 2020). Similar strategies have given promising results for CLAPP (Illing et al., 2021; Zihan et al., 2026).

What we will do: Following the suggestion, we will discuss biologically plausible learning rules for RNNs in the Discussion.

Reviewer #2 (Public review):

This is a very interesting manuscript, which proposes a novel idea on how cortical networks may learn useful representations of sensory stimuli. The model implementing this idea is thoroughly tested in multiple experimental paradigms. The manuscript is very clearly written. I feel it may have a significant impact on our understanding of cortical circuitry.

Reviewer #3 (Public review):

This paper presents Recurrent Predictive Learning (RPL), a self-supervised model conceptually similar to Joint-Embedding Predictive Architecture (JEPA) models. RPL sequentially observes dynamic scenes to predict subsequent observations. A central claim of the work is that the model’s trained representations are simultaneously invariant and equivariant to transformations, such as movement properties that emerge without explicit supervision. These representational qualities are demonstrated through three experiments utilizing two simulated datasets and one naturalistic dataset. Furthermore, the latent embeddings are qualitatively compared with neural data, showing that the model reproduces the successor representation observed in human V1 and the local/global oddball effect in the monkey Prefrontal Cortex.

The paper addresses a fundamental question relevant to both computational neuroscience and machine vision: how the brain learns representations that are simultaneously invariant and equivariant to transformations. The manuscript is well-written, easy to follow, and supported by clear visualizations.

While JEPA-style models have recently gained significant traction in the artificial intelligence community, this paper nicely bridges the gap to neuroscience. By framing these architectures as a theory for visual learning in the brain, the authors provide valuable insights into how predictive frameworks can explain cortical processing.

The qualitative alignment with V1 and PFC data is a particularly strong contribution, as it offers a potential mechanistic explanation for observed neural phenomena through the lens of selfsupervised learning.

(3.1) The central claim, that both invariance and equivariance emerge spontaneously, requires further scrutiny (see Ghaemi et al., NeurIPS, 2025; Garrido et al., arXive, 2024). In particular, the synthetic ”moving animal” dataset used in this paper may be too simple to fully support this claim. In latent space prediction, a model must predict both the scene content and the dynamics of movement. Because movement (whether ego-motion or external) is often highly uncertain (or multi-modal), predictive models in naturalistic settings often ”collapse” toward learning purely invariant representations, ignoring the hard-to-predict dynamics. In the provided simulations, the movements are extremely predictable. In more complex scenarios, the model would likely prioritize content (invariance) over dynamics (equivariance) unless aided by action-conditioning or explicit factor estimation (Zhang et al., ICLR, 2026). The authors’ results in Figure 5 using naturalistic video seem to reflect this limitation, given the lower performance on the naturalistic videos compared to the synthetic datasets.

We thank the reviewer for the feedback. We agree that further validation on more complex datasets would strengthen the claims, and we take this point seriously. If the reviewer has any suggestions for a specific alternative dataset, we would welcome any recommendations.

Regarding the mouse video data specifically, we realized that this is a suboptimal benchmark rather than a shortcoming of our method. The culprit presumably is that the mice remain largely stationary, leading to a heavily imbalanced velocity distribution peaked near zero (Supplementary Fig. S9). This imbalance makes equivariance evaluation unreliable regardless of the learning algorithm. For example, end-to-end supervised training results in an R² of 0.19 compared to 0.08 ± 0.02 for RPL.

Regarding the moving animal dataset, we note that the dynamics are not trivial from an SSL perspective: unlike moving MNIST (Srivastava et al., 2015), the dataset includes changes in scale and orientation, both features that invariance-focused SSL models can easily ignore, yet RPL recovers reliably. For example, this discrepancy can be seen in Supplementary Table S1 where we compare to InfoNCE and CPC. That said, we acknowledge the reviewer’s broader concern and will seek to validate RPL on more complex datasets.

While it would be nice to compare to related work by Ghaemi et al. (2024), this study used 3DIEBench (Garrido et al., 2023). Unfortunately, 3DIEBench’s reliance on pair-based representations with annotated but random augmentations (such as rotations or color changes) precludes the possibility of smooth latent traversals that would be required for RPL to learn from the same dataset. We will look into whether it is computationally feasible to adapt or regenerate a similar dataset that meets the requirements for temporal prediction.

Regarding stochasticity, we agree that predictive learning in latent space is most natural in approximately deterministic settings, whereas real world sensory information often comprises non-deterministic elements. While a deeper treatment of such stochastic environments is beyond the scope of the present manuscript, it will be the focus of ongoing and future work. Regarding ongoing work, it is worth mentioning that in recent work from our group (Hauri et al., 2026), we have demonstrated that RPL’s core objective can replace the reconstruction loss in Dreamer, achieving competitive performance in complex, stochastic environments. While we did not systematically evaluate equivariance in this study, the results suggests that representation-space predictive learning is viable beyond the deterministic regime.

What we will do: We will make the point about the real-world mouse video dataset being a poor benchmark and include the additional R² values to show that. Further, we will try to identify or generate alternative datasets to back the equivariance claims and discuss our findings in the light of previous work, e.g., Ghaemi et al. (2024). Moreover, we will sharpen our discussion of our model’s limitations in stochastic settings and highlight notable connections to related work.

(3.2) The framing of the RPL model as an entirely new theory of representation learning is slightly overstated. The focus on prediction in representation space rather than input space is the defining characteristic of JEPA and various other Self-Supervised Learning (SSL) models, even sequential prediction. While this paper clarifies the connection between these AI frameworks and cortical circuits, the work would be strengthened by more explicitly positioning RPL within the context of existing JEPA-style models and prior SSL theories of the visual system.

Thanks for raising this point. We are unsure what the reviewer refers to. We did not frame our work as ”an entirely new theory of representation learning,” as the reviewer suggests. In fact, we highlight quite the opposite already in the title of our article, which reads: “Understanding neural circuit principles for representation learning through joint-embedding predictive architectures.” We do not claim novelty over JEPA as an ML paradigm, we adopt it precisely because it provides a principled, non-generative framework for predictive representation learning, and our goal is to develop a circuit level instantiation that accounts for neural circuit computation. We already discuss a body of previous work of self-supervised learning and JEPAs at length. Since the reviewer did not specify what they are missing, we will briefly reiterate what is already there.

Our contribution is a theory of representation learning in the brain, built on JEPAs as the underlying ML framework. The Title and Introduction already position our work quite explicitly this way. Specifically, we mention prior work on JEPAs (CPC, BYOL, SimSiam, I-JEPA, seq-JEPA, V-JEPA, V-JEPA 2), while noting that “most JEPAs developed in machine learning are poor models of cortical computation” because of their reliance on negative sampling, transformers, masking, static images, and/or known parametrized transformations, and motivate RPL as the minimal candidate that “must instead rely on recurrent neural dynamics, learn from streaming sensory input without masking, support both invariant and equivariant representations, and reproduce key neurophysiological observations.”

The Discussion (“Relation to previous modeling work”) further details the specific novelties of RPL relative to existing sequential JEPA-style and SSL models like CPC (Oord et al., 2018), V-JEPA (Bardes et al., 2024), V-JEPA 2 (Assran et al., 2025), seq-JEPA (Ghaemi et al., 2024). In brief:

RPL is a recurrent JEPA based on RNN dynamics, not transformers, and learns from streaming sensory input without masking or random negative sampling;

It explicitly compares three prediction-error topologies (RPL vs. invariance learning vs. contextprediction; Fig. 2, Suppl. Fig. S2, S6) and shows that asymmetric recurrent prediction is essential for jointly learning invariant and equivariant representations;

Importantly, it does so via pure temporal prediction without access to underlying transformations, a property shared by very few JEPAs. The closest exception is VJ-VCR (Drozdov et al., 2024) which uses an explicit variance-covariance regularization (VCReg) in a JEPA, which we will cite in the revised manuscript;

It provides the first hierarchical JEPA optimizing local prediction errors at multiple levels (h-RPL, Fig. 8), as envisioned by LeCun (2022) but not previously implemented;

It connects directly to neurophysiological data: successor-like representations in human V1 and abstract sequence representations in macaque PFC, which provides qualitative correspondence between JEPA components and cortical activity that the existing JEPA literature, focused on ML benchmarks, does not address.

Finally, our article already includes a discussion paragraph on recent self-supervised learning models in the context of the brain where we discuss work by Nejad et al. (2025) and Asabuki et al. (2025). Most other SSL theories of the visual system rely on static images and recognition tasks (Yerxa et al., 2024; Margalit et al., 2024). However, there are two studies that include temporal prediction objectives and are worth mentioning with more details: First, Bakhtiari et al. (2021) show that representations similar to ventral and dorsal pathways in the visual system can emerge in a two-pathway encoder architecture within the CPC model. Second, Niu et al. (2024) use a “straightening” objective together with VCReg as a practical model of the perceptual straightening hypothesis (H´enaff et al., 2019). Though not a JEPA (i.e., has no predictor network), it can decode equivariant factors in a sequential MNIST dataset where only single factors change throughout a video.

What we will do: We will carefully review our discussion of previous work and further discuss Drozdov et al. (2024), Bakhtiari et al. (2021), and Niu et al. (2024) in the revised manuscript.

(3.3) A significant challenge in latent-space SSL is avoiding “representational collapse” (where the model provides a trivial constant output). While the paper alludes to JEPAlike solutions, it lacks a detailed explanation (in both the text and the architectural schematics) of the specific technique used to prevent collapse. Consequently, it is difficult to evaluate the authors’ claim of “biological plausibility,” as the biological equivalents of common machine learning techniques (such as stop gradient) are not discussed.

Thanks for pointing this out. Our model avoids collapse through the asymmetric stop-grad / predictor architecture. It does not require an EMA, when the predictor learns with a faster learning rate than the rest of the network (see also our response to Point P1.3).

The use of stop-grad suggests that a circuit learning with RPL needs to compute a vector-based instructive learning signal. While we do not explicitly model the circuit level mechanisms of how this could be implemented in the brain, excitation-inhibition balance is one possibility (Rossbroich et al., 2025). Finally, differences in learning rate can be implemented both structurally or functionally in the brain (see Liu et al. (2025) for instance), or activity normalization is suggested as a canonical computation in biological neural circuits (Carandini et al., 2012).

What we will do: We will make sure to discuss these putative biological mechanisms in the revised manuscript.

