DOI: 10.1038/s41467-025-66727-3

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DOI: 10.1038/s41467-025-66727-3

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DOI: 10.1038/s41467-025-66727-3

Resource: RRID:BDSC_7415

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DOI: 10.1038/s41467-025-66727-3

Resource: RRID:BDSC_7000

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DOI: 10.1038/s41467-025-66727-3

Resource: RRID:BDSC_31390

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DOI: 10.1038/s41467-025-66727-3

Resource: RRID:BDSC_6797

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DOI: 10.1038/s41467-025-66727-3

Resource: RRID:BDSC_27587

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DOI: 10.1038/s41467-025-66727-3

Resource: RRID:BDSC_67032

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DOI: 10.1038/s41467-025-66727-3

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DOI: 10.1038/s41467-025-66727-3

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DOI: 10.1038/s41467-025-66727-3

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DOI: 10.1038/s41467-025-66727-3

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What is this?

DOI: 10.17912/micropub.biology.000811

Resource: ChIP-X Enrichment Analysis 3 (RRID:SCR_023159)

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DOI: 10.12688/openresafrica.15820.1

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What is this?

DOI: 10.1093/nar/gkaf1514

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What is this?

DOI: 10.1038/s41593-025-02162-3

Resource: CellChat (RRID:SCR_021946)

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DOI: 10.1038/s41593-025-02162-3

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DOI: 10.1038/s41593-025-02162-3

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DOI: 10.1038/s41467-025-67232-3

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What is this?

DOI: 10.1016/j.celrep.2025.116854

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What is this?

DOI: 10.1007/s11548-025-03562-3

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What is this?

The core argument

The essay argues that polling has become less reliable at the same time that it has become more powerful, and that this combination distorts democratic politics.

Polls:

increasingly fail to accurately measure public opinion

yet increasingly determine who gets attention, legitimacy, money, debate access, and media coverage

How Trump fits in

The piece opens with Donald Trump claiming he has no pollster and doesn’t tailor his message to polls. Lepore calls this disingenuous:

Trump may not have had a traditional campaign pollster

but his rise depended heavily on polls for visibility and validation

polls got him into debates, dictated stage placement, and fueled media coverage

So Trump is described as “a creature of the Sea of Polls,” not above it

Why modern polls are broken

The article explains in detail why polling has deteriorated:

1. People don’t answer anymore

Response rates used to be 60–90%

Now they’re often in the single digits

Most Americans refuse poll calls, creating non-response bias

1. Technology & law made it worse

Fewer landlines

Cell-phone autodialing is illegal

Internet polls are self-selected and skew younger and more liberal

Mixed-method polling still doesn’t work well

1. Samples are tiny and fragile

National election polls often rely on ~1,000–2,000 people

Statistical “weighting” tries to fix bias, but the lower the response rate, the shakier the results

Why polls now matter more than ever

Despite being unreliable, polls are used to:

decide who qualifies for debates

determine media attention

shape fundraising and momentum

create “winners” and “losers” long before anyone votes

Fox News using polls to select debate participants is presented as a major example of polling replacing democratic processes.

Historical background

The essay gives a history of polling:

Early “straw polls” by newspapers

The rise of George Gallup in the 1930s

Polling claimed to represent “the will of the people” scientifically

But:

Early polls systematically excluded Black Americans, the poor, and the disenfranchised

Polling mirrored and amplified existing inequalities

What was presented as “public opinion” was often the opinion of a privileged subset

Deeper philosophical critique

Lepore raises a fundamental question:

What if measuring public opinion isn’t good for democracy at all?

Key ideas:

Polls treat public opinion as the sum of individual answers, ignoring how opinions are formed socially

Polls can create opinion rather than measure it

Constant polling shifts politics from deliberation and leadership to reacting to numbers

Bottom line

The piece isn’t just saying “polls are inaccurate.”

It’s saying:

Polls shape reality instead of describing it

They weaken representative democracy

They reward spectacle, momentum, and media attention over governance

And they increasingly substitute statistical artifacts for actual voting

Must be confident and very knowledgeable in what you are writing about.

I believe learning how to write in a variety of ways can help you be more creative and make it a little less stressful when having to do writing assignments.

it's important to know the different ways of writing, otherwise we can make mistakes in writing assignments. Not only in the way of writing but the way you read or speak.

R0:

EDITOR:

The reviewers agree that your manuscript addresses an important topic. They have also raised a number of well-justified concerns and points requiring clarification. I hope that you see these as opportunities to further improve your manuscript such that it may be accepted for publication.

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The author wrote this manuscript quite well. However, there are some suggestions for improving it better including,

Abstract: The abstract is written well. However, the results showed about self-stigma representing at 49% so this result should be suggested in conclusion as well.

Introduction: The introduction is organised and written well. However, there are some suggestions about referencing that should be revised along with Vancouver style and the journal format, such as (6)(7)(8), should be (6-8) or (Mbuthia et al., 2020) should be a number of reference. Another point, the abbreviation of drug-resistant TB (DR-TB) should be the same with DRTB in table 2 (page 10). Moreover, in terms of objective of the study, it should be written clearly. The author stated in line 98-100 (page 3), but it seems like expected outcomes rather than its objectives.

Methodology: Ethics statement: It is a clear statement, however, the date of approval should be presented as well to ensure the data were collected after approval.

Study population: The author stated that the target population comprised people with TB who were on treatment and were 15 years and above. However, the results showed that there were some participants aged under 15 years (0-14) as well. Thus, the author should revise and make it correct.

Sampling procedures: The author stated that the data were collected in 12 regions, however there were only 11 regions stated in line 139-140.

Sample size: The author showed some details of sample size calculation that met 421 persons (line 147). However, there were only 367 participants recruited to this study which is less than the appropriate sample size calculated. So, the author needs to explain more details about the sample size. It should be 421 as the result of calculation with the appropriate formula. Moreover, if there are sources of the number used to calculate, the reference needed to be stated as well.

Eligibility criteria: In terms of inclusion and exclusion criteria, the author stated that all people with TB aged 15 years and older would be recruited to the research and all people who were below the age of 15 years were excluded. This is the main point that needs to be clarified because in the results, there are some participants aged 0-14 years as stated before. Moreover, in terms of ethics, participants aged less than 18 years cannot sign the consent form by themselves, their parents should sign the consent form. So, the author needs to revise and clarify further.

Data collection tools and procedures: There is no information about the questionnaire well. The questionnaire should be clarified the details, especially the items used to categorise into "no stigma and stigma (binary). If you use only one item, it should not be appropriate to categorise. This is an important point of this study that needs to be explained. As well as, if the questionnaire was conducted by previous researchers, it should be cited correctly. Moreover, the author stated in this part that stigma was assessed using a set of standardised questions rated on a five-point Likert scale (0 = Strongly disagree to 4 = Strongly agree), while, in page 6, the author used (1 = Strongly agree to 5 = Strongly disagree) as well as the data were categorised in to 5 groups staring from 1.00 - 5.00. Please check the details again.

Data analysis: The Cronbach's alpha value needs to be presented with the exact value instead of >=0.70 that it will present the reliability of tools better. Moreover, the author stated in line 181 and table 2 "TB type" but in the conclusion, the author used "treatment type". So, this point needs to be the same. For the binary classification, the author needs to explain more details about how to categorise into 2 groups: no stigma and stigma. In terms of inferential statistics, the binary logistic regression and multiple logistic regression were not used and shown in the results. So, the author needs to revise about this point again.

Results: The sum of percentage, the details in Table 1 & 2 showed the percentage of each variable, which is good. However, the author needs to check the sum of each variable should be 100%. The author may use two decimal points for presenting the percentage. Moreover, some sub-variables which there is no data (0) does not need to present in the table. Please find the details in the attached file.

In table 2, inferential statistics, the author stated in data analysis that binary logistic regression and multiple logistic regression would be used to analyse to identify the predictors. However, in the results, there is no any results based on these statistics. So, the author needs to revise about the statistics stated in data analysis. Moreover, about chi-square, the author needs to check the assumption of chi-square because No cell should have an expected frequency < 1, and at least 80% of cells should have expected frequencies of 5 or more. So, if it does not meet the assumption, its results might be wrong.

Aged 0-14, the author stated in the methodology that the participants need to be 15 years or over. So, please check the data again.

Some words should be changed for example in line 213 from prevented to obstructed. Moreover, about abbreviation "DSTB and DRTB", for DSTB, the author did not state before so it needs to be mentioned in previous part first before using in this part. As well as, DRTB, the author used DR-TB in line 79 so it needs to be the same, with or without -.

Discussion: The author wrote this part quite well, however, the author needs to check about the number of percentage presented in this part again. Moreover, citation should be revise and rewrite following the format.

References: In terms of references, the author should check the format of Vancouver style referencing in both in-cited and references part again. As well as, the author needs to check the format along with the journal format. For example, (xx) to [xx]. Please revise and rewrite following the formats.

Reviewer #2: Please see my report. I think the manuscript transition from a dissertation to a paper is incomplete. Please review my report for details. I raise concerns regarding the sampling, statistical analysis and the conclusions made regarding the results.

Reviewer #3: This cross-sectional study tackles an important global health problem, TB-related stigma among people with TB. This study has significant merits including 367 people living with tuberculosis sampled over one year; and captures various contexts, specifically 180 health facilities across 11counties aiming for a nationally representative survey of TB-related stigma in Kenya. Two hundred and twenty-eight patients provided information regarding TB-related stigma, of whom 24 reported experiencing TB-related stigma.

Several areas remain unclear to me and require further clarification, elaboration or consideration for reformatting.

1. The referencing style used is inconsistent. e.g. introduction section lines 60-61(Mbuthia et al. 2020), whereas other areas have a different style that is numbered. Consider reformatting for consistency.

2. Previous research on TB-related stigma measurement and its implications to TB related outcomes in the Kenyan context has not been highlighted.

3. Ethics statement section could be aligned for consistent formatting with other text sections of the manuscript.

4. The sample size calculation could be further clarified for the readers to judge its robustness. a. Is there a proportion of TB-related stigma assumed from a previous study? b. What is the rationale of a 90% response rate? – Lines 147 to 150. c. What was the actual response rate?

5. From the manuscript, the sample size calculated was 421 TB patients, but only 367 are reported and 228 TB patients provide information related to TB stigma. These patients were sampled over one year from 180 health facilities across 11 counties in Kenya. a. Further clarification on the sampling frame is needed. b. How many patients were sampled per health facility? Was there any gender consideration per health facility? c. How were the 12 regions chosen and how do they relate to the current national or programmatic divisions? d. It is indicated that one county was chosen from the 12 regions but only 11 counties included.

6. Elaborating on the tool and procedures used is needed for the readers to judge the robustness of the methodology used. This information is crucial in the methods section. Lines 164-165: “Stigma was assessed using a set of standardized questions rated on a five-point Likert scale (0 = Strongly Disagree to 4 = Strongly Agree).”

(i) What is the set of standardized questions? (ii) What tool was used? (iii) Has this tool been previously used in the literature? (iv) Has the tool been previously used in the Kenyan context? (v) Is this a validated tool? (vi) In what language/s were the questions asked? (vii) Who administered the survey? Provide relevant references.

These details are missing in the methods section; and need to be considered for inclusion in the main text and/or supplementary material based on journal guidelines.

1. What do the authors think could be the implications of handling neutral scores as missing? Lines 189-190: ‘Responses with a "Neutral" score were treated as missing in the binary variable.’ Please elaborate and describe the possible limitation.

2. Lines 192 to 194: “Variables with p-values <0.05 in bivariate analysis were entered into a multivariate logistic regression model to identify independent predictors of TB-related stigma” – Do the authors mean a multivariable logistic regression model?