(3.4) Recent work has shown that the capacity (size) of the predictor significantly influences the learned representations in a JEPA-type world model (Gorrido et al., 2024). In simpler scenarios, a large enough predictor can allow a model to ”memorize” dynamics rather than learning generalized equivariant features. It would be beneficial to see how the ratio of predictor size to encoder size affects the emergence of these features.

Thanks for raising this concern. We don’t observe noticeable difference in position and velocity decoding when changing the width or depth of the MLP predictor in the moving animals data. However, performance on rotation speed and orientation decoding scales with the changes in width, but not depth of the predictor. This analysis excludes the effect of integrator’s capacity as it directly affects the dimensionality of the representations, even though it also effectively contributes to prediction computation in RPL.

What we will do: We will include a figure how how task performance varies with the predictor’s width and depth.

Methodological Clarifications

(3.5) The authors mention a contrastive learning comparison but provide few details. Since contrastive learning is primarily a technique to avoid collapse, it would be a more rigorous baseline if implemented within the same architecture as RPL to isolate the effect of the predictive objective.

Thanks for the question. We already use the same network model as in RPL for the contrastive predictive learning (InfoNCE) baseline in Supplementary Table S1 and mentioned in the main text (l.164).

What we will do: We will mention the architecture of the non-linear predictor used for InfoNCE baseline in Methods more explicitly.

(3.6) In the PFC data comparison (Figure 7f), there appears to be a discrepancy where the local and global conditions show nearly identical results in PFC, while different dynamics in the model. It is unclear if this is a visualization error or a genuine model deviation.

Thanks for picking up on this subtlety in the experimental results. To clarify, it is a model deviation but an interesting one. The local and global responses do look quite similar in the original PFC data. They differ in that the global oddball (xY|xx and xx|xY) response has a secondary peak that encodes the presence of the global oddball, whereas the initial response is actually dominated by local oddball encoding (xY vs xx). Concretely, this results in the response to the xx|xY condition only showing up weakly in the data and at a time lag with respect to the initial local oddball response. Our model, however, does not show the transient initial response to local oddballs in the decoding direction for global oddballs. In a sense, the network model encodes the global oddball concept more robustly than is seen in the PFC data. That said, whether this indicates a genuine difference in representational strategies that needs to be further accounted for, or whether it is an issue stemming from limited sub-sampling of PFC neurons, remains unclear.

(3.7) The criteria for selecting specific model variables for comparison with V1 versus PFC are not explicitly defined. Clarification is needed on whether the same latent variables were used for both brain regions or if different layers were selected.

To clarify, the successor-like representations in human V1 and abstract representations in macaque PFC are two different experiments, so each has different latent variables requiring different RPL models. The architecture used for each experiment is detailed in Methods and the criteria for selecting each architecture was the simplest that should work given the task complexity. Throughout the paper, all representation analysis is done on the output of integrator (c) unless said otherwise. We hope this resolves the confusion.

References

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Digital Media

• As a Digital Media Consultant, you will answer general questions about digital media equipment, services, and spaces.
• You will find accurate answers by researching online or asking a full-time Educational Technologies Librarian

Take this out

As a Digital Media Consultant, you will welcome, orient, and help visitors with digital media technologies, services, and spaces.

Robertson Media Center Front Desk

I would take this one out. Being on the 3rd floor, we're probably not the first people visitors encounter.

For the "Take a guess" interaction above: Make the question both more related to RMC and not a trick question. Something like - At the Robertson Media Center front desk, student workers only check media equipment in and out of the library. - False, While checking out equipment is an important duty of digital media consultants, it is only a part of what student workers do. Digital media consultants help orient visitors, support patrons' use of our various studios, and troubleshoot technology.

A new question could be something like - Which is NOT a responsibility of Digital Media Consultants? A) Orient visitors B) Check in and out books (correct) C) Check in and out digital media equipment D) Support patrons in media studios E) troubleshoot technological problems

Also - I would take out the type in questions, swapping them for true/false or multiple choice. Having to type in the exact word they are looking for doesn't really check the student's understanding, it just incentivizes them to copy and paste the answers

hmmmm this is sort of true, but definitely not the number one benefit of working for the RMC. Take it out? Or move it to the bottom of the list?

digital media and technology skills

Digital Media Consultant

This may create a legal risk if terms such as “recycled” and “sustainably sourced” are not clearly defined and supported with reliable evidence. Environmental claims should be specific, verifiable, and transparent so consumers understand what standards or certifications are used.

This claim is more specific because it includes a target, percentage, scope, and baseline year. However, consumers may still need clearer explanation of how the reduction will be achieved, especially across scope 3 emissions, which are linked to suppliers and the wider value chain.

The ethical concern is that this claim may make consumers believe that continued fashion consumption has limited environmental impact. Although the target sounds positive, the brand should clearly show whether emission reductions are enough to balance the environmental effects of producing and selling more fashion items.

This may raise a greenwashing concern because the statement connects business growth with sustainability. In fast fashion, growth can increase production, consumption, and waste. The brand needs to clearly explain how it can expand while genuinely reducing environmental impact.

This statement may be misleading because it presents the whole business as economically, socially, and environmentally sustainable. Although the page provides some targets, the wording is very broad and may make consumers assume that the entire business model is already sustainable. More specific explanation is needed to show how this applies across all products, stores, suppliers, and operations.

This text exposes H&M' "Conscious Collection" case of execution-style greenwashing, where vague buzzwords and green visual cues are used without transparent data to mislead consumers. Ethically, the brand capitalizes on genuine environmental concerns for financial profit while hiding behind a highly destructive, high-waste fast-fashion business model. This structural hypocrisy shifts from a moral issue to a severe legal liability, as evidenced by the Norwegian Consumer Authority's ruling against H&M. This case serves as a critical regulatory precedent, proving that deceptive sustainability claims now carry actionable legal risks, including official sanctions, immediate consumer boycotts and long-term reputational ruin.

eLife Assessment

This valuable study examines how the prelimbic cortex represents learned and generalized threat over time and identifies potentially distinct stable and dynamic subnetworks that may support these functions. The work is conceptually interesting and is strengthened by the longitudinal calcium imaging approach and the inclusion of key control groups. However, the evidence supporting the claims is incomplete, particularly because the interpretations regarding inference, time-dependent representational change, and the dissociation of neural activity from freezing behavior extend beyond what is currently established by the data.

Reviewer #1 (Public review):

Summary:

The authors combine discriminative auditory fear conditioning with longitudinal in vivo calcium imaging to ask how prelimbic (PL) representations of learned and generalized threat evolve across recent and remote memory time points. Using two different CS+ frequencies and a no-shock control group, they report that PL population activity tracks graded behavioral generalization, that population similarity is highest for tones eliciting strong threat responding, and that distinct subnetworks can be identified that appear to encode tone-specific sensory features versus learned threat-related response structure.

To my knowledge, this may be the first study to comprehensively examine neural encoding of fear generalization in prelimbic cortex (PL). The manuscript is ambitious and technically interesting, and several aspects are potentially important. In particular, the suggestion that neurons showing graded, learning-related response patterns become selectively stabilized over time is intriguing. The inclusion of two CS+ training conditions and a no-shock control also strengthens the case that at least some of the reported effects are related to associative learning rather than simple sensory differences. However, in its current form, the manuscript does not yet fully support the strength of the conceptual claims. Several issues limit confidence in the interpretation, including the possibility that repeated testing itself contributes to changes across days, uncertainty about the relationship between neural activity and freezing behavior, limited quantitative documentation of longitudinal cell registration, and a number of problems in figure clarity and statistical framing. Overall, the study contains promising observations, but the claims should be narrowed, and several analyses or controls would be needed to fully support the proposed framework.

Detailed Comments

(1) A general concern is that the repeated test procedure itself may contribute to extinction. Because the animals are exposed to multiple CS frequencies across multiple test days, and each tone is presented three times per session, some of the reported changes in behavior and neural activity across days could reflect extinction or repeated nonreinforced retrieval rather than the passage of time per se. This is especially relevant given that the manuscript makes claims about recent versus remote representations and representational drift over 30 days. At a minimum, the authors should discuss this limitation explicitly and temper claims about time-dependent changes. Ideally, they would include a control group in which animals are tested only once or twice (e.g., at an early and later time point with fewer CS frequencies), or a reduced-frequency testing design that minimizes extinction while still allowing evaluation of recent versus remote memory.

(2) More generally, some of the reported learning-related neural differences may be driven by behavioral differences, particularly freezing, rather than by learning or generalization per se. For example, animals that freeze more to certain frequencies may show corresponding neural response differences simply because freezing alters PL activity. The authors should examine this possibility more directly. Analyses testing whether recorded cells encode freezing behavior, or whether tone frequency-related neural differences remain robust when comparing high- and low-freezing epochs, would help determine whether the reported effects reflect learned stimulus value rather than behavioral state differences.

(3) A central feature of the manuscript is the analysis of neural response properties over an extended period of time, up to 30 days after learning. However, aside from a brief mention in the Methods that spatial registration was used, the manuscript provides very little quantitative information about this critical aspect of the study. The paper would be strengthened by including explicit metrics describing longitudinal cell tracking, such as the number and proportion of ROIs retained across all sessions, distributions of spatial-footprint correlations or centroid distances across days, and representative examples of matched imaging fields over time. Without this information, it is difficult to assess how strongly the longitudinal claims are supported.

(4) The text states that "Figs. 1c and 1d show GCaMP6f expression in PL, representative calcium footprints, and activity traces". However, the figure as presented does not clearly show all of these elements, at least not in a way that matches the description in the Results. The correspondence between text and figure should be corrected.

(5) The labeling of Figure 2a is insufficient for interpretation. The legend states that the panel shows raster plots of sound responsiveness, but the axes and scaling are not clearly defined. It is not clear from the figure what the x-axis represents, whether the y-axis corresponds to individual neurons, where the CS period occurs, or what the activity scale at the right denotes. Also, the term 'rasters' implies that spikes were analyzed. It seems that the spike inference approach (CASCADE) was only used for later analyses. Perhaps 'heat-plot' would be more accurate here? Generally, this figure should be annotated more clearly so that the reader can understand it without referring back to the Methods.

(6) In relation to Figure 3, the analysis of population-averaged responses across tone frequencies is useful, but the manuscript would be stronger with additional statistical analyses across time and across groups. For example, if the authors want to argue that learning induces graded changes in neural responses and that these evolve across time, they should directly compare within-group responses across days and also compare matched frequencies between the conditioned groups and the no-shock controls. These analyses would help establish whether the observed differences are genuinely learning dependent and whether they change significantly over time.

(7) The inclusion of two different CS+ frequencies and a no-shock control is a strength of the study and substantially improves the interpretation that graded neural responses are related to learning and generalization rather than to simple sensory processing or passage of time. That said, I am not entirely comfortable with the use of the term "inference" throughout the manuscript. What is being measured here appears closer to sensory generalization than inference in a stronger cognitive sense. The current task does not clearly require that animals infer hidden structure or stimulus value through abstract reasoning; rather, the generalized stimulus may simply be treated as similar to the conditioned cue. The terminology should therefore be reconsidered or softened.

(8) I also found the use of the term "valence" somewhat problematic. The manuscript appears to use valence to refer to graded responding across tones with different aversive significance, but valence typically refers more broadly to distinctions between appetitive and aversive value. Here, terms such as "threat value," "aversive value," may be more precise. The authors should consider revising this language throughout.

Reviewer #2 (Public review):

Summary:

The following points are those that occurred to me across readings of the paper. They are listed in what I take to be the order of their significance. Many of the points relate to the loose use of language and invocation of concepts that are not warranted, given the study design and results obtained.

Major Comments:

(1) The concept of ensemble turnover is interesting - the way it is introduced and discussed implies some type of spontaneous change in the neural underpinnings of fear discrimination and generalization in the PL. But, of course, every trial involves an opportunity to learn about the threat CS or the generalization test stimuli, and I am troubled by the thought that stability in the neural underpinnings of fear discrimination and generalization will actually reflect the level of defensive behaviours evoked on different trial types and/or the discrepancy between those behaviours and the outcome of a given trial in the generalization test. That is, stability in the neural underpinnings may be related to an animal's certainty or uncertainty in the contingency between a stimulus and danger; or, put another way, an animal's confidence that danger will or won't occur given the presence of some stimulus. This is not uninteresting. It is, however, not considered anywhere in the paper, which is overloaded with references to inferred threat values and integration of information across different types of stimuli. The protocol is not one that requires inference about anything or integration across anything.

(2) I appreciate the link to Gu and Johansen in paragraph 3 of the Introduction, but the type of generalization under investigation here is not the same as the type of 'generalization' studied by Gu and Johansen [who used a sensory preconditioning protocol]. Nonetheless, the authors have forced the language used by Gu and Johansen into their paper, and this has created tension [at least for this reader] as the concepts introduced by Gu and Johansen [inference, integration] are simply not relevant given the generalization protocol used here. Here are a few examples of points where the tension might interfere with a reader's understanding:

a. 'We hypothesized that generalization to novel stimuli depends on stable subnetwork organization that enables comparisons between learned and inferred valence, as well as population-level features that reduce variability across related representations.'

I understand the words in the hypothesis, but can't form a representation of what is being said because of the reference to terms that stand in need of clarification [inferred valence, variability across related representations], but, ultimately, won't be clarified. This needs to be re-expressed so that the reader can appreciate what is being said.

b. 'Our results show that stable cortical subnetworks integrate the emotional "gist" of memory and inferred valence for novel cues over time, despite ongoing ensemble reorganization, and that population-level firing rate similarity across stimulus presentations determines threat generalization.'

Again, what does this mean? How is the gist of a memory integrated with inferred valence for novel cues over time? The statement simply doesn't make sense. This needs to be rewritten for clarity.

c. 'In CS⁺15 mice, positively modulated sound-responsive neurons exhibited graded tone activity reflecting the contingency learned valence as well as the inferred valence of novel tones across testing days...'.

Can this be rewritten as 'In CS⁺15 mice, positively modulated sound-responsive neurons exhibited graded activity to the tone CS and its variants that were used to assess generalization.'? The overloading of the text with references to 'contingency learned valence' and 'inferred valence' is unnecessary and makes it much harder to understand what has been shown in the results.

(3) Re the same passage of text as in 2c:

Is it the case that these neurons are simply tracking the expression of freezing to the various tones? The same question applies to the results obtained for the CS+3 mice. If this is the case, then why should the results be taken to support the banner statement that 'Sound-modulated PL population responses encode learned and inferred valence' - these analyses do not support that statement. And, as indicated, I don't believe that the language of learned and inferred valence is appropriate to such statements, given the nature of the protocol used and results obtained. It is a study looking at how populations of neurons in the PL respond during presentations of auditory stimuli that were subject to discriminative conditioning, and during tests of generalized freezing to other [intermediate] auditory stimuli.

(4) It is stated that:

'In no-shock controls, although both positive and negative responses were present, population activity was not modulated by tone frequency or valence'.

What does this mean? I can understand that population activity was not modulated by tone frequency. But what does it mean to say that it was not modulated by valence? Why should it have been when none of the tones were conditioned in this group and, hence, mice were responding to all the tones equally? And given that this is true, I don't understand the use of 'valence' here, or the subsequent statements in this paragraph that 'graded responses require associative learning' and that 'PL population responses encode graded sound-valence associations that reflect both learning and inference, closely matching behavioral generalization.' The latter statement is particularly unwarranted and, again, highlights a major issue with the paper. It could and should be rewritten as 'PL population responses reflect behavioral generalization.' There is nothing in the additional language that adds to the reader's understanding of what has been shown. The reference to 'graded sound-valence associations that reflect both learning and inference' is completely unwarranted, given the nature of this study. It is anathema to the vast literature on stimulus generalization. If the authors wished to make statements of this sort, they should have taken a different approach, perhaps using protocols like those featured in Gu and Johansen.

(5) The section titled, 'Consistently active neurons preserve valence representations as newly recruited neurons sharpen remote memory traces' ends with the following summary:

'Together, these results indicate that consistently active neurons maintain stable representations of learned and inferred sound associations across time, whereas neurons recruited after conditioning progressively acquire graded tuning at later retrieval stages. This dynamic refinement suggests that cortical memory representations become increasingly selective during systems consolidation, while a stable neuronal subpopulation preserves the core emotional content of the memory.'

Once again, the summary is not in keeping with the results obtained. The 'dynamic refinement' of representations is far more likely to reflect the repeated testing across days 1, 15, and 30 rather than anything to do with systems consolidation - at the very least, it is the simplest interpretation of the results. The impact of repeated testing is evident in the sharpening of generalization gradients over time, which is contrary to what is otherwise observed in the literature - the incredibly well -documented broadening of generalization gradients with time. Given this impact of repeated testing, surely the changes in the neuronal population that underlie performance are more likely to reflect the learning that occurs on days 1, 15, and 30, which is reflected in reduced freezing to the non-conditioned tones. If this is a reasonable take on the results, then I don't see the basis for invoking systems consolidation at all, and I don't see the basis for inferring a stable neuronal subpopulation that preserves the emotional content of the memory. Rather, non-reinforced presentations of 'never-reinforced' tones result in recruitment of additional neurons that result in suppression of freezing responses to those stimuli.

(6) In the section titled, 'Population vector similarity at stimulus onset determines degree of generalization', it is stated that:

'Because population similarity peaked shortly after stimulus onset, we quantified similarity during the first 5 s after tone onset relative to the CS⁺. In CS⁺15 mice, population similarity was highest for 15/15 and 15/11 tone pairs with no differences between them.'

Isn't this consistent with the view that the population response in the PL simply reflects the level of freezing? Freezing to the 15-15 and 15-11 tones is most likely to be similar on their first presentation prior to the effects of extinction on the 11 Hz tone; hence the results obtained. That is, these results appear to clearly indicate that neuronal responses in the PL reflect the degree of stimulus generalization, as evidenced in freezing behavior. Given all that we know about the involvement of the PL in expressing fear responses, it is not appropriate to claim that 'population vector similarity at stimulus onset *determines* the degree of generalization. The PL responses simply reflect the varying levels of performance displayed to the different types of tones. What have I missed that could be taken to support additional statements?

Later in the same section, it is stated that 'population-level similarity at stimulus onset scales with behavioral threat generalization and is maximal for tones associated with robust threat responses.' For simplicity and, therefore, clarity, this should be rewritten as 'population-level similarity at stimulus onset reflects behavioral threat generalization.'

(7) In the section titled, 'Different subnetworks encode acoustic versus learned properties of sound association', it is stated that:

'Our previous analyses show that learned and inferred associations are represented at the population level. However, these results do not resolve whether graded responses arise from pooled activity of frequency-selective neurons or from subnetworks encoding integrated learned valence across tones.'

What does it mean to say 'integrated learned valence across tones'? As it presently stands, the meaning of the phrase is unclear. It only makes sense if one supposes that generalized freezing responses to the 11 and 7 kHZ tones reflect separate associations between those tones and the aversive foot shock US. This supposition is inconsistent with the rich literature on generalization of Pavlovian conditioned fear responses. Specifically, it is inconsistent with the many theories of fear generalization, which attribute the reduction in fear as one moves away from the specific conditioned stimulus to a decrement in the ability of the test stimulus to activate the trained CS-US association. My strong impression is that the authors would do well to ground their findings in theories of stimulus/fear generalization, of which there are many. This would better serve the results obtained [and the reader's appreciation of them] - at present, the unnecessary invocation of concepts does very little to enhance the reader's appreciation or understanding of what has been found in the study.

(8) Another example of what has been a common theme in this review :

'...we hypothesized that the PL active ensemble segregates into functionally distinct subnetworks: one encoding tone-specific sensory features with dynamic characteristics, and another responding to all frequencies encoding stable core memory content and inferred emotional valence.'

What does it mean to say 'all frequencies encoding stable core memory content and inferred emotional valence'? Do the authors mean to say '...and another that tracks freezing/defensive responses regardless of whether they were elicited by the trained CS or one of the generalization test stimuli'?

(9) It is stated that - 'Graded clusters encode emotional valence but constitute only a fraction of the active population; yet valence coding at the population level remains accurate and precise. This indicates that neurons newly recruited into the population-likely frequency-selective and organized within learning-independent clusters-can be shaped by associative processes through modulation of firing activity.'