3. In the results section, 10 participants are aged 0-14 years however, one of the study inclusion criteria is that participants should be aged 15 years and above. Further clarification is needed.

4. Are the age group categories shown in Table 1 meaningful? Would other summary descriptive statistics for age central tendency and dispersion be considered to provide more information about patient characteristics.

5. The term “Pagan” in Table 1 may be considered derogatory – consider an alternative word.

6. Several other participant characteristics would be important to understand TB-related stigma, including: a) the type of tuberculosis; b) the timing of treatment for the TB patient at which this survey was being performed; c) disclosure of a TB diagnosis; among others. There is existing global, regional and particularly Kenyan literature that supports the importance of these particular characteristics. Consider including these in Table 1.

7. Lines 228-230: “Out of 367 participants, 228 individuals with TB shared their experiences regarding stigma. Among them, 24 (11%) reported experiencing TB-related stigma, while 204 (89%) did not. The remaining 139 participants did not provide an opinion and were excluded from the bivariate analysis.” a. Based on this statement, it is not clear what the procedures for study participation were. The study was to assess TB-related stigma, but 139 participants did not provide an opinion. Please elaborate the study procedures for the readers to gain clarity. b. What are the characteristics of the individuals of TB patients who did not share their experiences regarding stigma? c. Were they different from those who did?

8. Clarification is needed regarding the proportions of stigma provided in different sections of the manuscript. TB-related stigma dimensions in Figure 2 report relatively high TB-related stigma levels (49% for self-stigma, 68% of community-level stigma); compared to the overall TB-related stigma reported as 11% and also shown in Table 2.

9. Consider including whether the type of TB was pulmonary or not, in Table 2. This is not clear.

10. Data analysis:

Lines 171-175: “Exploratory factor analysis (EFA) was conducted to test the internal consistency and construct validity of the stigma scale in the Kenyan context. Cronbach’s alpha was calculated to assess internal reliability, with values ≥0.7 indicating acceptable consistency. The principal components extraction method was used to identify underlying factors, with factor loadings ≥0.4 considered acceptable.”

• Although this section is included in the data analysis methods section, there is no data in the manuscript to support this. Please provide this information if it is available.

Lines 182-186: “Stigma-related responses covering domains such as guilt, fear, social avoidance, and disclosure concerns were numerically encoded (1 = Strongly Agree to 5 = Strongly Disagree). Scores were aggregated row-wise per participant to generate a mean stigma score, which was then categorized as follows: 1.00–1.49: Strongly Disagree, 1.50–2.49: Disagree, 2.50–3.49: Neutral, 3.50–4.49: Agree and 4.50–5.00: Strongly Agree.”

• Similarly, although this section is included in the data analysis methods section, there is no data in the manuscript to support this. Stigma is reported as a binary variable and not continuous. Please provide this information if it is available.

Lines 192-194: Variables with p-values <0.05 in bivariate analysis were entered into a multivariate logistic regression model to identify independent predictors of TB-related stigma. Outputs are presented in Table 1 and Table 2 of the Results section.

• Again, this section is included in the data analysis methods section, but there is no data in the manuscript to support this. No results are provided for multivariable logistic regression in Table 1 or Table 2. Please provide this information if it is available.

Was there a justification of including age group instead of age as a continuous variable instead in the data analyses models used?

Was the sample size calculated powered to determine the factors associated with TB-related stigma?

1. Results, Discussion and Conclusion. The main confusion for me is around denominators and the respective proportions related to TB stigma that have been presented. Clarification on this is needed.

2. Study limitations need to be acknowledged.

A jersey shorts, socks , footbol shoes.

Trustworthy, responsible, hardworking.

Dependable, Responsible, Hardworking, Thoughtful, Extrovert

The 3-5 adjectives I want my reciever to describe me as is dependable, organized, approachable, and driven.

I would want my receiver to describe me as hard working, timely, and trustworthy.

Le phénomène du bavardage scolaire : Analyse et perspectives

Synthèse Exécutive

Ce document présente une analyse approfondie du phénomène de bavardage scolaire, un enjeu souvent sous-estimé qui affecte de manière significative le climat de classe et la réussite des élèves.

Basée sur une étude combinant une revue de la littérature scientifique et une enquête de terrain menée auprès d'élèves de 4ème, cette synthèse met en lumière la complexité du bavardage, les perceptions divergentes qu'il suscite et l'efficacité limitée des interventions basées uniquement sur la prise de conscience individuelle.

L'analyse théorique révèle que le bavardage a évolué, passant d'un "chahut traditionnel" structuré à un désordre plus "anomique" et généralisé.

Les travaux de chercheurs comme Florence Ehnuel et Alain Courneloup soulignent un décalage fondamental entre les perceptions des différents acteurs : les élèves le banalisent souvent comme une interaction sociale normale, les parents le perçoivent avec une faible gravité, tandis que les enseignants le vivent comme une source de déprofessionnalisation et d'impuissance.

Les causes identifiées sont multiples, incluant l'ennui, le besoin d'interaction sociale, la pression des pairs et un cadre scolaire parfois perçu comme trop rigide.

L'enquête de terrain, réalisée via un questionnaire suivi d'entretiens individuels, confirme ces constats. Une majorité d'élèves bavards ne se sentent pas personnellement dérangés par le bruit et estiment que leurs propres conversations ne nuisent pas à leurs camarades, se croyant capables de parler et d'écouter simultanément.

L'outil de questionnement a permis une prise de conscience modérée chez environ la moitié des participants, mais n'a entraîné un changement de comportement durable que pour une minorité.

La crainte de sanctions demeure le levier externe le plus efficace, tandis que la motivation interne reste fragile.

En conclusion, la lutte contre le bavardage scolaire ne peut se résumer à des sanctions disciplinaires.

Elle exige une approche globale qui intègre la compréhension des perceptions des élèves, la mise en place de cadres clairs et co-construits, et l'adoption de stratégies pédagogiques actives pour réduire l'ennui.

Si la prise de conscience est une étape nécessaire, elle s'avère insuffisante sans un accompagnement structuré et des règles appliquées avec constance.

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I. Cadre Théorique du Bavardage Scolaire

Définition et Évolution du Phénomène

Le bavardage scolaire est défini comme toute prise de parole non autorisée par l'enseignant durant un temps de cours. Il constitue un phénomène social complexe qui perturbe la transmission des savoirs et le climat d'apprentissage.

L'analyse sociologique de Jacques Testanière (1967) offre une perspective historique sur l'évolution du désordre en classe. Il distingue :

• Le chahut traditionnel : Une "anomalie normale" et collective, souvent ritualisée, qui visait à tester l'autorité de l'enseignant tout en renforçant la cohésion du groupe d'élèves.

• Le chahut anomique : Une forme de désordre plus généralisée, individualiste et sans règles, qui exprime une mauvaise intégration de l'élève au système pédagogique. Le bavardage contemporain s'apparente davantage à cette seconde forme, caractérisée par une multitude de conversations parallèles plutôt qu'une confrontation unifiée.

Perceptions Divergentes des Acteurs

L'une des difficultés majeures dans la gestion du bavardage réside dans le profond décalage de perception entre les différents acteurs de la communauté éducative, comme le démontre l'ouvrage de Florence Ehnuel, « Le bavardage : Parlons-en enfin ! ».

| Acteur | Perception du Bavardage | | --- | --- | | Élèves | Considéré comme une interaction sociale normale et un "non-acte". Beaucoup estiment pouvoir écouter et parler en même temps. Il est souvent justifié par l'ennui, le besoin d'échanger avec les pairs ou le désintérêt pour la matière. | | Enseignants | Vécu comme une nuisance majeure, un manque de respect, et une source de fatigue et de culpabilité. Les réactions varient de la tolérance à la sanction systématique, en passant par un sentiment d'impuissance. | | Parents | Souvent perçu comme un problème mineur, non comparable à l'insolence ou aux mauvais résultats. Certains y voient même un signe de "vitalité" ou d'"aisance relationnelle". | | Didacticiens | Interprété comme une forme de résistance à la norme scolaire, une pratique sociale d'échange, une échappatoire face aux difficultés d'apprentissage, ou un symptôme du décalage entre la culture scolaire et la culture jeune. |

Causes et Motivations du Bavardage

La littérature identifie plusieurs facteurs expliquant la prévalence du bavardage :

• Facteurs Pédagogiques : L'ennui provoqué par un cours jugé trop lent ou inintéressant est une cause majeure. Comme le souligne Alain Courneloup, "un élève qui s'ennuie est un élève qui va trouver à s'occuper".

• Facteurs Sociaux : Le besoin d'interaction avec les pairs est fondamental à l'adolescence. Le groupe agit comme un "médiateur" entre l'individu et les adultes. Répondre à un camarade est souvent perçu comme une obligation sociale pour ne pas le "vexer" ou trahir une amitié.

• Facteurs Sociétaux : La "génération du zapping" est habituée à un environnement bruyant et à la multi-activité. Le silence peut être perçu comme angoissant par certains élèves.

• Facteurs Institutionnels : L'absence de règles claires ou le manque de constance dans l'application des sanctions par les enseignants peut créer un cadre propice au développement du bavardage.

Conséquences et Enjeux

Les méfaits du bavardage sont souvent sous-estimés. Il ne s'agit pas d'un simple désagrément sonore.

• Sur les apprentissages : Le bavardage est une "forme d'absentéisme" intellectuel.

Même si l'élève est physiquement présent, son attention est détournée, ce qui nuit à la concentration, à la compréhension et à la mémorisation.

• Sur le climat de classe : Le bruit constant génère de la fatigue et de la tension pour l'enseignant et pour les élèves qui souhaitent travailler.

Il ralentit le rythme du cours et peut créer un sentiment d'impunité.

• Sur le parcours de l'élève : À long terme, le bavardage persistant, lorsqu'il est le symptôme d'un désintérêt plus profond, peut être un indicateur de risque de décrochage scolaire.

François Dubet, dans « La galère », décrit comment le désengagement scolaire peut mener à des trajectoires de marginalisation.

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II. Enquête de Terrain sur la Prise de Conscience des Élèves

Objectif et Méthodologie de l'Étude

L'enquête visait à déterminer si un outil de questionnement pouvait amener des élèves de 4ème à prendre conscience de l'ampleur et des conséquences de leur propre bavardage, et si cette prise de conscience pouvait induire un changement de comportement. L'expérimentation s'est déroulée en trois phases :

1. Phase 1 : Administration d'un questionnaire en ligne (Google Forms) à 52 élèves pour évaluer leurs pratiques et perceptions.

2. Phase 2 : Une période de plusieurs semaines pour observer d'éventuels changements.

3. Phase 3 : Entretiens individuels avec un échantillon de 8 élèves pour mesurer l'impact de l'intervention.

Principaux Résultats du Questionnaire (N=52, dont 35 "bavards")

L'analyse s'est concentrée sur les 35 élèves s'identifiant comme discutant en cours "de temps en temps", "assez" ou "tout le temps".

• Auto-perception des élèves bavards :

◦ Un paradoxe central : Une grande majorité des élèves bavards (65,7%) déclarent ne pas être dérangés par le bruit en classe.   

◦ La rationalisation du multitâche : Plus de la moitié (54,3%) estiment que leurs propres discussions ne gênent "pas du tout" leurs camarades. La raison principale invoquée (68,4%) est leur conviction de pouvoir "parler à [leur] voisin et écouter le professeur en même temps". 

◦ Les motivations sociales avant tout : La raison principale du bavardage est d'avoir "des choses importantes à dire à leurs amis" (45,7%), devant les difficultés de concentration (40%) et le désintérêt pour la matière (34,3%).

• Conscience de l'Impact :

◦ Un effet modéré : Le questionnaire a permis à 51,4% des élèves de prendre "un peu" conscience des conséquences de leurs conversations.