What does this mean? Are the authors trying to say that - 'Some clusters of PL neurons track freezing responses. In spite of the fact that these are only a fraction of the total active neuronal population, the population-level response of PL neurons also tracks the levels of fear to the trained tone and its variants used in the test for generalization.' If this is what one wants to say, then the final statement in the reproduced section does not follow. That is, there is no indication that 'neurons newly recruited into the population-likely frequency-selective and organized within learning-independent clusters-can be shaped by associative processes through modulation of firing activity.' As noted, the characteristics of other ensembles that become active across the repeated tests on days 1, 15, and 30 are more likely to reflect learning from non-reinforcement that occurs within and across those sessions. Perhaps this is what is meant by the phrase, 'shaped by associative processes'? If so, it should be stated explicitly instead of left to the reader to work out.

(10) The following points all relate to the Discussion and reiterate many of the points above.

a. 'A subset of neurons remains consistently active across sessions, preserving core components of the memory trace and supporting inference of emotional valence for novel sounds, while neurons recruited after conditioning progressively acquire valence selectivity at remote time points.'

'Inference of emotional valence' is unclear and unwarranted for all of the reasons provided above regarding the use of language.

b. '...Our data reconcile these views by demonstrating that cortical representations of emotional valence emerge rapidly after learning and persist within stable subnetworks, even as the broader population undergoes substantial turnover. This architecture preserves core mnemonic content while allowing flexibility in the surrounding ensemble.'

These statements assume that the PL neuronal responses reflect something more than the levels of freezing behavior to the different stimuli; what are the grounds for this assumption?

c. 'Importantly, these subnetworks encode both learned contingencies and the inferred valence of novel stimuli along a graded representational axis, suggesting that strong recurrent connectivity provides a stable scaffold for emotional memory representations.'

What is a graded representational axis, and what part of the first statement suggests that 'strong recurrent connectivity provides a stable scaffold for emotional memory representations'? If the authors' goal was to make statements about emotional memory representations vis-à-vis emotional memory content, they should have used protocols that allowed them to probe such content. The auditory fear conditioning protocol used here [followed by tests for generalization to other auditory stimuli that differ in frequency from the conditioned tone] is not one that lends itself to analysis of emotional memory representations or content.

d. 'Dynamic tone-selective responsive neurons emerge independently of learning, as they are present in both control and experimental mice, reflecting pre-existing PL sensory-driven properties (Hockley & Malmierca, 2024; Zikopoulos & Barbas, 2006).'

Maybe. They are also likely to have developed as a consequence of the repeated testing on days 1, 15, and 30, which involved intermixed exposures to the tones of different frequencies. That is, rather than 'pre-existing PL sensory-driven properties', the responses of these neurons might reflect the emergence of discrimination between the various tones across testing, and greater suppression of freezing to the non-trained tones compared to the trained tone across the various test intervals.

Reviewer #3 (Public review):

Summary:

Normandin et al. explore the coding of stimuli predicting an aversive event in the prelimbic cortex. Stimuli could either be explicitly paired, explicitly unpaired, or novel but with an inferred association with the aversive event (generalization). Long-term tracking of GCaMP-positive neurons allowed them to examine how coding evolves out to a month following training. In general, they found two types of ensemble codes. One was ensembles coding for each stimulus independently, but with enhanced responding to the one eliciting a freezing response. The other was ensembles that responded to all stimuli in proportion to their similarity to the stimulus paired with the aversive event, either increasing or decreasing their activation with the degree of freezing elicited by a stimulus. Importantly, this second set of ensembles was more stable across days, potentially providing a memory trace.

Strengths:

(1) The authors track ensembles in prelimbic cortex over long time scales, providing valuable information on the consolidation of neural codes.

(2) Neural coding of generalization is examined, which is under-examined in the field.

Weaknesses:

(1) Difficult to determine if responses treated as encoding stimulus valence are driven instead by the behavior that the stimulus elicits, freezing.

(2) The study implies that the identified ensembles are causally related to valence memory, but no experimental interventions are performed to justify this.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors combine discriminative auditory fear conditioning with longitudinal in vivo calcium imaging to ask how prelimbic (PL) representations of learned and generalized threat evolve across recent and remote memory time points. Using two different CS+ frequencies and a no-shock control group, they report that PL population activity tracks graded behavioral generalization, that population similarity is highest for tones eliciting strong threat responding, and that distinct subnetworks can be identified that appear to encode tone-specific sensory features versus learned threat-related response structure.

To my knowledge, this may be the first study to comprehensively examine neural encoding of fear generalization in prelimbic cortex (PL). The manuscript is ambitious and technically interesting, and several aspects are potentially important. In particular, the suggestion that neurons showing graded, learning-related response patterns become selectively stabilized over time is intriguing. The inclusion of two CS+ training conditions and a no-shock control also strengthens the case that at least some of the reported effects are related to associative learning rather than simple sensory differences. However, in its current form, the manuscript does not yet fully support the strength of the conceptual claims. Several issues limit confidence in the interpretation, including the possibility that repeated testing itself contributes to changes across days, uncertainty about the relationship between neural activity and freezing behavior, limited quantitative documentation of longitudinal cell registration, and a number of problems in figure clarity and statistical framing. Overall, the study contains promising observations, but the claims should be narrowed, and several analyses or controls would be needed to fully support the proposed framework.

Detailed Comments

(1) A general concern is that the repeated test procedure itself may contribute to extinction. Because the animals are exposed to multiple CS frequencies across multiple test days, and each tone is presented three times per session, some of the reported changes in behavior and neural activity across days could reflect extinction or repeated nonreinforced retrieval rather than the passage of time per se. This is especially relevant given that the manuscript makes claims about recent versus remote representations and representational drift over 30 days. At a minimum, the authors should discuss this limitation explicitly and temper claims about time-dependent changes. Ideally, they would include a control group in which animals are tested only once or twice (e.g., at an early and later time point with fewer CS frequencies), or a reduced-frequency testing design that minimizes extinction while still allowing evaluation of recent versus remote memory.

We agree with the reviewer that repeated testing is an inherent limitation of longitudinal memory studies and may itself contribute to some neural changes across sessions. However, several aspects of our behavioral design and results argue against extinction or repeated nonreinforced retrieval as the primary drivers of the observed effects. Importantly, discrimination ratios remained stable or increased across time rather than progressively diminishing as would be expected under extinction (this new analysis will be added to the resubmission). Nevertheless, we will address this important point in the Discussion and explicitly acknowledge that repeated retrieval may contribute to some component of the observed representational changes.

(2) More generally, some of the reported learning-related neural differences may be driven by behavioral differences, particularly freezing, rather than by learning or generalization per se. For example, animals that freeze more to certain frequencies may show corresponding neural response differences simply because freezing alters PL activity. The authors should examine this possibility more directly. Analyses testing whether recorded cells encode freezing behavior, or whether tone frequency-related neural differences remain robust when comparing high- and low-freezing epochs, would help determine whether the reported effects reflect learned stimulus value rather than behavioral state differences.

We thank the reviewer for raising this important point, which was also noted by the other reviewers. To address this issue, we will implement Reviewer 3’s suggested Generalized Linear Model (GLM) analysis using inferred spiking activity derived from the Ca2+ signals, with both tone identity and freezing behavior included as predictors. Because freezing behavior varies across trials whereas stimulus identity is fixed, this approach will allow us to dissociate their respective contributions to neuronal activity. If, after accounting for freezing behavior, responsive neurons continue to exhibit graded coding consistent with inferred threat value, this would strengthen the interpretation that the identified ensembles reflect generalization gradients related to aversive value rather than freezing behavior alone. Otherwise, we will adjust the conclusions according to the interpretation that freezing itself drives the generalization gradients.

(3) A central feature of the manuscript is the analysis of neural response properties over an extended period of time, up to 30 days after learning. However, aside from a brief mention in the Methods that spatial registration was used, the manuscript provides very little quantitative information about this critical aspect of the study. The paper would be strengthened by including explicit metrics describing longitudinal cell tracking, such as the number and proportion of ROIs retained across all sessions, distributions of spatial-footprint correlations or centroid distances across days, and representative examples of matched imaging fields over time. Without this information, it is difficult to assess how strongly the longitudinal claims are supported.

We thank the reviewer for this suggestion. We will include measures of registration quality in the resubmission.

(4) The text states that "Figs. 1c and 1d show GCaMP6f expression in PL, representative calcium footprints, and activity traces". However, the figure as presented does not clearly show all of these elements, at least not in a way that matches the description in the Results. The correspondence between text and figure should be corrected.

We will correct correspondence between text and Figure.

(5) The labeling of Figure 2a is insufficient for interpretation. The legend states that the panel shows raster plots of sound responsiveness, but the axes and scaling are not clearly defined. It is not clear from the figure what the x-axis represents, whether the y-axis corresponds to individual neurons, where the CS period occurs, or what the activity scale at the right denotes. Also, the term 'rasters' implies that spikes were analyzed. It seems that the spike inference approach (CASCADE) was only used for later analyses. Perhaps 'heat-plot' would be more accurate here? Generally, this figure should be annotated more clearly so that the reader can understand it without referring back to the Methods.

Thank you for this suggestion. We will clarify the labelling of the Figure 2a and call the graphs “activity-plots”.

(6) In relation to Figure 3, the analysis of population-averaged responses across tone frequencies is useful, but the manuscript would be stronger with additional statistical analyses across time and across groups. For example, if the authors want to argue that learning induces graded changes in neural responses and that these evolve across time, they should directly compare within-group responses across days and also compare matched frequencies between the conditioned groups and the no-shock controls. These analyses would help establish whether the observed differences are genuinely learning dependent and whether they change significantly over time.

We will redo the Statistics of Figure 3 to take into account the following variables: group (CS15, CS3, no shocks), frequency (3, 7, 11, 15), and day of testing (2, 15, 30).

(7) The inclusion of two different CS+ frequencies and a no-shock control is a strength of the study and substantially improves the interpretation that graded neural responses are related to learning and generalization rather than to simple sensory processing or passage of time. That said, I am not entirely comfortable with the use of the term "inference" throughout the manuscript. What is being measured here appears closer to sensory generalization than inference in a stronger cognitive sense. The current task does not clearly require that animals infer hidden structure or stimulus value through abstract reasoning; rather, the generalized stimulus may simply be treated as similar to the conditioned cue. The terminology should therefore be reconsidered or softened.