Seuls 5 élèves (14,3%) ont jugé cette prise de conscience "nécessaire" ou "essentielle". 

◦ Lien avec les résultats scolaires contesté : Les avis sont partagés quant à l'impact du bavardage sur les notes. 37,5% pensent que leurs discussions n'ont "pas d'impact" sur leurs résultats.

• Volonté de Changement :

◦ Une faible envie d'arrêter : Plus de la moitié des élèves bavards n'ont pas l'intention de mettre fin à leurs discussions, considérant que ce n'est "pas si bavard que ça" ou que c'est "plus fort que moi".  

◦ Le poids des sanctions : La "sanction de la part du professeur" est identifiée comme la pression extérieure la plus efficace pour les inciter à diminuer leurs bavardages.   

◦ Des résolutions fragiles : Malgré tout, 16 élèves sur 35 ont décidé de "prendre une résolution" pour se modérer.

Résultats des Entretiens Individuels (N=8)

Les entretiens menés quelques semaines après le questionnaire ont permis de nuancer les résolutions prises.

• Un Impact Limité sur le Comportement Réel : Seuls 3 des 8 élèves interrogés ont déclaré avoir effectivement diminué leur niveau de bavardage. Pour les autres, la situation était "pareille" voire "accentuée".

• La Persistance des Habitudes : Le changement de comportement s'est avéré difficile.

Le placement en classe (proximité avec un ami) reste un facteur déterminant.

Plusieurs élèves reconnaissent que malgré leur bonne volonté, l'habitude reprend le dessus.

• Un Acte Anormal mais Inévitable : La majorité des élèves interrogés conviennent qu'il n'est "pas normal" de discuter en classe.

Cependant, cette reconnaissance intellectuelle ne se traduit que rarement par une auto-discipline efficace, illustrant le fossé entre la conscience d'une règle et sa mise en application.

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III. Synthèse et Recommandations Stratégiques

Synthèse des Constats

1. Le fossé perceptuel comme obstacle majeur : Le principal frein au changement est que les élèves bavards ne perçoivent majoritairement pas leur comportement comme une nuisance, ni pour eux-mêmes ni pour les autres.

La croyance erronée en leur capacité à effectuer plusieurs tâches à la fois est une rationalisation puissante.

2. L'insuffisance de la prise de conscience seule : L'enquête démontre qu'une intervention visant à provoquer une prise de conscience interne, bien qu'utile, est insuffisante pour modifier durablement les comportements.

La volonté de changer est souvent volatile et rapidement supplantée par les habitudes et la dynamique sociale de la classe.

3. L'importance persistante du cadre externe : Les facteurs externes, notamment la clarté des règles et la constance dans l'application des sanctions, restent des leviers d'action déterminants pour la majorité des élèves.

Pistes de Réflexion et Stratégies d'Intervention

En s'appuyant sur les apports de la littérature et les résultats de l'enquête, plusieurs stratégies peuvent être envisagées pour une gestion plus efficace du bavardage.

• Co-construire les règles de vie (Courneloup) : Impliquer les élèves dans l'élaboration des règles de communication en classe.

Cet exercice de citoyenneté permet de rendre les règles plus explicites et de favoriser l'adhésion en montrant qu'elles servent l'intérêt collectif.

• Établir un cadre clair et constant (Ehnuel) : Dès le début de l'année, l'enseignant doit définir clairement ses attentes en matière de silence et de prise de parole.

La constance est cruciale : les élèves identifient rapidement les enseignants dont les avertissements ne sont pas suivis d'effets.

• Adopter une pédagogie active (Courneloup) : Pour contrer l'ennui, il est essentiel de varier les modalités de travail. Alterner les exposés magistraux avec des exercices, des travaux de groupe structurés, et des mises en commun permet de canaliser l'énergie des élèves et de réduire les temps morts propices au bavardage.

• Utiliser la communication non verbale (Courneloup) : Un regard appuyé, un doigt sur la bouche ou un déplacement silencieux vers un groupe d'élèves est souvent plus efficace et moins perturbateur pour le reste de la classe qu'une réprimande verbale à voix haute.

• Privilégier le dialogue individuel (Ehnuel) : En cas de bavardage récurrent d'un élève, une discussion en aparté à la fin du cours peut être bénéfique.

Elle permet de comprendre les raisons du comportement (difficultés, anxiété, etc.) et de responsabiliser l'élève sans l'humilier publiquement.

Synthèse sur la Consommation d'Alcool en France : Enjeux, Conséquences et Stratégies

Résumé Exécutif

Ce document synthétise les enjeux majeurs liés à la consommation d'alcool en France, en se basant sur les analyses d'experts et des témoignages.

L'alcool demeure la deuxième cause de mortalité évitable dans le pays, avec 49 000 décès par an, juste après le tabac.

Bien que la consommation globale soit en baisse, elle reste à un niveau excessivement élevé, posant un problème de santé publique majeur.

Les conséquences de cette consommation sont multiples : sanitaires (cancers, maladies cardiovasculaires), sociales (destruction de familles, stigmatisation des abstinents) et personnelles (perte de contrôle, addiction).

La jeunesse est particulièrement vulnérable, avec une prévalence alarmante du "binge drinking" qui compromet le développement cérébral et augmente le risque de dépendance à l'âge adulte.

Face à ce constat, plusieurs stratégies sont débattues : une prévention jugée insuffisante, notamment en milieu scolaire ; des initiatives comme le "Dry January" portées par la société civile face à un État réticent ; l'émergence d'un marché dynamique de boissons sans alcool comme alternative ; et un cadre réglementaire et fiscal (Loi Évin, taxes) considéré comme moins dissuasif que celui appliqué au tabac, soulevant des questions sur l'influence des lobbys.

Analyse Approfondie des Thématiques Clés

L'Ampleur du Problème de l'Alcool en France

La situation de la consommation d'alcool en France est marquée par une dualité : une tendance à la baisse sur le long terme mais des niveaux qui restent parmi les plus préoccupants en Europe.

• Bilan Humain : L'alcool est directement responsable de 49 000 décès chaque année, ce qui en fait un enjeu de santé publique de premier ordre.

• Le Paradoxe Français : La formule "On boit moins, mais on boit trop" résume la situation. La consommation moyenne a diminué, mais elle excède toujours les seuils de risque recommandés.

• Repères de Consommation : Depuis 2017, Santé Publique France recommande de ne pas dépasser deux verres par jour, et pas tous les jours.

L'Organisation Mondiale de la Santé (OMS) va plus loin en affirmant qu'il n'existe aucune consommation d'alcool sans risque.

Conséquences Sanitaires et Sociales

Les impacts de l'alcool sont profonds et touchent toutes les sphères de la vie de l'individu et de la société. L'âge moyen où les complications graves apparaissent est de 56 ans, mais les dommages peuvent survenir bien plus tôt.

| Type de Conséquence | Description détaillée | | --- | --- | | Sanitaires Aiguës | Liées à l'ivresse : accidents de la voie publique, accidents domestiques, chutes, traumatismes et mises en danger diverses. | | Sanitaires Chroniques | Pathologies graves se développant sur le long terme : cancers (digestifs, foie, sphère ORL), maladies digestives et maladies cardiovasculaires. | | Sociales et Familiales | L'alcool est décrit comme un "ravage dans une famille". L'impact sur les enfants de parents alcooliques est particulièrement dévastateur, créant des perturbations profondes. | | Personnelles | Conséquences professionnelles, financières et judiciaires (retraits de permis, gardes à vue pour bagarre). | | Culturelles | L'alcool est banalisé et associé à la convivialité ("bon vivant"). Inversement, la sobriété est stigmatisée, les non-buveurs étant perçus comme "pas fun", "chiants" ou même "malades". |

Le Mécanisme de l'Addiction et les Facteurs de Vulnérabilité

L'addiction à l'alcool est un processus insidieux qui s'installe progressivement.

1. La "Belle Rencontre" : La consommation débute souvent par la recherche d'effets psychotropes agréables (désinhibition, plaisir).

2. La Perte de Contrôle : Progressivement, l'individu perd la maîtrise de sa consommation (quantité, fréquence, temps consacré). C'est un critère central de l'addiction.

3. La Poursuite Malgré les Conséquences : Le signe définitif de l'addiction est la continuation de la consommation alors même que la personne constate les conséquences négatives sur sa santé, sa famille ou son travail.

Le Dr Delphine Moisan souligne que tout le monde n'est pas égal face à ce risque. Plusieurs facteurs de vulnérabilité individuelle existent :

• Génétiques : Antécédents familiaux d'addiction.

• Biologiques : Différences dans le fonctionnement du "circuit de la récompense" cérébral.

• Psychologiques : Faible estime de soi, sensibilité au stress, introversion.

• Psychiatriques : Comorbidités comme la dépression, la bipolarité ou l'hyperactivité.

• Environnementaux : Traumatismes vécus, contexte social.

L'Alcoolisation des Jeunes : Le Phénomène du "Binge Drinking"

La consommation d'alcool chez les jeunes représente une préoccupation majeure en raison de ses risques spécifiques.

• Définition : Le "Binge Drinking" (ou Alcoolisation Ponctuelle Importante) consiste à consommer une grande quantité d'alcool en un temps très court. Le seuil est fixé à 5 verres ou plus par occasion pour un adolescent.

• Statistiques Inquiétantes :

◦ 36 % des jeunes de 17 ans ont connu un épisode de binge drinking le mois précédant l'enquête.    ◦ 15 % des élèves de 4ème et 3ème rapportent des épisodes similaires.

• Risques Spécifiques :

◦ Développement Cérébral : Le cerveau est en maturation jusqu'à l'âge de 25 ans. L'exposition précoce à l'alcool perturbe ce développement.    ◦ Risque d'Addiction Future : Il est prouvé que plus la consommation d'alcool commence tôt, plus le risque de développer une dépendance à l'âge adulte est élevé.

Stratégies de Réduction et Alternatives

Face à ce tableau, différentes approches sont mises en œuvre ou envisagées, avec des niveaux d'implication variables des acteurs publics et privés.

• Prévention : La sénatrice Cathy Apourceau-Poly dénonce un manque de moyens pour la prévention, notamment dans les établissements scolaires qui voient le nombre d'infirmières et d'assistantes sociales diminuer.

• Initiatives Citoyennes : Le "Dry January", une campagne d'origine britannique invitant à un mois sans alcool, a été adoptée par 1 million de Français. Fait notable, cette initiative n'est pas portée par l'État français, qui s'est rétracté, mais par la société civile.

• Le Marché des Boissons Sans Alcool : Ce secteur est en pleine expansion, avec l'ouverture d'une trentaine de caves spécialisées en France.

◦ Clientèle : Entre 70 % et 80 % des clients de ces caves sont des consommateurs d'alcool qui cherchent à réduire leur consommation, notamment en semaine.   

◦ Adoption : Plus de 25 % des Français déclarent consommer des boissons sans alcool, un chiffre qui monte à 41 % chez les 26-35 ans. 

◦ Usage Thérapeutique : Pour les personnes dépendantes, ces boissons peuvent être une aide mais aussi un risque, le goût pouvant déclencher une envie de consommer de l'alcool.

Le Rôle des Pouvoirs Publics et de la Réglementation

L'action de l'État est jugée ambivalente et souvent insuffisante par les experts interrogés.

• Fiscalité Comportementale : La taxation est un levier efficace mais sous-utilisé pour l'alcool, contrairement au tabac (un paquet de cigarettes comporte 75 % de taxes).

L'exemple de l'Écosse, où une surtaxe a entraîné une baisse de 13 % de la mortalité liée à l'alcool, démontre le potentiel de cette mesure.

• Législation : La Loi Évin est jugée "pas satisfaisante" et ne va pas assez loin.

• Publicité : Contrairement au tabac, la publicité pour l'alcool reste autorisée, ce qui est considéré comme un problème majeur.