We thank the reviewer for appreciating the strengths of the experimental design and for this thoughtful suggestion regarding terminology. We agree that the term “inference” may overstate the cognitive processes engaged by the current task. Accordingly, we will revise the terminology throughout the manuscript to describe these effects as graded generalization of threat value across stimuli.

(8) I also found the use of the term "valence" somewhat problematic. The manuscript appears to use valence to refer to graded responding across tones with different aversive significance, but valence typically refers more broadly to distinctions between appetitive and aversive value. Here, terms such as "threat value," "aversive value," may be more precise. The authors should consider revising this language throughout.

We will correct the language and use “threat value”.

Reviewer #2 (Public review):

Summary:

The following points are those that occurred to me across readings of the paper. They are listed in what I take to be the order of their significance. Many of the points relate to the loose use of language and invocation of concepts that are not warranted, given the study design and results obtained.

Major Comments:

(1) The concept of ensemble turnover is interesting - the way it is introduced and discussed implies some type of spontaneous change in the neural underpinnings of fear discrimination and generalization in the PL. But, of course, every trial involves an opportunity to learn about the threat CS or the generalization test stimuli, and I am troubled by the thought that stability in the neural underpinnings of fear discrimination and generalization will actually reflect the level of defensive behaviours evoked on different trial types and/or the discrepancy between those behaviours and the outcome of a given trial in the generalization test. That is, stability in the neural underpinnings may be related to an animal's certainty or uncertainty in the contingency between a stimulus and danger; or, put another way, an animal's confidence that danger will or won't occur given the presence of some stimulus. This is not uninteresting. It is, however, not considered anywhere in the paper, which is overloaded with references to inferred threat values and integration of information across different types of stimuli. The protocol is not one that requires inference about anything or integration across anything.

We thank the reviewer for these important points, which we address in further detail below.

Ongoing learning during test sessions: The reviewer correctly notes that unreinforced test presentations may constitute extinction-learning trials and that some neural changes across days could therefore reflect ongoing learning rather than spontaneous ensemble reorganization. However, new analyses indicate that extinction is unlikely to be the primary driver of our findings. Discrimination ratios do not decay over time; instead, they either sharpen or remain stable across sessions (new analyses to be included in the resubmission). These results argue against robust extinction as the primary source of the neural changes observed across sessions. This interpretation is also consistent with the strength of our conditioning protocol, which used 10 CS+ shock pairings and 10 CS− no-shock pairings specifically to minimize extinction across repeated testing sessions. Nevertheless, we acknowledge that the current design cannot fully dissociate time-dependent consolidation from retrieval-induced plasticity, and we will explicitly discuss this limitation in the revised Discussion.

Stability reflecting behavioral consistency: We agree this alternative cannot be fully excluded. However, the cluster stability analyses assess identity at the level of response profile across all four frequencies, not response magnitude alone. Tone-selective clusters, which also show consistent behavioral correlates (firing rate correlates with threat-value, Fig. S8), do not show equivalent profile stability, suggesting that the stability of graded clusters is not simply a consequence of behavioral consistency. This point will be added to the Discussion in the resubmission.

Language of "inference" and "integration": The reviewer is correct that responses to novel tones are consistent with graded stimulus generalization. We will substantially revise the manuscript to replace "inference" and "integration" with more precise language describing graded frequency generalization gradients.

(2) I appreciate the link to Gu and Johansen in paragraph 3 of the Introduction, but the type of generalization under investigation here is not the same as the type of 'generalization' studied by Gu and Johansen [who used a sensory preconditioning protocol]. Nonetheless, the authors have forced the language used by Gu and Johansen into their paper, and this has created tension [at least for this reader] as the concepts introduced by Gu and Johansen [inference, integration] are simply not relevant given the generalization protocol used here. Here are a few examples of points where the tension might interfere with a reader's understanding:

We thank the reviewer for these specific and constructive criticisms. We will revise the manuscript throughout to remove or redefine terms like "inferred valence" and "integration," replacing them with clearer, more accurate descriptions of gradient generalization of threat value. Below we address each point raised by the reviewer regarding terminology clarifications.

(a) 'We hypothesized that generalization to novel stimuli depends on stable subnetwork organization that enables comparisons between learned and inferred valence, as well as population-level features that reduce variability across related representations.'

I understand the words in the hypothesis, but can't form a representation of what is being said because of the reference to terms that stand in need of clarification [inferred valence, variability across related representations], but, ultimately, won't be clarified. This needs to be re-expressed so that the reader can appreciate what is being said.

The hypothesis will be rewritten as: "We hypothesized that generalization to tones acoustically similar to the CS+ and CS− depends on the emergence of stable ensembles encoding threat value, and that population-level response similarity across stimuli would correlate with the degree of behavioral fear generalization, consistent with prior work in auditory cortex [1]."

(b) 'Our results show that stable cortical subnetworks integrate the emotional "gist" of memory and inferred valence for novel cues over time, despite ongoing ensemble reorganization, and that population-level firing rate similarity across stimulus presentations determines threat generalization.'

Again, what does this mean? How is the gist of a memory integrated with inferred valence for novel cues over time? The statement simply doesn't make sense. This needs to be rewritten for clarity.

The summary statement will be rewritten: "Our results show that stable cortical sub-ensembles preserve the emotional content of the fear memory over time, despite ongoing ensemble reorganization, and that population-level firing rate similarity in response to tones associated with threat correlates with the degree of behavioral threat generalization."

(c) 'In CS⁺15 mice, positively modulated sound-responsive neurons exhibited graded tone activity reflecting the contingency learned valence as well as the inferred valence of novel tones across testing days...'.

Can this be rewritten as 'In CS⁺15 mice, positively modulated sound-responsive neurons exhibited graded activity to the tone CS and its variants that were used to assess generalization.'? The overloading of the text with references to 'contingency learned valence' and 'inferred valence' is unnecessary and makes it much harder to understand what has been shown in the results.

We will adopt the reviewer's suggested rewording: "In CS+15 mice, positively modulated sound-responsive neurons exhibited graded activity to the tone CS and its variants that were used to assess generalization."

We will systematically review the entire manuscript to ensure consistency with this revised framing.

(3) Re the same passage of text as in 2c:

Is it the case that these neurons are simply tracking the expression of freezing to the various tones? The same question applies to the results obtained for the CS+3 mice. If this is the case, then why should the results be taken to support the banner statement that 'Sound-modulated PL population responses encode learned and inferred valence' - these analyses do not support that statement. And, as indicated, I don't believe that the language of learned and inferred valence is appropriate to such statements, given the nature of the protocol used and results obtained. It is a study looking at how populations of neurons in the PL respond during presentations of auditory stimuli that were subject to discriminative conditioning, and during tests of generalized freezing to other [intermediate] auditory stimuli.

The reviewer is correct that the graded population responses observed in PL could reflect freezing behavior across tone frequencies rather than encoding an abstract threat-value representation. This important concern was also raised by other reviewers. To address it directly, we will follow Reviewer 3’s suggestion and implement a Generalized Linear Model (GLM) using inferred spiking activity derived from the Ca2+ signals, with both tone identity and freezing behavior included as predictors. This analysis will allow us to dissociate the respective contributions of tone frequency and freezing to the graded neural responses. Based on the outcome of this analysis, we will revise and appropriately adjust our conclusions.

In addition, we will revise the section heading and surrounding text to remove the terminology of “learned and inferred valence.” Instead, the findings will be described more conservatively as: “PL population responses reflect behavioral generalization to auditory stimuli following discriminative fear conditioning.”

(4) It is stated that:

'In no-shock controls, although both positive and negative responses were present, population activity was not modulated by tone frequency or valence'.

What does this mean? I can understand that population activity was not modulated by tone frequency. But what does it mean to say that it was not modulated by valence? Why should it have been when none of the tones were conditioned in this group and, hence, mice were responding to all the tones equally? And given that this is true, I don't understand the use of 'valence' here, or the subsequent statements in this paragraph that 'graded responses require associative learning' and that 'PL population responses encode graded sound-valence associations that reflect both learning and inference, closely matching behavioral generalization.' The latter statement is particularly unwarranted and, again, highlights a major issue with the paper. It could and should be rewritten as 'PL population responses reflect behavioral generalization.' There is nothing in the additional language that adds to the reader's understanding of what has been shown. The reference to 'graded sound-valence associations that reflect both learning and inference' is completely unwarranted, given the nature of this study. It is anathema to the vast literature on stimulus generalization. If the authors wished to make statements of this sort, they should have taken a different approach, perhaps using protocols like those featured in Gu and Johansen.

The reviewer is correct that controls do not form threat associations; however, these animals still could respond differentially to distinct frequencies, something that is not reflected in the data. We will correct the section indicating that distinct neutral frequencies do not produce graded responses: "graded responses require associative learning" will be retained but reframed simply as: "graded frequency-dependent population responses were absent in animals that did not receive fear conditioning." The concluding statement of the paragraph will be rewritten as: "PL population responses reflect behavioral generalization to acoustically similar stimuli following discriminative conditioning," in line with the reviewer's suggestion.

(5) The section titled, 'Consistently active neurons preserve valence representations as newly recruited neurons sharpen remote memory traces' ends with the following summary:

'Together, these results indicate that consistently active neurons maintain stable representations of learned and inferred sound associations across time, whereas neurons recruited after conditioning progressively acquire graded tuning at later retrieval stages. This dynamic refinement suggests that cortical memory representations become increasingly selective during systems consolidation, while a stable neuronal subpopulation preserves the core emotional content of the memory.'

Once again, the summary is not in keeping with the results obtained. The 'dynamic refinement' of representations is far more likely to reflect the repeated testing across days 1, 15, and 30 rather than anything to do with systems consolidation - at the very least, it is the simplest interpretation of the results. The impact of repeated testing is evident in the sharpening of generalization gradients over time, which is contrary to what is otherwise observed in the literature - the incredibly well -documented broadening of generalization gradients with time. Given this impact of repeated testing, surely the changes in the neuronal population that underlie performance are more likely to reflect the learning that occurs on days 1, 15, and 30, which is reflected in reduced freezing to the non-conditioned tones. If this is a reasonable take on the results, then I don't see the basis for invoking systems consolidation at all, and I don't see the basis for inferring a stable neuronal subpopulation that preserves the emotional content of the memory. Rather, non-reinforced presentations of 'never-reinforced' tones result in recruitment of additional neurons that result in suppression of freezing responses to those stimuli.

We respectfully disagree with the reviewer’s interpretation. While repeated testing cannot be entirely excluded as a contributing factor, several lines of evidence suggest that it cannot fully account for our observations.

Regarding extinction: discrimination ratios between CS+ and all other frequencies either remained stable or increased over time (new analysis included in resubmission), indicating that animals continued to discriminate threat value across the testing period rather than showing the progressive suppression expected under extinction — the opposite of what we observe.

Regarding the recruitment of new neurons: repeated non-reinforced tone exposure would be expected to produce stimulus-specific adaptation — characterized by reduced, less discriminative neural responsiveness and flatter tuning profiles [2]— not the progressive sharpening we observe. The same would be expected if these neurons represent or are associated with new extinction learning.

Finally, sharpening of generalization gradients during repeated within-subjects testing has been reported previously [3], suggesting that successive exposures may promote more precise discrimination in some cases. Consistent with this, discrimination learning has also been shown to narrow or sharpen fear generalization gradients rather than broaden them [4], supporting the idea that discriminative conditioning enhances stimulus specificity during testing. Although we cannot exclude the possibility that more extended training could eventually broaden the generalization gradient, under the training parameters and temporal window used in our study, the data support a progressive sharpening of the gradient over time. In the revised Discussion, we will present systems consolidation as the primary interpretive framework and further elaborate on why repeated testing is unlikely to account for the full pattern of behavioral and neural findings reported here.

(6) In the section titled, 'Population vector similarity at stimulus onset determines degree of generalization', it is stated that:

'Because population similarity peaked shortly after stimulus onset, we quantified similarity during the first 5 s after tone onset relative to the CS⁺. In CS⁺15 mice, population similarity was highest for 15/15 and 15/11 tone pairs with no differences between them.'

Isn't this consistent with the view that the population response in the PL simply reflects the level of freezing? Freezing to the 15-15 and 15-11 tones is most likely to be similar on their first presentation prior to the effects of extinction on the 11 Hz tone; hence the results obtained. That is, these results appear to clearly indicate that neuronal responses in the PL reflect the degree of stimulus generalization, as evidenced in freezing behavior. Given all that we know about the involvement of the PL in expressing fear responses, it is not appropriate to claim that 'population vector similarity at stimulus onset *determines* the degree of generalization. The PL responses simply reflect the varying levels of performance displayed to the different types of tones. What have I missed that could be taken to support additional statements?

The GLM analysis described in our response to reviewers 1 and 3 will directly address the contribution of freezing. We will report these results in the resubmission and revise the interpretive language in the manuscript accordingly.

However, regarding the analysis of population vector similarity, we need to clarify a point of confusion. The reviewer states “Freezing to the 15-15 and 15-11 tones is most likely to be similar on their first presentation prior to the effects of extinction on the 11 Hz tone; hence the results obtained”. The similarity vectors were calculated by correlating activity across all tone presentations within each testing day, not only the first two presentations. In Fig. 4, “Early” and “Late” refer to the order of a tone within a trial, which we will clarify more explicitly in the resubmission. Notably, repeated-measures analyses did not reveal any effect of the time variable (Fig. 4e,f), indicating that similarity across tone presentations remained high for tones associated with high threat value. Importantly, our data showed no evidence that responses to 11 kHz or 15 kHz in the CS15 group, or to 3 kHz in the CS3 group, exhibited extinction-like patterns at either the behavioral or neural level. Therefore, the persistence of high population similarity across time provides additional evidence against extinction as the primary explanation for our findings.

We will remove the word "determines" from the manuscript, as our data cannot conclusively establish a causal relationship.

Later in the same section, it is stated that 'population-level similarity at stimulus onset scales with behavioral threat generalization and is maximal for tones associated with robust threat responses.' For simplicity and, therefore, clarity, this should be rewritten as 'population-level similarity at stimulus onset reflects behavioral threat generalization.'

We will make this correction.

(7) In the section titled, 'Different subnetworks encode acoustic versus learned properties of sound association', it is stated that:

'Our previous analyses show that learned and inferred associations are represented at the population level. However, these results do not resolve whether graded responses arise from pooled activity of frequency-selective neurons or from subnetworks encoding integrated learned valence across tones.'

What does it mean to say 'integrated learned valence across tones'? As it presently stands, the meaning of the phrase is unclear. It only makes sense if one supposes that generalized freezing responses to the 11 and 7 kHZ tones reflect separate associations between those tones and the aversive foot shock US. This supposition is inconsistent with the rich literature on generalization of Pavlovian conditioned fear responses. Specifically, it is inconsistent with the many theories of fear generalization, which attribute the reduction in fear as one moves away from the specific conditioned stimulus to a decrement in the ability of the test stimulus to activate the trained CS-US association. My strong impression is that the authors would do well to ground their findings in theories of stimulus/fear generalization, of which there are many. This would better serve the results obtained [and the reader's appreciation of them] - at present, the unnecessary invocation of concepts does very little to enhance the reader's appreciation or understanding of what has been found in the study.

We thank the reviewer for raising this point. The phrase "integrated learned valence across tones" refers specifically to a subpopulation of neurons that respond to all four frequencies in a graded manner, with response magnitude scaling according to threat value. This is distinct from tone-selective neurons, which respond preferentially to a single frequency. The neurons responding to all tones in a graded manner are present only in conditioned animals and not in no-shock controls, demonstrating that their graded response profile is shaped by associative learning.

We agree, however, that the phrase "integrated learned valence" is unnecessarily opaque and we will replace it with more precise language: these neurons will be described as showing graded frequency-dependent responses whose magnitude scales with threat value. We believe this subpopulation represents a genuinely novel finding that complements the behavioral generalization literature by identifying a specific neural substrate for the generalization gradient within PL.

(8) Another example of what has been a common theme in this review:

'...we hypothesized that the PL active ensemble segregates into functionally distinct subnetworks: one encoding tone-specific sensory features with dynamic characteristics, and another responding to all frequencies encoding stable core memory content and inferred emotional valence.'

What does it mean to say 'all frequencies encoding stable core memory content and inferred emotional valence'? Do the authors mean to say '...and another that tracks freezing/defensive responses regardless of whether they were elicited by the trained CS or one of the generalization test stimuli'?

As stated in our previous responses, in the resubmission we will determine the contribution of freezing. If we find that freezing predicts graded neural responses, we will adjust the language of the manuscript.

(9) It is stated that - 'Graded clusters encode emotional valence but constitute only a fraction of the active population; yet valence coding at the population level remains accurate and precise. This indicates that neurons newly recruited into the population-likely frequency-selective and organized within learning-independent clusters-can be shaped by associative processes through modulation of firing activity.'

What does this mean? Are the authors trying to say that - 'Some clusters of PL neurons track freezing responses. In spite of the fact that these are only a fraction of the total active neuronal population, the population-level response of PL neurons also tracks the levels of fear to the trained tone and its variants used in the test for generalization.' If this is what one wants to say, then the final statement in the reproduced section does not follow. That is, there is no indication that 'neurons newly recruited into the population-likely frequency-selective and organized within learning-independent clusters-can be shaped by associative processes through modulation of firing activity.' As noted, the characteristics of other ensembles that become active across the repeated tests on days 1, 15, and 30 are more likely to reflect learning from non-reinforcement that occurs within and across those sessions. Perhaps this is what is meant by the phrase, 'shaped by associative processes'? If so, it should be stated explicitly instead of left to the reader to work out.

We thank the reviewer for highlighting the lack of clarity in this passage and agree that the original phrasing was insufficiently precise. What we intended to convey is that only a subset of PL neurons displays graded tuning that tracks behavioral generalization across tones. Nevertheless, despite constituting only a fraction of the total active population, this graded coding is also reflected at the population level. Therefore, we suggest that neurons recruited into the active population after conditioning — likely frequency-selective neurons — contribute to the graded population responses through changes in their firing-rate activity, which is modulated by threat value (Fig. S8). We will rewrite this passage in the resubmission to make this interpretation explicit rather than leaving it to the reader to infer.

Regarding the reviewer's suggestion that the characteristics of newly recruited neurons more likely reflect learning from non-reinforced exposures during repeated test sessions, we respectfully maintain that this interpretation is difficult to reconcile with two aspects of our data. First, graded-response neurons are absent in no-shock controls that are exposed to nonreinforced repeated testing. Second, as detailed in our responses to previous points, the progressive sharpening of population responses over time is inconsistent with what would be expected from repeated non-reinforced exposure, which would more plausibly produce broader or flatter tuning profiles.

We agree that the phrase "shaped by associative processes" was ambiguous and will replace it with explicit language clarifying that we refer to fear conditioning as the associative process driving the emergence of graded responses, rather than any learning occurring during the test sessions themselves.

(10) The following points all relate to the Discussion and reiterate many of the points above. 

(a) 'A subset of neurons remains consistently active across sessions, preserving core components of the memory trace and supporting inference of emotional valence for novel sounds, while neurons recruited after conditioning progressively acquire valence selectivity at remote time points.'

'Inference of emotional valence' is unclear and unwarranted for all of the reasons provided above regarding the use of language.

We will modify the language as stated in the prior points.

(b) '...Our data reconcile these views by demonstrating that cortical representations of emotional valence emerge rapidly after learning and persist within stable subnetworks, even as the broader population undergoes substantial turnover. This architecture preserves core mnemonic content while allowing flexibility in the surrounding ensemble.'

These statements assume that the PL neuronal responses reflect something more than the levels of freezing behavior to the different stimuli; what are the grounds for this assumption?

We will incorporate new analysis (GLM) to better address this point and conclusions.

(c) 'Importantly, these subnetworks encode both learned contingencies and the inferred valence of novel stimuli along a graded representational axis, suggesting that strong recurrent connectivity provides a stable scaffold for emotional memory representations.'

What is a graded representational axis, and what part of the first statement suggests that 'strong recurrent connectivity provides a stable scaffold for emotional memory representations'? If the authors' goal was to make statements about emotional memory representations vis-à-vis emotional memory content, they should have used protocols that allowed them to probe such content. The auditory fear conditioning protocol used here [followed by tests for generalization to other auditory stimuli that differ in frequency from the conditioned tone] is not one that lends itself to analysis of emotional memory representations or content.

We thank the reviewer for this comment and agree that both phrases require clarification or revision.