La régulation est de plus complexifiée par les publicités provenant de l'étranger via les réseaux sociaux.

Témoignages et Études de Cas

Le Parcours de Marine : De l'Addiction à la Sobriété

Le témoignage de Marine, 31 ans, illustre le cheminement vers la dépendance et la possibilité d'en sortir.

• Historique : Consommation initiée à 15 ans, qui s'intensifie avec le "binge drinking" durant ses études supérieures, où l'alcool devient une "béquille" sociale indispensable.

• Le Déclic : Une période de convalescence post-opératoire, marquée par une consommation excessive par ennui, lui fait prendre conscience de son problème.

• La Transition : Elle entame un "Dry January", réduit drastiquement sa consommation, se met au sport, puis décide d'arrêter totalement.

• Les Bénéfices : En 475 jours, elle a économisé près de 3 000 €, évité 171 000 calories et a perdu beaucoup de poids.

• Les Défis Sociaux : Elle se heurte à la pression sociale et à l'image de la personne "rabat-joie". Son témoignage met en lumière la difficulté d'une sobriété choisie mais subie :

"Je préférerais être capable de boire un verre de vin de temps en temps mais j'en suis vraiment pas capable donc je préfère ne rien boire du tout."

Ressources et Informations Utiles

Plusieurs dispositifs d'aide et d'information sont disponibles pour les personnes souhaitant évaluer ou réduire leur consommation.

• Ligne d'écoute : 09 80 980 930

• Site d'information : alcool-info-service.fr

• Outil d'auto-évaluation : alcoometre.fr

Analyse du Microlycée de Sénart : Une Approche Pédagogique Alternative pour les Décrocheurs Scolaires

Synthèse

Le microlycée de Sénart est un établissement public qui incarne une approche pédagogique radicalement différente, conçue pour rescolariser les jeunes de 17 à 26 ans ayant quitté le système traditionnel.

Face au phénomène national de 100 000 décrocheurs annuels, cette structure offre une "seconde chance" à 90 élèves, en s'attaquant aux causes profondes de leur déscolarisation : phobie scolaire, harcèlement, problèmes psychologiques ou mauvaise orientation.

La méthode du microlycée repose sur trois piliers fondamentaux : la flexibilité, la confiance et la co-construction.

Le cadre scolaire est volontairement assoupli : les retards sont tolérés, il n'y a pas de sanctions, et certaines règles des lycées classiques sont levées, comme l'interdiction du téléphone en cours ou de la cigarette (par dérogation).

Les classes à effectifs réduits (neuf élèves) permettent une relation enseignant-élève intime et familière, caractérisée par le tutoiement et un suivi proactif, comme les appels quotidiens aux absents pour les encourager.

L'évaluation est entièrement réinventée pour ne plus être une source de jugement destructeur.

Les notes sur 20 sont remplacées par des pourcentages de réussite que les élèves peuvent discuter, voire négocier, avec leurs professeurs.

Ce système de "co-construction" vise à faire de l'évaluation un outil d'apprentissage, renforçant l'autonomie et la confiance de l'élève.

De même, le conseil de classe est transformé en un format de "speed dating" où chaque élève échange directement avec ses professeurs, devenant ainsi un acteur central de son parcours.

Les parcours d'élèves comme Romain, Lola et Léo témoignent de l'efficacité de cette approche. Ils illustrent la capacité de l'établissement à reconstruire des jeunes brisés par le système traditionnel, en leur redonnant le goût d'apprendre et en leur permettant de se réconcilier avec l'école et avec eux-mêmes.

Le dispositif inclut également un soutien crucial aux familles, via des groupes de parole, qui partagent leur désarroi et leur soulagement.

Malgré un taux d'abandon de 20% en cours d'année, le microlycée parvient à mener 7 élèves sur 10 jusqu'au baccalauréat, prouvant la pertinence de son modèle atypique.

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1. Contexte et Mission du Microlycée de Sénart

Le microlycée de Sénart, ouvert en septembre 2000, est l'une des 61 structures publiques en France dédiées à la rescolarisation des élèves décrocheurs.

Chaque année, 100 000 jeunes quittent le lycée sans diplôme, soit 9% des élèves.

L'établissement accueille 90 de ces jeunes, âgés de 17 à 26 ans, de la seconde à la terminale.

Les raisons du décrochage sont multiples et complexes :

• Phobie scolaire

• Refus du système éducatif traditionnel

• Problèmes psychologiques

• Harcèlement scolaire

• Mauvaise orientation

La mission principale de l'établissement est de "réapprendre à aimer l'école" à ces jeunes en leur offrant un cadre bienveillant et des méthodes alternatives.

2. Une Approche Pédagogique Fondée sur la Confiance et la Flexibilité

Le programme de l'Éducation Nationale est suivi à la lettre, mais les méthodes d'enseignement et le cadre de vie scolaire sont radicalement différents de ceux d'un lycée traditionnel.

2.1. Un Cadre Souple et Non-Punitif

L'objectif est de dédramatiser l'école en supprimant les sources de stress et de conflit.

• Absence de Sanctions : Les retards sont autorisés et il n'y a pas de sanctions disciplinaires. Comme le souligne une enseignante, "on a d'autres moyens aussi de faire en sorte que ces jeunes puissent raccrocher".

• Tolérance et Flexibilité : Les élèves peuvent utiliser leur téléphone portable pour écouter de la musique en cours. La cigarette est autorisée dans des zones dédiées, sur dérogation de l'inspection académique.

• Suivi Proactif : Une professeure, Christine, appelle chaque jour les dizaines d'élèves absents, non pas pour les réprimander, mais pour les encourager à revenir. "Je t'appelle pour t'encourager à revenir à l'école [...] courage".

2.2. La Relation Enseignant-Élève

Le rapport entre les professeurs et les élèves est au cœur du dispositif.

• Effectifs Réduits : Les classes ne comptent que neuf élèves, favorisant une interaction directe et personnalisée.

• Proximité et Familiarité : Le tutoiement est la norme et les élèves sont appelés par leur prénom. Un élève explique : "Ça apporte plutôt je me trouve plus à l'aise en fait avec les profs [...] on commence à prendre confiance".

• Enseignants Volontaires et Formés : Les 14 professeurs sont tous volontaires et suivent des formations spécifiques pour encadrer ces élèves. Une professeure de français, Emmanuel, témoigne de la valeur de son travail : "Ce qu'il m'apporte de plus fondamental, c'est un sens profond à ce que je fais [...] j'ai l'impression de pouvoir accompagner ces jeunes jusqu'à une reprise de confiance en eux".

3. L'Évaluation Réinventée : De la Sanction à la Co-construction

L'un des aspects les plus innovants du microlycée est sa redéfinition complète du système de notation, souvent vécu comme un "jugement de la personne" dans le système classique.

• Pas de Note sur 20 : Les copies ne sont pas notées sur 20 mais reçoivent un pourcentage de réussite.

• La Co-construction : L'évaluation n'est pas un verdict final mais le début d'un dialogue. L'élève peut discuter son résultat avec le professeur.

Emmanuel Catinois, professeure de français, précise : "C'est ce qu'on appelle la co-construction, c'est on construit ensemble l'évaluation. L'idée c'est que l'évaluation, ça ne doit pas être un coup prêt, une note qui sanctionne, mais une note sur laquelle on peut s'appuyer, une note qui aide".

• Le Droit à l'Erreur : Les élèves ont la possibilité de retravailler une partie d'un devoir pour améliorer leur score. Romain, un élève, apprécie cette méthode : "Je peux le refaire, je peux me reprendre et elle va accepter [...] au moins on peut justifier au lieu de perdre des points bêtement".

4. Parcours d'Élèves : Portraits de la Reconstruction

Les profils des élèves sont variés, issus de tous les milieux sociaux, et illustrent les défis auxquels le microlycée répond.

| Élève | Âge | Parcours Avant le Microlycée | Situation au Microlycée | | --- | --- | --- | --- | | Romain | 17 | Ancien "premier de la classe", il tombe en dépression après le divorce de ses parents. Déscolarisé pendant deux ans. | Est devenu le meilleur élève de sa classe. Se réconcilie avec l'école grâce à la nouvelle approche de l'évaluation et des conseils de classe. | | Lola | \- | Déscolarisée pendant deux ans après avoir été harcelée au collège en raison de son homosexualité. A fait une tentative de suicide et a été hospitalisée six mois en psychiatrie. | A réappris à "aimer les lycées, à aimer les cours". Malgré d'importantes lacunes scolaires, l'équipe pédagogique parie sur elle en la faisant passer en classe supérieure. | | Léo | 19 | Décrit comme "je-m'en-foutiste", il a été renvoyé de plusieurs lycées. A décroché pendant deux ans, vivant la nuit et faisant des petits boulots. | Participe activement en cours, a pris le rôle d' "intendant café" et s'est réconcilié avec le français, écrivant désormais des chansons. |

5. Le Rôle Crucial des Familles

Le microlycée reconnaît que le décrochage scolaire est une épreuve pour toute la famille et intègre les parents dans son dispositif.

• Groupe de Parole : Un professeur anime des réunions régulières pour les parents, leur permettant de partager leur "désarroi" et de se soutenir mutuellement.

• Témoignages Émouvants : Les parents expriment un immense soulagement.

◦ Claude, père de Lola : "On est complètement paumé [...] Aujourd'hui [...] regardez c'est le sourire, Lola elle a un soir magnifique".    ◦ Laurence, mère de Romain : "Vous nous avez sauvé [...] le Romain de l'année dernière [...] à se poser des grosses questions est-ce qu'il va pas franchir une autre étape. Et puis aujourd'hui où je retrouve un môme de 17 ans vraiment bien dans ses baskets [...] c'est le jour et la nuit, on respire enfin".

• Forte Demande : L'établissement est perçu comme une "bouée de sauvetage". Chaque année, 40 familles postulent mais 20 candidatures doivent être refusées faute de place.

6. Le Conseil de Classe : Un Modèle de Transparence et d'Implication

Le traditionnel conseil de classe à huis clos est remplacé par un format innovant, conçu pour rendre l'élève acteur de son parcours.

• Format "Speed Dating" : Chaque élève rencontre individuellement chaque professeur pendant trois minutes pour discuter de ses résultats et de ses appréciations.

• Transparence Totale : "Il ne faut pas qu'il y ait des choses qui se disent sans la présence des élèves, rien n'est secret".

• Implication de l'Élève : Les élèves valident ou non les commentaires des professeurs. Romain explique : "On a l'interaction avec le prof et il nous met son commentaire devant nous et on valide ou pas [...] Ça m'apporte que je vois l'avis du prof en face de moi et qu'il fasse pas derrière mon dos".

• Renforcement de la Confiance : Cette pratique est jugée essentielle pour associer des élèves "adultes" (plus de 17 ans) à la construction de leur scolarité, ce qui "permet la confiance et le raccrochage".

7. Résultats, Défis et Perspectives

Le modèle du microlycée de Sénart, bien qu'exigeant, affiche des résultats probants.

• Taux de Réussite : 7 élèves sur 10 qui poursuivent leur scolarité au microlycée décrochent leur baccalauréat.

• Taux d'Abandon : Le parcours reste difficile, et 20% des élèves abandonnent en cours d'année.

• Défis Pédagogiques : L'équipe doit gérer des écarts de niveau considérables, comme celui de Lola qui a un niveau de 5ème en langues. L'établissement fait alors "le pari de la seconde chance" en adaptant ses décisions pour ne pas décourager les élèves malgré leurs lacunes.

• Transformation Personnelle : Au-delà du succès scolaire, l'établissement permet aux élèves de se reconstruire, de reprendre confiance et de développer de nouveaux projets, à l'image de Léo qui compose des chansons : "Apprendre, me cultiver et revenir avec des phrases de prof de français".

3 Misconceptions About Using Index Cards #officesupplies #indexcards<br /> by [[Shannon Medisky]] on YouTube<br /> accessed on 2026-01-14T09:21:52

Shannon Medisky appreciates the "fidget friendl[iness]" of index cards which help her work out nervous energy and reduce her stress.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

SECTION A - Evidence, Reproducibility, and Clarity Summary The study investigates the neurodevelopmental impact of trisomy 21 on human cortical excitatory neurons derived from induced pluripotent stem cells (hiPSCs). Key findings include a modest reduction in spontaneous firing, a marked deficit in synchronized bursting, decreased neuronal connectivity, and altered ion channel expression-particularly a downregulation of voltage‐gated potassium channels and HCN1. These conclusions are supported by a combination of in vitro calcium imaging, electrophysiological recordings, viral monosynaptic tracing, RNA sequencing, and in vivo transplantation with two‐photon imaging.

Major Comments • Convincing Nature of Key Conclusions: The study's conclusions are generally well supported by a diverse set of experimental approaches. However, certain claims regarding the intrinsic properties of the excitatory network would benefit from further qualification. In particular, the assertion that reduced synchronization is solely attributable to altered ion channel expression might be considered somewhat preliminary without additional corroborative experiments.

1.1) We agree with the reviewer and now write in the abstract: 'Together, these findings demonstrate long-lasting impairments in human cortical excitatory neuron network function associated with Trisomy 21 .' And in the Introduction: 'Collectively, the observed changes in ion channel expression, neuronal connectivity, and network activity synchronization may contribute to functional differences relevant to the cognitive and intellectual features associated with Down syndrome.'

• One major limitation of the current experimental design is the reliance on predominantly excitatory neuronal cultures derived from hiPSCs. Although the authors convincingly demonstrate differences in network synchronization and connectivity between trisomic (TS21) and control neurons, the almost exclusive focus on excitatory cells limits the physiological relevance of the in vitro network. In the developing cortex, interneurons and astrocytes play crucial roles in modulating network excitability, synaptogenesis, and plasticity. Therefore, incorporating these cell types-either through co-culture systems or through directed differentiation protocols that yield a more heterogeneous neuronal population-could help to determine whether the observed deficits are intrinsic to excitatory neurons or are compounded by a lack of proper inhibitory regulation and glial support. 1.2) Thank you for this thoughtful comment. We agree that interneurons and astrocytes are crucial for network function. To clarify, astrocytes are generated in this culture system, as we previously reported in our characterisation of the timecourse of network development using this approach (Kirwan et al., Development 2025). However, our primary goal was to first isolate and define the cell-autonomous defects intrinsic to TS21 excitatory neurons, minimizing the complexity introduced by additional neuronal types. This focused approach was chosen also because engineering a stable co-culture system with reproducible excitatory/inhibitory (E/I) proportions is a significant undertaking that extends beyond the scope of this initial investigation, and has proven challenging to date for the field. By establishing this foundational phenotype, our work complements prior studies on interneuron and glial contributions. Future studies building on this work will be essential to dissect the more complex, non-cell-autonomous effects within a heterogeneous network. Importantly, since our initial submission, two highly relevant preprints have emerged-including a notable study from the Geschwind laboratory at UCLA (Vuong et al., bioRxiv, 2025; Risgaard et al., bioRxiv, 2025), as well as our own complementary study Lattke et al, under revision, that highlight widespread transcriptional changes in excitatory cells of the human fetal DS cortex, providing strong validation for our central findings. This convergence of results from multiple groups underscores the timeliness and importance of our work.

• Furthermore, the assessment of neuronal connectivity via pseudotyped rabies virus tracing, while innovative, has inherent limitations. The quantification of connectivity as a ratio of red-to-green fluorescence pixels may be influenced by differential viral infection efficiencies, variations in the expression levels of the TVA receptor, or even by the lower basal activity levels observed in TS21 cultures. Complementary approaches-such as electron microscopy for synaptic density analysis or functional connectivity measurements using multi-electrode arrays (MEAs)-could provide additional structural and functional insights that would validate the rabies tracing data. 1.3) Thank you for this constructive feedback. While we cannot formally exclude that TS21 cells might express the TVA receptor at lower levels due to generalized gene dysregulation, we infected all WT and TS21 cultures in parallel using identical virus preparations and titers to minimize technical variability. Crucially, we also addressed the potential confound of differential basal activity by performing the rabies tracing under TTX incubation (see Suppl. Fig. 7), which blocks network activity and ensures that viral spread reflects structural connectivity alone.

While complementary methods like EM or MEA could provide additional insight, they fall outside the scope of the current study. We are confident that our rigorous controls validate our use of the rabies tracing method to assess structural connectivity.

• Qualification of Claims: Some conclusions, particularly those linking specific ion channel dysregulation (e.g., HCN1 loss) directly to network deficits, might be better presented as preliminary. The authors could temper their language to indicate that while the evidence is suggestive, the mechanistic link remains to be fully established. 1.4) We have revised the text to more clearly indicate that the link between HCN1 dysregulation and network deficits is correlative and remains to be fully established. While our ex vivo recordings suggest altered Ih-like currents consistent with reduced HCN1 expression, we now present these findings as preliminary and hypothesis-generating, pending further functional validation. We write in the discussion: However, further targeted functional validation will be needed to confirm a causal link.

• Need for Additional Experiments: Additional experiments that could further consolidate the current findings include: o Inclusion of Inhibitory Neurons or Co-culture Systems: Incorporating interneurons or astrocytes would help determine whether the observed deficits are solely intrinsic to excitatory neurons. See 1.2 o Alternative Connectivity Assessments: Complementing the rabies virus tracing with electron microscopy or multi-electrode array (MEA) recordings would add structural and functional validation of the connectivity differences. See 1.3 o Extended Temporal Profiling: Monitoring network activity over a longer developmental window would clarify whether the observed deficits represent a delay or a permanent alteration in network maturation. 1.5) In vivo we were able to track the cells for up to five months post-transplantation supporting the interpretation of a permanent alteration.

• Reproducibility and Statistical Rigor: The methods and data presentation are largely clear, with adequate replication and appropriate statistical analyses. Nonetheless, a more detailed description of the experimental replicates, particularly regarding the viral tracing and in vivo transplantation studies, would enhance reproducibility. The availability of raw data and scripts for calcium imaging analysis would also further support independent verification. We thank the reviewer for these suggestions and we now provide a more detailed description of replicates. We also add the raw data.

Minor Comments • Experimental Details: Minor revisions could include clarifying the infection efficiency and expression levels of the viral constructs used in connectivity assays to rule out technical variability.

See 1.3

• Literature Context: The authors reference prior studies appropriately; however, integrating a brief discussion comparing their findings with alternative DS models (e.g., organoids or other hiPSC-derived systems) would improve contextual clarity. We thank the reviewer for this helpful suggestion. We have now added a brief discussion comparing our findings with those reported in alternative Down syndrome models, including brain organoids and other hiPSC-derived systems. This addition helps to contextualize our results within the broader field and highlights the unique strengths and limitations of our in vitro and in vivo xenograft approach. We write: 'Our findings align with and extend previous studies using alternative Down syndrome models, such as brain organoids and other hiPSC-derived systems. Organoid models have provided valuable insights into early neurodevelopmental phenotypes in DS, including altered interneuron proportions (Xu et al Cell Stem Cell 2019) but also suggest that variability across isogenic lines can overshadow subtle trisomy 21 neurodevelopmental phenotypes (Czerminski et al Front in Neurosci 2023). However, these systems often lack the structural complexity, vascularization, and long-term maturation achievable in vivo. By using a xenotransplantation model, we were able to assess the maturation and functional properties of human neurons within a physiologically relevant environment over extended time frames, offering complementary insights into DS-associated circuit dysfunction (Huo et al Stem Cell Reports 2018; Real et al., 2018).

• Presentation and Clarity: Figures are generally clear,.But the manuscript contains a minor labeling error. On page 13, the figure is erroneously labeled as "Fig6A", whereas, based on the context and corresponding data, it should be "Fig5A". I recommend that the authors correct this mistake to ensure consistency and avoid potential confusion for readers. Thank you for pointing this out. This has been corrected in the revised manuscript.

Reviewer #1 (Significance (Required)):

SECTION B - Significance • Nature and Significance of the Advance: The work offers a substantial conceptual advance by providing a mechanistic link between trisomy 21 and impaired neuronal network synchronization. Technically, the study integrates state-of-the-art imaging, electrophysiology, and transcriptomic profiling, thereby offering a multifaceted view of DS-related neural dysfunction. Clinically, the findings have the potential to inform future therapeutic strategies targeting network connectivity and ion channel function in Down syndrome.

We thank the reviewer for this very supportive comment.

• Context in the Existing Literature: The study builds on previous observations of altered network activity in DS patients and DS mouse models (e.g., altered EEG synchronization and reduced synaptic connectivity). It extends these findings to human-derived neuronal models, thus bridging a gap between clinical observations and molecular/cellular mechanisms. Relevant literature includes studies on DS neurodevelopment and the role of ion channels in synaptic maturation. • Target Audience: The reported findings will be of interest to researchers in neurodevelopmental disorders, Down syndrome, and ion channel physiology. Additionally, the study may attract the attention of those working on hiPSC-derived models of neurological diseases, as well as clinicians interested in the pathophysiology of DS. • Keywords and Field Contextualization: Keywords: Down syndrome, trisomy 21, neuronal connectivity, synchronized network activity, hiPSC-derived cortical neurons, ion channel dysregulation.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary The manuscript by Peter et al., reports on the neuronal activity and connectivity of iPSC-derived human cortical neurons from Down syndrome (DS) that is caused by caused by trisomy of the human chromosome 21 (TS21). Major points: Although the manuscript is potentially interesting, the results appear somehow preliminary and need to be corroborated by control experiments and quantifications of effects to fully sustain the conclusions. (1) The authors have not assessed the percentage of WT and TS21 cells that acquire a neuronal or glia identity in their cultures. Indeed, the origin of alterations in network activity and connectivity observed in TS21 neurons could simply derive from reduced number of neurons arising from TS21 iPSC. Alternatively, the same alteration in network activity and connectivity could derive from a multitude of other factors including deficits in neuronal development, neurite extension, or intrinsic electrophysiological properties. In the current version of the manuscript, none of these has been investigated. 2.1) We thank the reviewer for this thoughtful comment. In response, we included an in vivo characterization of cell-type proportions at the same time points where we observed network activity defects using in vivo calcium imaging (see Supplementary Fig. 6).

Previous work has identified several cellular and molecular phenotypes in human cells, postmortem tissue, and mouse models-including those mentioned by the reviewer. In this study, our focus was on investigating neural network activity, intrinsic electrophysiological properties both in vitro and in vivo, and preliminary bulk RNA sequencing. We have also independently measured cell proportions in the human fetal cortex and conducted a more extensive transcriptomic analysis of Ts21 versus control cells in a separate study (Lattke et al., under revision). We observed a reduction of RORB/FOXP1-expressing Layer 4 neurons in the human fetal cortex at midgestation, as well as increased GFAP+ cells, reduced progenitors and a non significant reduction of Cux2+ cells in late stage DS human cell transplants, along with a gene network dysregulation specifically affecting excitatory neurons (Lattke et al., under revision). Here, we provide complementary findings, demonstrating reduced excitatory neuron network connectivity in vitro and decreased neural network synchronised activity in both in vitro and in vivo models (see also 2.8). We agree with the reviewer that this could be for a number of reasons, both cell autonomous (channel expression and/or function) or non-autonomous (connectivity and/or network composition - as reflected in differences in proportions of SATB2+ neurons generated in TS21 cortical differentiations).