By "graded representational axis" we intended to convey that PL population activity varies systematically as a function of stimulus similarity to the conditioned tone — that is, population responses are not categorical but scale continuously with spectral proximity to the CS+. We agree this was not clearly stated and will revise the manuscript accordingly.

Regarding recurrent connectivity, we agree with the reviewer that nothing in our data directly measures or manipulates connectivity between neurons. This statement was intended as a speculative interpretive hypothesis in the Discussion, motivated by the established literature linking strong recurrent connectivity in prefrontal circuits to stable population-level representations [5]. However, we acknowledge that invoking it in this context, without direct evidence, risks overstating our conclusions. We will revise this sentence to make its speculative nature explicit and ground it more carefully in the cited literature rather than presenting it as an inference from our own data.

In summary, we will ensure our conclusions will be restricted to population-level coding of learned threat value and its generalization across auditory frequencies. We will revise the relevant passages in the Discussion to ensure that speculative interpretations regarding emotional memory content are either removed or clearly flagged as speculative hypotheses.

(d) 'Dynamic tone-selective responsive neurons emerge independently of learning, as they are present in both control and experimental mice, reflecting pre-existing PL sensory-driven properties (Hockley & Malmierca, 2024; Zikopoulos & Barbas, 2006).'

Maybe. They are also likely to have developed as a consequence of the repeated testing on days 1, 15, and 30, which involved intermixed exposures to the tones of different frequencies. That is, rather than 'pre-existing PL sensory-driven properties', the responses of these neurons might reflect the emergence of discrimination between the various tones across testing, and greater suppression of freezing to the non-trained tones compared to the trained tone across the various test intervals.

We thank the reviewer for this point. Our interpretation that these neurons reflect pre-existing PL sensory-driven properties was based on the observation that tone-selective responses were present in control animals that never received conditioning, consistent with prior reports of sensory responsiveness in PL cortex ([6, 7]. Because these responses emerge from the first time we expose mice to the intermediate frequencies, they cannot be explained by repeated exposure. Moreover, we did not observe progressive refinement, emergence of discrimination-like changes, or suppression of responding to non-reinforced tones in control mice. This difference between conditioned and control animals indicates that repeated tone exposure alone is not sufficient to produce the observed dynamics — associative learning is necessary. We therefore maintain that the tone-selective responses of these neurons reflect pre-existing sensory-driven properties of PL cortex that are present independently of conditioning history.

In summary, we thank the reviewer for suggesting clarifications to our interpretation, for raising the possibility that freezing behavior may contribute to graded neural responses, and for raising the question of whether repeated tone exposure may contribute to the properties of neurons recruited after conditioning. In the revised manuscript, we will include additional analyses to better dissociate the contributions of freezing behavior and tone identity, clarify passages that were insufficiently precise, and include a paragraph in the Discussion addressing potential alternative explanations alongside our own interpretation of the data.

Reviewer #3 (Public review):

Summary:

Normandin et al. explore the coding of stimuli predicting an aversive event in the prelimbic cortex. Stimuli could either be explicitly paired, explicitly unpaired, or novel but with an inferred association with the aversive event (generalization). Long-term tracking of GCaMP-positive neurons allowed them to examine how coding evolves out to a month following training. In general, they found two types of ensemble codes. One was ensembles coding for each stimulus independently, but with enhanced responding to the one eliciting a freezing response. The other was ensembles that responded to all stimuli in proportion to their similarity to the stimulus paired with the aversive event, either increasing or decreasing their activation with the degree of freezing elicited by a stimulus. Importantly, this second set of ensembles was more stable across days, potentially providing a memory trace.

Strengths:

(1) The authors track ensembles in prelimbic cortex over long time scales, providing valuable information on the consolidation of neural codes.

(2) Neural coding of generalization is examined, which is under-examined in the field.

We thank the reviewer for appreciating our design to track ensembles over time and the relevance of studying the neural substrates of generalization.

Weaknesses:

(1) Difficult to determine if responses treated as encoding stimulus valence are driven instead by the behavior that the stimulus elicits, freezing.

We thank the reviewer for this thoughtful and constructive comment. We agree that an alternative interpretation is that the graded-response ensembles may partially reflect freezing-related activity rather than mnemonic or salience-related representations of the conditioned stimuli themselves. In the revision, we will acknowledge that prior work has identified PL neurons that encode freezing independently of stimulus identity or associative content. Furthermore, we will implement the reviewer’s suggested generalized linear model (GLM) approach using inferred spiking activity derived from the Ca2+ signals. Specifically, we will include both stimulus identity and freezing behavior as predictors. Because freezing varies across trials whereas stimulus presentation is fixed, this analysis will allow us to dissociate the relative contributions of stimulus-related versus freezing-related activity to the graded neuronal responses. We thank the reviewer for this excellent suggestion.

If graded stimulus coding remains significant after accounting for freezing behavior, this would strengthen the interpretation that these ensembles encode learned salience or associative properties of the stimuli rather than behavioral output alone. Conversely, if freezing explains a substantial proportion of the variance, we will revise our interpretation accordingly.

(2) The study implies that the identified ensembles are causally related to valence memory, but no experimental interventions are performed to justify this.

We appreciate the reviewer's point. We agree that our data are correlational in nature and that establishing a causal relationship between identified ensembles and valence memory would require experimental interventions such holographic two-photon manipulations, which are beyond the scope of the present study but represent an important direction for future work.

To provide an indirect link between ensemble organization and behavior within the constraints of the current dataset, we will examine inter-individual variability in the revised manuscript. Specifically, we will test whether the proportion of neurons participating in stable graded-response ensembles versus dynamic stimulus-specific ensembles predicts individual differences in freezing behavior and fear generalization across retrieval sessions. If animals with a higher proportion of stable graded-response neurons show stronger discrimination and less generalization to non-conditioned tones, this would strengthen the association between ensemble organization and behavioral outcome, while remaining correlational in interpretation.

We will modify the manuscript terminology accordingly, replacing causal language with phrasing that accurately reflects the associative nature of our conclusions.

References

(1) Aschauer, D.F., et al., Learning-induced biases in the ongoing dynamics of sensory representations predict stimulus generalization. Cell Rep, 2022. 38(6): p. 110340.

(2) Kato, H.K., S.N. Gillet, and J.S. Isaacson, Flexible Sensory Representations in Auditory Cortex Driven by Behavioral Relevance. Neuron, 2015. 88(5): p. 1027–1039.

(3) Vervliet, B., et al., Generalization gradients in human predictive learning: Effects of discrimination training and within-subjects testing. Learning and Motivation, 2011. 42(3): p. 210–220.

(4) Dunsmoor, J.E. and K.S. LaBar, Effects of discrimination training on fear generalization gradients and perceptual classification in humans. Behav Neurosci, 2013. 127(3): p. 350–6.

(5) Mante, V., et al., Context-dependent computation by recurrent dynamics in prefrontal cortex. Nature, 2013. 503(7474): p. 78–84.

(6) Hockley, A. and M.S. Malmierca, Auditory processing control by the medial prefrontal cortex: A review of the rodent functional organisation. Hear Res, 2024. 443: p. 108954.

(7) Zikopoulos, B. and H. Barbas, Prefrontal projections to the thalamic reticular nucleus form a unique circuit for attentional mechanisms. J Neurosci, 2006. 26(28): p. 7348–61.

In the story of Joseph, gender and heroism are connected through power, temptation, and morality. Joseph is presented as honorable because he resists the advances of Potiphar’s wife and remains loyal to his values. His heroic identity is connected with self-control, wisdom, and faith rather than physical strength or war. Potiphar’s wife, however, is often presented as dangerous and emotional, reflecting a common pattern in ancient stories where female desire becomes a source of conflict and punishment. This creates a strong contrast between the “pure” male hero and the woman who is blamed for temptation.

Different retellings of Joseph’s story focus on different aspects of the characters. Some versions portray Potiphar’s wife as manipulative and sinful, while others present her with more sympathy and complexity. The language used in religious and historical retellings reflects the values of the culture and time in which they were written. In many traditional versions, masculine virtue is connected with discipline and leadership, while female desire is treated as threatening to social and moral order. CC BY 4.0

es kann zu Überverkäufen kommen --> ist allerdings nicht das einzige Problem im E-Commerce! Weitere Herausforderungen sind: - Skalierung/Expansion ohne hohen Ressourhenaufwand - Datenchaos kann zu Überverkäufen und anderen Fehlern führen - Veraltete Tech-Stacks blocken Automatisierungen - Insellösungen --> teuer und komplex

den deutschen Markt (sonst Dopplung oben)

Finde die Aussage schwierig, denn Preise steigen können aufgrund des modularen Aufbaus ja schnell wachsen.

was sind Einzelunternehmer? Ich würde nur von KMU sprechen

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ggf. noch ergänzen/Vorteil: Neben den großen Playern, führt PlentyONE auch Nischen- und länderspezifische Marktplätze

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Macht es vielleicht hier Sinn, ein längeres Zitat einzusetzen? Die anderen Zitate sind zum Teil deutlich länger

E-Commerce Germany Awards 2026

konnte das Bild leider nicht markieren, aber macht es vielleicht Sinn, einen Screenshot der deutschen Homepage einzubinden, statt der englischen seite? Oder habt ihr das bewusst gewählt?

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In the story of Phaedra and Hippolytus, gender plays an important role in the construction of both desire and heroism. Hippolytus is presented as pure, disciplined, and devoted to Artemis, rejecting love and sexuality. His identity as a heroic male figure is connected with control, chastity, and distance from women. Phaedra, however, is controlled by forbidden desire and emotional suffering. This creates a contrast between masculine self-control and feminine passion that appears often in ancient literature. At the same time, Phaedra is also trapped by the expectations of honor and shame inside a patriarchal society.

Different versions of the myth present Phaedra differently. In some versions she appears manipulative and dangerous, while in Euripides’ version she becomes more tragic and human because Aphrodite curses her with desire. This changes how readers judge her actions. The language and focus of each retelling reflect the values of the culture and time in which the story was written or translated. Ancient and later versions often place blame on female desire, while male heroism remains connected to purity, honor, and public reputation. CC BY 4.0

STF Súmula 442 - A inscrição do contrato de locação no Registro de Imóveis, para a validade da cláusula de vigência contra o adquirente do imóvel, ou perante terceiros, dispensa a transcrição no Registro de Títulos e Documentos.

Observe que o direito de vigência da locação, na hipótese de alienação do imóvel a terceiros, tem 3 requisitos: - Existir no contrato de locação a cláusula de vigência; - Haver averbação do contrato de locação na matrícula do imóvel. - Locação por prazo determinado.

Com isso, inexistindo algum dos requisitos acima, não haverá direito à vigência.

É preciso autorização do cônjuge para a validade de contrato de locação superior a 10 anos.

O locatário não precisará pagar a multa de rescisão se esta for motivada por transferência do locatário a localidade diversa a mando do empregador, devendo o locador ser notificado em 30 dias.

Possibilidades de reforço ou substituição da garantia da locação

Nos contratos ajustados por mais de 30 meses, ocorrendo a prorrogação, o locador poderá denunciar o contrato sem qualquer justificativa.

Nos contratos ajustados por prazo inferior a 30 meses, ocorrendo a prorrogação, somente o locador poderá retomar o imóvel e denunciar o contrato com as motivações prevista neste artigo.

Embora ocorra a prorrogação do contrato por prazo indeterminado, ou seja, por prazo superior a 30 meses, o locador somente poderá denunciar o contrato com motivação prevista em lei, não se aplicando a denúncia vazia, via de regra, para os contratos ajustados por prazo superior a trinta meses.

O direito de preferência deve ser exercido em até 30 dias, sob pena de caducidade, após a notificação do locador sobre a venda do imóvel.

Nesses casos, em que o contrato dura menos de 30 meses, decorrido o prazo, o contrato não se extingue, mas sim se prorroga.

• Daí se incluir, entre os deveres do locatário, o de permitir a vistoria do imóvel pelo locador, o que não traduz turbação da posse. Mas é evidente que o locador poderia abusar do seu direito, marcando visitas repetidas, com curto prazo de intervalo, ou em horários inconvenientes, que perturbassem a privacidade, o descanso ou o lazer do locatário. Para se evitar este comportamento, que constrangeria o locatário, levando-o, até mesmo, a encerrar a locação, a lei condicionou a vistoria à combinação prévia do dia e da hora em que se realizará. Igual obrigação tem o locatário, quando o locador pretender alienar o imóvel, e, para isto, precisa mostrá-lo aos eventuais pretendentes. Esta disposição não constava da lei anterior, e, graças a esta omissão, felizmente agora suprida, muitos locatários dificultavam a venda, impedindo visitas dos candidatos. Se os locatários agora assim procederem, estarão cometendo infração legal e sujeitando-se à ação de despejo. Não havendo acordo entre locador e locatário, quanto aos dias e horários das visitas, caberá ao juiz designá-los, de acordo com seu prudente arbítrio, respeitando os costumes do lugar e as peculiaridades de cada caso concreto. (Sylvio Capanema de Souza, Lei do Inquilinato Comentada Artigo por Artigo, 10ª edição, Rio de Janeiro, Ed. Forense, p. 142).

SEÇÃO III

Da locação não residencial

Art. 51. Nas locações de imóveis destinados ao comércio, o locatário terá direito a renovação do contrato, por igual prazo, desde que, cumulativamente:

• I - o contrato a renovar tenha sido celebrado por escrito e com prazo determinado;

• II - o prazo mínimo do contrato a renovar ou a soma dos prazos ininterruptos dos contratos escritos seja de cinco anos;

• III - o locatário esteja explorando seu comércio, no mesmo ramo, pelo prazo mínimo e ininterrupto de três anos.

Art. 9º A locação também poderá ser desfeita:

• I - por mútuo acordo;

• II - em decorrência da prática de infração legal ou contratual;

• III - em decorrência da falta de pagamento do aluguel e demais encargos;

• IV - para a realização de reparações urgentes determinadas pelo Poder Público, que não possam ser normalmente executadas com a permanência do locatário no imóvel ou, podendo, ele se recuse a consenti - las.

Ordem de preferência na alienação do imóvel locado: - Sublocatário tem preferência ao locatário; - Locatário tem preferência ao terceiro; - Condomínio tem preferência ao locatário.

AGRAVO REGIMENTAL NO RECURSO ESPECIAL. AÇÃO INDENIZATÓRIA. DIREITO DE PREFERÊNCIA. AVERBAÇÃO DO CONTRATO NO REGISTRO IMOBILIÁRIO. PRESCINDIBILIDADE. - 1. Nos termos da jurisprudência desta Corte, a inobservância do direito de preferência do locatário na aquisição do imóvel enseja o pedido de perdas e danos, que <u>não se condiciona ao prévio registro do contrato de locação</u> na matrícula imobiliária. Precedentes. - 2. Agravo regimental não provido. (AgRg no REsp n. 1.356.049/RS, relator Ministro Ricardo Villas Bôas Cueva, Terceira Turma, julgado em 25/2/2014, DJe de 28/2/2014.)

PROCESSUAL CIVIL. NEGATIVA DE PRESTAÇÃO JURISDICIONAL. NÃO OCORRÊNCIA. CIVIL. LOCAÇÃO. DIREITO DE PREFERÊNCIA. EFEITOS OBRIGACIONAL E REAL. PLEITO INDENIZATÓRIO E DE ADJUDICAÇÃO COMPULSÓRIA DO IMÓVEL. CONTRATO DE LOCAÇÃO NÃO AVERBADO NO CARTÓRIO DE REGISTRO DE IMÓVEIS POR FALHA DO LOCADOR. IRRELEVÂNCIA. INEXISTÊNCIA DE DIREITO DE REAVER O BEM. MANUTENÇÃO DO ARESTO RECORRIDO. - 1. Afasta-se a alegada negativa de prestação jurisdicional quando o acórdão recorrido, integrado por julgado proferido em embargos de declaração, dirime, de forma expressa, congruente e motivada, as questões suscitadas nas razões recursais. - 2. O art. 27 da Lei n. 8.245/91 prevê os requisitos para que o direito de preferência seja exercido pelo inquilino que tenha interesse em adquirir o imóvel locado em igualdade de condições com terceiros, sendo certo que, em caso de inobservância de tal regramento pelo locador, poderá o locatário fazer jus a indenização caso comprove que tinha condições de comprar o bem nas mesmas condições que o adquirente. - 3. Além dos efeitos de natureza obrigacional correspondentes ao direito a perdas e danos, o desrespeito à preempção do locatário pode ter eficácia real consubstanciada no direito de adjudicação compulsória do bem, uma vez observados os ditames do art. 33 da Lei do Inquilinato. - 4. O direito real à adjudicação do bem somente será exercitável se o locatário a) efetuar o depósito do preço do bem e das demais despesas de transferência de propriedade do imóvel; b) formular referido pleito no prazo de 6 (seis) meses do registro do contrato de compra e venda do imóvel locado adquirido por terceiros; c) promover a averbação do contrato de locação assinado por duas testemunhas na matrícula do bem no cartório de registro de imóveis, pelo menos 30 (trinta) dias antes de referida alienação. - 5. Impõe-se a obrigação legal de averbar o contrato de locação para possibilitar a geração de efeito erga omnes no tocante à intenção do locatário de fazer valer seu direito de preferência e tutelar os interesse de terceiros na aquisição do bem imóvel. - 6. Ainda que obstada a averbação do contrato de locação por falha imputável ao locador, não estaria assegurado o direito à adjudicação compulsória do bem se o terceiro adquirente de boa-fé não foi cientificado da existência de referida avença quando da lavratura da escritura de compra e venda do imóvel no cartório de registro de imóveis. - 7. Recurso especial conhecido e desprovido.

(REsp n. 1.554.437/SP, relator Ministro João Otávio de Noronha, Terceira Turma, julgado em 2/6/2016, DJe de 7/6/2016.)

Locatário tem direito à indenização: - Acaso o locador não renove a locação em decorrência de proposta de terceiro; - Acaso o locador, em três meses após recebimento do imóvel, não der a destinação declarada para não renovar a locação.

Acaso a ação renovatória seja improcedente em decorrência de oferta de terceiro, a sentença fixará a indenização devida ao locatário (art. 52, § 3º).

A indenização será devida tanto pelo locador, quanto pelo terceiro proponente, de forma solidária.

O locatário tem direito de preferência na compra do imóvel que locatário pretenda vender. Acaso haja preterição, o locatário ainda possui meios para obter a propriedade do bem.

Para tanto, os requisitos para a constituição de Direito Real a favor do locatário:

• Requerer, em até 6 (seis) meses, o imóvel para si, contado da data do registro em cartório de imóveis;
• Depositar o valor do imóvel e demais despesas de transferências;
• Averbação junto à matrícula do imóvel do contrato de locação, desde que averbado em, no mínimo, 30 (trinta) dias da data da alienação junto à matrícula;

Observe que a lei estabelece que a averbação do contrato de locação na matrícula do imóvel tem 2 importantes efeitos: - Assegurar que eventual novo locatário observe o prazo de locação, proibindo a denúncia do contrato sem antes decorrer o prazo contratual; - Assegurar a aquisição do imóvel acaso haja preterição do locador quanto ao direito de preferência do locatário.

Por fim, cabível destacar que a averbação, enquanto manifestação da publicidade dos atos relativos a direitos reais, é essencial para geração de efeito erga omnes. Com efeito, para garantir o direito real de aquisição, é imprescindível a averbação do contrato de locação.

Lado outro, tratando-se da outra hipótese referente ao prejuízo do direito de preferência do locatário, o pleito de perdas e danos não se submete a registro público como condição.

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In this version of Beowulf, heroism is again connected with fame, war, leadership, and family bloodline. The male figures are described as strong rulers and warriors, showing how masculinity in the poem is tied to honor and power. Hrothgar receives “glory of war,” which presents battle and military success as important qualities of the heroic male identity. At the same time, the queen is described mainly as a “helpmate,” showing how women are often placed in supportive roles around male heroes rather than being central figures themselves.

Francis Barton Gummere’s translation uses formal and poetic language such as “glory of war” and “chieftain of clansmen.” The elevated style creates a legendary and heroic atmosphere. Compared to the Morris and Wyatt translation, Gummere’s wording feels slightly clearer and more direct, but both translations glorify masculine power and warfare. The language also reflects older cultural values where men are associated with leadership and public honor, while women are connected with loyalty, marriage, and support inside the heroic society. CC BY 4.0