(2) Electrophysiological properties of TS21 and WT neurons at day 53/54 in vitro indicate an extremely immature stage of development (i.e. RMP between -36 and -27 mV with most of the cells firing a single action potential after current injection) in the utilized culture conditions: This is far from ideal for in vitro neuronal-network studies. Finally, reduced activity of HCN1 channels should be confirmed by specific recordings isolating or blocking the related current.

2.2) Thank you for this thoughtful comment. We have also conducted ex vivo electrophysiological recordings and found that the neurons exhibit relatively immature properties, consistent with the known slow developmental trajectory of human neuron cultures. In light of this and the absence of direct confirmatory evidence, we now refer to the observed reduction in HCN1 as preliminary.

Main points highlighting the preliminary character of the study. 1) In Figure 1 immunofluorescence images of the neuronal differentiation markers (Tbr1, Ctip2 and Tuj1) are showed. However, no quantification of the percentage of cells expressing these markers for WT and TS21 neurons is reported. On the other hand, simple inspection of the representative images clearly seams to indicate a difference between the two genotypes, with TS21 cultures showing lower number of cells expressing neuronal markers. This quantification should be corroborated by a similar staining for an astrocyte marker (GFAP, but not S100b since is triplicated in DS). This is an extremely important point since it is obvious that any change in the percentage of neurons (or the neuron/astrocyte ratio) in the cultures will strongly affect the resulting network activity (shown in Figure 2) and the connectivity (showed in Figure 4). Possibly, the quantification should be done at the same time points of the calcium imaging experiments.

2.3) See 2.1. We included an in vivo characterization of cell-type proportions at the same time points where we observed network activity defects using in vivo calcium imaging. (see Supplementary Fig. 6).

2) In Figure 2 the authors show some calcium imaging traces of WT and TS21 cultures at different time points. However, they again do not show any quantification of neuronal activity. A power spectra analysis is shown in Supplementary Figure 2, but only for WT cultures, while in Supplementary Figure 3 a comparison between WT and Ts21 power spectra is done, but only at the 50 day time point, while difference in synchrony are assessed at 60 days. At minimum, the author should include in main Figure 2 the quantification of the mean calcium event rate and mean event amplitude at the different time points and the power spectra analysis for both WT and TS21 cultures at the same timepoints.

2.4) We thank the reviewer for this comment. We now add the power spectra analysis in the main Figure 2 and quantification of the mean calcium burst rate and mean event amplitude in SuppFig. 4.

Of note, the synchronized neuronal activity is present in WT cultures at day 60, but totally lost at subsequent time-points (70 and 80 days). The results of this later time points are different from previous data from the same lab (Kirwan et al., 2015). How might these data be explained? It would be important to rule out any potential issues with the health of the culture that could explain the loss of neuronal activity.It would be beneficial to check cell viability at the different time points to exclude possible confounding factors ? A propidium staining or a MTT assay would strongly improve the soundness of the calcium data.

2.5) We thank the reviewer for this important observation. The difference from the findings reported in Kirwan et al., 2015 is due to the use of a different neuronal differentiation medium in the current study (BrainPhys versus N2B27). BrainPhys medium supports robust early network activity compared to N2B27 (onset before day 60 in BrainPhys, post-day 60 in N2B27), resulting in an earlier decline in synchrony at later stages (day 70-80 in BrainPhys, compared with day 90-100 in N2B27). Importantly, in our in vivo xenograft model, burst activity is sustained up to at least 5 months post-transplantation (mpt), indicating that the neurons retain the capacity for network activity over extended periods in a more physiological environment. We adapted the text accordingly.

3) In Figure 3 there is no quantification of the number and/or density of transplanted neurons for WT and TS21, but only representative images. As above, inspection of the representative images seems to show a decrease in cells labeled by the Tbr1 neuronal marker for TS21 cells. Moreover, the in vivo calcium imaging of transplanted WT and TS21 cells lacks most of the quantification normally done in calcium imaging experiments. Are the event rate and event amplitude different between WT and TS21 neurons ? The measure of neuronal synchrony by mean pixel correlation is not well explained, but it looks somehow simplistic. Neuronal synchrony can be more precisely measured by cross-correlation analysis or spike time tiling coefficients on the traces from single-neuron ROI rather than on all pixels in the field of view, as apparently was done here.

2.6) We thank the reviewer for these valuable points. We now include quantification of the number and density of transplanted neurons for both WT and Ts21 grafts in Extended Data Figure 5 (see 2.1).

Regarding the in vivo calcium imaging, we appreciate the reviewer's suggestion to include additional standard metrics. We have quantified the event rate in Real et al 2018. These analyses reveal that Ts21 neurons show a reduction in event rate.

We agree that our initial description of the synchrony analysis using mean pixel correlation was not sufficiently detailed. We have now clarified this in the Methods and Results, and we acknowledge its limitations. Importantly, we note that the reduced synchronisation is a highly consistent phenotype, observed across at least six independent donor pairs, different differentiation protocols, and both in vitro (and in two independent labs) and in vivo settings. As suggested, future studies using ROI-based approaches-such as cross-correlation or spike-time tiling coefficients-would provide a more refined characterization of synchrony at the single-neuron level (Sintes et al, in preparation). We now include this point in the discussion.

4) The results on reduced neuronal connectivity in Figure 3 look very striking. However, these results should be accompanied by control experiments to verify the number of neuronal cells and neurite extension in WT and Ts21 cultures. These two parameters could indeed strongly influence the results. As the cultures appear to grow in clusters, bright-field images and TuJ1 staining of the cultures will also greatly help to understand the degree of morphological interconnection between the clusters.

We now add Tuj1 staining in Supplementary figure 10.

5) The authors performed RNA-seq experiments on day 50 cultures. Why the authors do not show the complete differential gene expression analysis, but only a small subset of genes? A comprehensive volcano plot and the complete list of identified genes with logFC and FDR values would be helpful. If possible, comparison of the present data (particularly on KCN and HCN expression changes) with published and publicly available expression datasets of other human or human Down syndrome iPSC-derived neurons or human Down syndrome brains will greatly increase the soundness of the present findings. In addition, the gene ontology (GO) results are mentioned in the text, but are not presented. Showing the complete GO analysis for both up and downregulated genes will help the reader to better understand the RNA-seq results. Notably, the results shown in Supplementary Figure on GRIN2A and GRIN2B expression (with values of 300-700 counts versus 2000-4000 counts, respectively) clearly indicate that in both WT and TS21 cultures the NMDA developmental switch has not occurred yet at the 50 days timepoint.

We now show volcano plots in Supplementary Fig. 11.

6) The measure of hyperpolarization-activated currents shown in Figure 5 lack proper control experiments. First, the hyperpolarizing current in TS21 cells do not reach a steady-state as the controls. The two curves are therefore hard to compare. To exclude possible difference in kinetic activation, the authors should have prolonged the current injection period (1-2 seconds). Second, to ultimately prove that such currents are mediated by HCN channels in WT cells the authors should perform some control experiments with a specific HCN blocker. A good example of a suitable protocol, with also current blockers to exclude all other possible current contributions, is the one reported in Matt et al Cell. Mol. Life Sci. 68, 125-137 (2011).

2.7) We thank the reviewer for this detailed and helpful comment. We agree that to definitively identify the recorded currents as Ih, it would be necessary to isolate them pharmacologically using specific HCN channel blockers and appropriate controls, such as those described in Matt et al., Cell. Mol. Life Sci. Unfortunately, due to current constraints, we no longer have access to the animals used in this study and cannot allocate the necessary time or resources, we are unable to perform the additional experiments at this stage.

However, our goal here was to use electrophysiological recordings as an indication of altered HCN channel activity, which we then support with molecular evidence. We now emphasize this point more clearly in the revised manuscript.

7) The manuscript lacks information on the statistical analysis used. Also, the numerosity of samples is not clear. Were the dots shown in some graph technical replicates from a single neuronal induction or were all independent neuronal inductions or a mix of the two ? Please clarify.

We now clarify the numbers in the Figure legend.

8) The method section lacks important information to guarantee reproducibility. Just a few examples: • Only electrophysiology methods for slice are reported, but not for in vitro culture.

We now clarify these details in the methods.

• Details on Laminin coating is lacking. What concentration was used ? Was poly-ornithine or poly-lysine used before Laminin coating ? We now clarify these details in the methods.

• How long cells were switched to BrainPhys medium before calcium imaging ? We now clarify these details in the methods.

Minor point/typos etc.

Introduction • Page 4 line 6: in the line "Trisomy 21 in humans commonly results in a range in developmental and morphological changes in the forebrain ..." "in" could be replaced by "of". We have fixed this. • Page 5 line 2: please remove "an" before the word "another". We have fixed this. • Page 5 line 2: please replace "ecitatory" with "excitatory". We have fixed this typo.

Results • Page 10 line 25: The concept of "pixel-wise" appears for the first time in this section and could be better introduced to facilitate the understanding of the experiment. • In the "results" section, page 11 line 1 and 4, references are made to "Figure 4D" and "4F," but these figures do not appear to be present in the figure section. Upon reviewing the rest of the section, the data seem to refer to "Figure 3D" and "3E." We have fixed this. Discussion • Page 15 line 20: please replace "synchronised" with "synchronized". We have fixed this typo. • Page 16 line 11: please replace "T21" with "TS21". We have fixed this typo. Methods • Page 19 line 12: "Pens/Strep" has to be replaced by Pen/Strep. We have fixed this typo. • Page 20 line 20: "Tocris Biocience" has to be replaced by "Tocris Bioscience". We have fixed this typo. • Page 21 line 2: "Addegene" has to be replaced by "Addgene". We have fixed this typo. Figures • Figure 3: the schematic experimental design (Fig. 3A) could be enlarged to match the width of the images/graphs below. We have fixed this. • Figure 5: the reviewer suggests resizing/repositioning the graphs in Fig. 1A so that they match the width of those below. We have fixed this. • Figure S1D: In all the figures of the paper, the respective controls for the TS21 1 and TS21 2 lines are labelled as "WT1/WT2," while in these graphs, they are called "Ctrl1" and "Ctrl2." To ensure consistency throughout the paper, it is suggested to change the names in these graphs. We have fixed this. • Figure S4L: The graph is not very clear, especially regarding the significance reported at -50 pA, please modify the graphical visualization and/or add a legend in the caption. We have fixed this.

Reviewer #2 (Significance (Required)):

Nature and significance of the advance for the field. The results presented in the manuscript are potentially interesting and useful, but not completely novel (currents deregulation has already been highlighted in mouse models of Down Syndrome).

2.8) We thank the reviewer for this comment. While we agree that current deregulation has been observed in mouse models of Down syndrome, the novelty and significance of our study lie in demonstrating these alterations directly in human neurons using both in vitro and in vivo xenograft models.

This is a critical advance because the human cortex has distinct developmental and functional properties not fully recapitulated in mice. In fact, three recent studies have already highlighted significant defects mainly in excitatory neurons within the fetal human DS cortex (Vuong et al., bioRxiv, 2025; Risgaard et al., bioRxiv, 2025; Lattke et al, under revision). Our work builds directly on these observations by providing, for the first time, an electrophysiological and network-level characterization of these human-specific deficits.

Our findings thus provide translationally relevant insight that is not merely confirmatory but extends previous work by grounding it in a human cellular context.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #2

Evidence, reproducibility and clarity

Summary

The manuscript by Peter et al., reports on the neuronal activity and connectivity of iPSC-derived human cortical neurons from Down syndrome (DS) that is caused by caused by trisomy of the human chromosome 21 (TS21).

Major points:

Although the manuscript is potentially interesting, the results appear somehow preliminary and need to be corroborated by control experiments and quantifications of effects to fully sustain the conclusions.

(1) The authors have not assessed the percentage of WT and TS21 cells that acquire a neuronal or glia identity in their cultures. Indeed, the origin of alterations in network activity and connectivity observed in TS21 neurons could simply derive from reduced number of neurons arising from TS21 iPSC. Alternatively, the same alteration in network activity and connectivity could derive from a multitude of other factors including deficits in neuronal development, neurite extension, or intrinsic electrophysiological properties. In the current version of the manuscript, none of these has been investigated.

(2) Electrophysiological properties of TS21 and WT neurons at day 53/54 in vitro indicate an extremely immature stage of development (i.e. RMP between -36 and -27 mV with most of the cells firing a single action potential after current injection) in the utilized culture conditions: This is far from ideal for in vitro neuronal-network studies. Finally, reduced activity of HCN1 channels should be confirmed by specific recordings isolating or blocking the related current.

Main points highlighting the preliminary character of the study.

1) In Figure 1 immunofluorescence images of the neuronal differentiation markers (Tbr1, Ctip2 and Tuj1) are showed. However, no quantification of the percentage of cells expressing these markers for WT and TS21 neurons is reported. On the other hand, simple inspection of the representative images clearly seams to indicate a difference between the two genotypes, with TS21 cultures showing lower number of cells expressing neuronal markers. This quantification should be corroborated by a similar staining for an astrocyte marker (GFAP, but not S100b since is triplicated in DS). This is an extremely important point since it is obvious that any change in the percentage of neurons (or the neuron/astrocyte ratio) in the cultures will strongly affect the resulting network activity (shown in Figure 2) and the connectivity (showed in Figure 4). Possibly, the quantification should be done at the same time points of the calcium imaging experiments.

2) In Figure 2 the authors show some calcium imaging traces of WT and TS21 cultures at different time points. However, they again do not show any quantification of neuronal activity. A power spectra analysis is shown in Supplementary Figure 2, but only for WT cultures, while in Supplementary Figure 3 a comparison between WT and Ts21 power spectra is done, but only at the 50 day time point, while difference in synchrony are assessed at 60 days. At minimum, the author should include in main Figure 2 the quantification of the mean calcium event rate and mean event amplitude at the different time points and the power spectra analysis for both WT and TS21 cultures at the same timepoints.

Of note, the synchronized neuronal activity is present in WT cultures at day 60, but totally lost at subsequent time-points (70 and 80 days). The results of this later time points are different from previous data from the same lab (Kirwan et al., 2015). How might these data be explained? It would be important to rule out any potential issues with the health of the culture that could explain the loss of neuronal activity.It would be beneficial to check cell viability at the different time points to exclude possible confounding factors ? A propidium staining or a MTT assay would strongly improve the soundness of the calcium data.

3) In Figure 3 there is no quantification of the number and/or density of transplanted neurons for WT and TS21, but only representative images. As above, inspection of the representative images seems to show a decrease in cells labeled by the Tbr1 neuronal marker for TS21 cells. Moreover, the in vivo calcium imaging of transplanted WT and TS21 cells lacks most of the quantification normally done in calcium imaging experiments. Are the event rate and event amplitude different between WT and TS21 neurons ? The measure of neuronal synchrony by mean pixel correlation is not well explained, but it looks somehow simplistic. Neuronal synchrony can be more precisely measured by cross-correlation analysis or spike time tiling coefficients on the traces from single-neuron ROI rather than on all pixels in the field of view, as apparently was done here.

4) The results on reduced neuronal connectivity in Figure 3 look very striking. However, these results should be accompanied by control experiments to verify the number of neuronal cells and neurite extension in WT and Ts21 cultures. These two parameters could indeed strongly influence the results. As the cultures appear to grow in clusters, bright-field images and TuJ1 staining of the cultures will also greatly help to understand the degree of morphological interconnection between the clusters.

5) The authors performed RNA-seq experiments on day 50 cultures. Why the authors do not show the complete differential gene expression analysis, but only a small subset of genes? A comprehensive volcano plot and the complete list of identified genes with logFC and FDR values would be helpful. If possible, comparison of the present data (particularly on KCN and HCN expression changes) with published and publicly available expression datasets of other human or human Down syndrome iPSC-derived neurons or human Down syndrome brains will greatly increase the soundness of the present findings. In addition, the gene ontology (GO) results are mentioned in the text, but are not presented. Showing the complete GO analysis for both up and downregulated genes will help the reader to better understand the RNA-seq results. Notably, the results shown in Supplementary Figure on GRIN2A and GRIN2B expression (with values of 300-700 counts versus 2000-4000 counts, respectively) clearly indicate that in both WT and TS21 cultures the NMDA developmental switch has not occurred yet at the 50 days timepoint.

6) The measure of hyperpolarization-activated currents shown in Figure 5 lack proper control experiments. First, the hyperpolarizing current in TS21 cells do not reach a steady-state as the controls. The two curves are therefore hard to compare. To exclude possible difference in kinetic activation, the authors should have prolonged the current injection period (1-2 seconds). Second, to ultimately prove that such currents are mediated by HCN channels in WT cells the authors should perform some control experiments with a specific HCN blocker. A good example of a suitable protocol, with also current blockers to exclude all other possible current contributions, is the one reported in Matt et al Cell. Mol. Life Sci. 68, 125-137 (2011).

7) The manuscript lacks information on the statistical analysis used. Also, the numerosity of samples is not clear. Were the dots shown in some graph technical replicates from a single neuronal induction or were all independent neuronal inductions or a mix of the two ? Please clarify.

8) The method section lacks important information to guarantee reproducibility. Just a few examples: - Only electrophysiology methods for slice are reported, but not for in vitro culture. - Details on Laminin coating is lacking. What concentration was used ? Was poly-ornithine or poly-lysine used before Laminin coating ? - How long cells were switched to BrainPhys medium before calcium imaging ?

Minor point/typos etc.

Introduction

• Page 4 line 6: in the line "Trisomy 21 in humans commonly results in a range in developmental and morphological changes in the forebrain ..." "in" could be replaced by "of".
• Page 5 line 2: please remove "an" before the word "another".
• Page 5 line 2: please replace "ecitatory" with "excitatory"

Results

• Page 10 line 25: The concept of "pixel-wise" appears for the first time in this section and could be better introduced to facilitate the understanding of the experiment.
• In the "results" section, page 11 line 1 and 4, references are made to "Figure 4D" and "4F," but these figures do not appear to be present in the figure section. Upon reviewing the rest of the section, the data seem to refer to "Figure 3D" and "3E."

Discussion

• Page 15 line 20: please replace "synchronised" with "synchronized".
• Page 16 line 11: please replace "T21" with "TS21".

Methods

• Page 19 line 12: "Pens/Strep" has to be replaced by Pen/Strep.
• Page 20 line 20: "Tocris Biocience" has to be replaced by "Tocris Bioscience".
• Page 21 line 2: "Addegene" has to be replaced by "Addgene".

Figures

• Figure 3: the schematic experimental design (Fig. 3A) could be enlarged to match the width of the images/graphs below.
• Figure 5: the reviewer suggests resizing/repositioning the graphs in Fig. 1A so that they match the width of those below.
• Figure S1D: In all the figures of the paper, the respective controls for the TS21 1 and TS21 2 lines are labelled as "WT1/WT2," while in these graphs, they are called "Ctrl1" and "Ctrl2." To ensure consistency throughout the paper, it is suggested to change the names in these graphs.
• Figure S4L: The graph is not very clear, especially regarding the significance reported at -50 pA, please modify the graphical visualization and/or add a legend in the caption.

Significance

Nature and significance of the advance for the field. The results presented in the manuscript are potentially interesting and useful, but not completely novel (currents deregulation has already been highlighted in mouse models of Down Syndrome).

Work in the context of the existing literature. This work follows the line of evidence that characterizes Down Syndrome in human neurons (Huo, H.-Q. et al. Stem Cell Rep. 10, 1251-1266 (2018); Briggs, J. A. et al. Etiology. Stem Cells 31, 467-478 (2013)), both in vitro and in xenotransplanted mice, by corrborating some important findings already found in animal models (Stern, S., Segal, M. & Moses, E. EBioMedicine 2, 1048-1062 (2015); Cramer, N. P., Xu, X., F. Haydar, T. & Galdzicki, Z. Physiol. Rep. 3, e12655 (2015); Stern, S., Keren, R., Kim, Y. & Moses, E. http://biorxiv.org/lookup/doi/10.1101/467522 (2018) doi:10.1101/467522.

Audience. Scientists in the field of pre-clinical biomedical research, especially those working on neurodevelopmental disorders and iPSC-based non-animal models.

Field of expertise. In vitro electrophysiology, Neurodevelopmental disorders, Down Syndrome, ips cells.

Reviewer #3 (Public review):

Summary:

The authors have identified novel dRTA causing SLC4A1 mutations and studied the resulting kAE1 proteins to determine how they cause dRTA. Based on a previous study on mice expressing the dRTA kAE1 R607H variant, the authors hypothesize that kAE1 variants cause an increase in intracellular pH which disrupts autophagic and degradative flux pathways. The authors clone these new kAE1 variants and study their transport function and subcellular localization in mIMCD cells. The authors show increased abundance of LC3B II in mIMCD cells expressing some of the kAE1 variants, as well as reduced autophagic flux using eGFP-RFP-LC3. These data, as well as the abundance of autophagosomes, serve as the key evidence that these kAE1 mutants disrupt autophagy. Furthermore, the authors demonstrate that decreasing the intracellular pH abrogates the expression of LC3B II in mIMCD cells expressing mutant SLC4A1. Lastly, the authors argue that mitochondrial function, and specifically ATP synthesis, is suppressed in mIMCD cells expressing dRTA variants and that mitochondria are less abundant in AICs from the kidney of R607H kAE1 mice. Overall, the authors provide evidence about how new kAE1 mutants may cause dRTA.

Strengths:

The authors cloned novel dRTA causing kAE1 mutants into expression vectors to study the subcellular localization and transport properties of the variants. The immunofluorescence images are generally of high quality and the authors do well to include multiple samples for all of their western blots.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary: 

This study is an evaluation of patient variants in the kidney isoform of AE1 linked to distal renal tubular acidosis. Drawing on observations in the mouse kidney, this study extends findings to autophagy pathways in a kidney epithelial cell line. 

Strengths: 

Experimental data are convincing and nicely done.

Thank you

Weaknesses: 

Some data are lacking or not explained clearly. Mutations are not consistently evaluated throughout the study, which makes it difficult to draw meaningful conclusions.

We have revised our manuscript to clarify some earlier explanations and provided rationale for focusing on specific variants throughout the study.

Reviewer #2 (Public review):

Context and significance: 

Distal renal tubular acidosis (dRTA) can be caused by mutations in a Cl-/HCO3- exchanger (kAE1) encoded by the SLC4A1 gene. The precise mechanisms underlying the pathogenesis of the disease due to these mutations are unclear, but it is thought that loss of the renal intercalated cells (ICs) that express kAE1 and/or aberrant autophagy pathway function in the remaining ICs may contribute to the disease. Understanding how mutations in SLC4A1 affect cell physiology and cells within the kidney, a major goal of this study, is an important first step to unraveling the pathophysiology of this complex heritable kidney disease. 

Summary: 

The authors identify a number of new mutations in the SLC4A1 gene in patients with diagnosed dRTA that they use for heterologous experiments in vitro. They also use a dRTA mouse model with a different SLC4A1 mutation for experiments in mouse kidneys. Contrary to previous work that speculated dRTA was caused mainly by trafficking defects of kAE1, the authors observe that their new mutants (with the exception of Y413H, which they only use in Figure 1) traffic and localize at least partly to the basolateral membrane of polarized heterologous mIMCD3 cells, an immortalized murine collecting duct cell line. They go on to show that the remaining mutants induce abnormalities in the expression of autophagy markers and increased numbers of autophagosomes, along with an alkalinized intracellular pH. They also reported that cells expressing the mutated kAE1 had increased mitochondrial content coupled with lower rates of ATP synthesis. The authors also observed a partial rescue of the effects of kAE1 variants through artificially acidifying the intracellular pH. Taken together, this suggests a mechanism for dRTA independent of impaired kAE1 trafficking and dependent on intracellular pH changes that future studies should explore. 

Strengths: 

The authors corroborate their findings in cell culture with a well-characterized dRTA KI mouse and provide convincing quantification of their images from the in vitro and mouse experiments

Thank you  

Weaknesses: 

The data largely support the claims as stated, with some minor suggestions for improving the clarity of the work. Some of the mutants induce different strengths of effects on autophagy and the various assays than others, and it is not clear why this is from the present manuscript, given that they propose pHi and the unifying mechanism

We have modified our manuscript to discuss the various strengths of the mutants and emphasize that alteration of cytosolic pH by kAE1 variants may not be the only mechanism leading to dRTA.  

Reviewer #3 (Public review):

Summary: 

The authors have identified novel dRTA causing SLC4A1 mutations and studied the resulting kAE1 proteins to determine how they cause dRTA. Based on a previous study on mice expressing the dRTA kAE1 R607H variant, the authors hypothesize that kAE1 variants cause an increase in intracellular pH, which disrupts autophagic and degradative flux pathways. The authors clone these new kAE1 variants and study their transport function and subcellular localization in mIMCD cells. The authors show increased abundance of LC3B II in mIMCD cells expressing some of the kAE1 variants, as well as reduced autophagic flux using eGFP-RFP-LC3. These data, as well as the abundance of autophagosomes, serve as the key evidence that these kAE1 mutants disrupt autophagy. Furthermore, the authors demonstrate that decreasing the intracellular pH abrogates the expression of LC3B II in mIMCD cells expressing mutant SLC4A1. Lastly, the authors argue that mitochondrial function, and specifically ATP synthesis, is suppressed in mIMCD cells expressing dRTA variants and that mitochondria are less abundant in AICs from the kidney of R607H kAE1 mice. While the manuscript does reveal some interesting new results about novel dRTA causing kAE1 mutations, the quality of the data to support the hypothesis that these mutations cause a reduction in autophagic flux can be improved. In particular, the precise method of how the western blots and the immunofluorescence data were quantified, with included controls, would enhance the quality of the data and offer more supportive evidence of the authors' conclusions. 

Strengths: 

The authors cloned novel dRTA causing kAE1 mutants into expression vectors to study the subcellular localization and transport properties of the variants. The immunofluorescence images are generally of high quality, and the authors do well to include multiple samples for all of their western blots.

Thank you

Weaknesses: 

Inconsistent results are reported for some of the variants. For example, R295H causes intracellular alkalinization but also has no effect on intracellular pH when measured by BCECF. The authors also appear to have performed these in vitro studies on mIMCD cells that were not polarized, and therefore, the localization of kAE1 to the basolateral membrane seems unlikely, based upon images included in the manuscript. Additionally, there is no in vivo work to demonstrate that these kAE1 variants alter intracellular pH, including the R607H mouse, which is available to the authors. The western blots are of varying quality, and it is often unclear which of the bands are being quantified. For example, LAMP1 is reported at 100kDa, the authors show three bands, and it is unclear which one(s) are used to quantify protein abundance. Strikingly, the authors report a nonsensical value for their quantification of LCRB II in Figure 2, where the ratio of LCRB II to total LCRB (I + II) is greater than one. The control experiments with starvation and bafilomyocin are not supportive and significantly reduce enthusiasm for the authors' findings regarding autophagy. There are labeling errors between the manuscript and the figures, which suggest a lack of vigilance in the drafting process.

The R295H variant was identified in a dRTA patient and as such, it was important to report it. However, this is the first mutation located in the amino-terminus of the protein, which may be involved in protein-protein interactions, so other mechanisms may cause dRTA for this variant. We have therefore modified our manuscript to state that alteration of cytosolic pH may not be the only mechanism leading to dRTA. At this time, we are not able to measure cytosolic pH in vivo and hope to be able to do it in the future.

In our revised manuscript, we also show cell surface biotinylation results supporting that plasma membrane abundance of the kAE1 S525F and R589H variants is not significantly different than WT in non-polarized mIMCD3 cells (Figure 3 A&B), in line with the predominant basolateral localization of the variants in polarized cells (Figure 1C). Therefore, these two mutant proteins are not mis-trafficked in non-polarized cells.  Finally, we have clarified which bands have been used for quantification and corrected quantifications (including ratio measurements).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) R295H is recessively inherited, whereas Y413H is dominantly inherited: this is interesting and may be linked to their cellular expression and function. Is this information known for the other mutations examined in this study? 

The S25F and R589H dRTA variants have both been reported to exhibit autosomal dominant inheritance. This information is now updated in lines 146 and 158-159.

(2) R589H expression levels are evaluated in the Western blot of Figure 1, but localization and activity are not examined in Figure 2. However, R589H is included in autophagy experiments shown in later figures. Similarly, mutant R607H is the subject of several experiments further into the manuscript, but no initial analysis is provided for this variant. 

Protein abundance and localization of the R589H mutant in mIMCD3 cells have been shown in our previous publication in Supplementary Fig 5D and Supplementary Fig 2J [1]. This now indicated on lines 158-159. Our previous paper also presented a detailed study of the R607H dRTA mutant, the mouse model corresponding to the human R589H mutation. This is now indicated on lines 70, 118-119 and 180. The present study builds upon those published findings.

(3) This inconsistency is confusing, detracts from the usefulness of the study, and makes the comparative analysis of mutations incomplete. It is difficult to extrapolate from published studies in MDCK1 cells, which show different results on trafficking. 

The mIMCD3 cell line, which more closely resembles the physiology of the mouse collecting duct than MDCK cells, was selected for this study and our previous one [1]. Accordingly, the results obtained are better aligned with in vivo evidence. In contrast, differences in mutant protein expression and localization observed in other cell lines, like the MDCK cells, are likely attributable to differences in their cellular origin. 

(4) In Figure 2, could the authors explain why total LC3B is graphed for the data shown in mouse lysates, whereas the ratio of bands is analysed for cell lysates? Both sets of data show the two LC3B bands.

Total LC3B levels were significantly increased in the mutant compared to WT; however, no significant difference was observed in the lipidation ratio. For this reason, that graph is not shown in the main paper but has been included in the Supplementary Figure 1D. 

(5) In Figure 3, representative fluorescence images should be shown for all cell lines.

We have now included representative immunofluorescence images for all cell lines in Figure 3C.

(6) pH effects: Suggest that steady state pHi (Figure 3E) and rate of alkalization (Figure 1F) would be more effective together in Figure 1. The authors should show data for the effect of nigericin on cytoplasmic pH in Figure 3. If the rate of alkalinization in the mutant cells is reduced, shouldn't the intracellular steady state pH be more acidic? A cartoon depicting the transporter activity in the cell and the expected changes in pHi would be helpful. Is there a way to activate/inhibit NHE1 and rescue the effect of the mutant kAE1? It is unclear if the link between the mutant kAE1 and mitochondrial ATP production is a consequence of the intracellular pH or an indirect effect.

We opted to keep the effect of nigericin on pHi in Supplementary Fig1A given that Figure 3 already contains 11 panels. Also, in intercalated cells, the kAE1 protein physiologically exports 1 molecule of bicarbonate in exchange of 1 chloride ion import hence a reduced transport activity would result in a more alkaline intracellular pH. To clarify this point, we have included a diagram in Figure 1E as suggested. However, to calculate the rate of intracellular alkalinisation, the transporter is functioning in the opposite direction, i.e. extruding chloride and importing bicarbonate (see methods protocol for transport assay). Therefore, in this assay (Figure 1G), a defective chloride/bicarbonate activity results in a reduced rate of intracellular alkalinisation rate. This is now explained on lines 169-172.

Disruption of NHE1 function would impair sodium homeostasis and as such, potentially affect the activity of other proteins associated with acid-base balance and autophagy in collecting duct cells. Therefore, any resulting effects may not be confidently attributed specifically to the mutant kAE1. With nigericin, we aimed to alter pHi while affecting the least possible other ion concentration. Due to space considerations, Figure 1 has been reorganised to include the rate of alkalinisation and pHi (panels F and G). 

Reviewer #2 (Recommendations for the authors):

(1) The authors could improve the readability of this manuscript for a general audience by clarifying and summarizing the respective phenotype(s)/effect(s) of the different mutants in some kind of table in the main figures. It is hard to keep track of the different disease mutants alongside the KI mouse mutations, as the text frequently discusses multiple mutants at a time. 

As requested, we added two tables (Supplementary Tables 1 & 2) in Supplementary files summarizing the data obtained in this study. We hope this will help the readership to keep track of each variant’s phenotype.

(2) The subtitle of the results section of Figure 2 should be reworded to reflect that  whole kidney lysates are used for the KI mice and not the other mutants.

As requested, the title in the Results section has been modified (lines 178-179).

(3) More discussion of why the different mutants cause different strengths of phenotypes should be included.

Different variants induce different degree of functional defects as seen in Figure 1F & G. The kAE1 R295H, the only amino acid substitution in the amino-terminal cytosol causing dRTA, does not affect the transporter’s function or cells’ pHi. Therefore, this variant may cause dRTA via a different pathway than transport-defective S525F or partially inactive R589H variants that both affect pHi. Our study does not exclude that dRTA may be caused by other defects than pHi alterations, including defective proteinprotein interactions. This discussion is now included in the manuscript on lines 386-391.

Reviewer #3 (Recommendations for the authors):

In general, I found the subject matter of this manuscript interesting and of value to the scientific community. The interpretation of the data and how much it supports the conclusion that "kAE1 variants increases pHi which alters mitochondrial function and leads to reduced cellular energy levels that eventually attenuate energy-dependent autophagic pathways" is largely incomplete. There are significant concerns about the quantification of Western blot data. Additionally, including the R607H variant in the in vitro experiments would improve the interpretation and extrapolation of in vitro data to the kidney.

We apologize for the confusion with R589H and R607H variants. The R607H mutant is the murine ortholog to the human R589H dRTA variation. To clarify this, we have added this information on line 180, in addition to lines 118-119 and line 70.

Suggestions:

(1) Can an anion replacement experiment be performed in the mIMCD cells (no Cl or no HCO3) to determine that bicarbonate transport through AE1 is responsible for the reduced ATP rates in Figure 5? Inclusion of WT +dox control would be helpful to convince the reader of the effects.

Because Seahorse real-time cell metabolism ATP rates measurements require specific and patented buffers with un-specified compositions, it was not possible to modify the Cl⁻ or HCO₃⁻ content during the ATP measurement assay. All cell lines, including empty vector cells (EV) were treated with doxycycline; thus, WT + dox was already included. The empty vector cell line treated with doxycycline allowed the exclusion of specific effects of doxycycline on mitochondrial activity as a control. This is now clarified in Figure 5 legend, lines 655-656.

(2) Can the authors measure pHi in fresh kidney sections from the R607H mouse?

Unfortunately, we are not currently able to measure pHi in fresh kidney sections and although we recognize it would benefit greatly to our study, establishing a new collaboration to perform this measurement would significantly delay the publication of this work; therefore, these results will not be available for the present manuscript. 

(3) Does pH 7.0 media have any effect on autophagy, as shown in Figure 3? Why was pH 6.6 selected?

The idea was to artificially acidify pHi in mutant cell lines (that have a steady state alkaline pHi) and assess whether this acidification corrects autophagy defects. We first determined that incubation in cell culture medium at pH 6.6 with 0.033 µM nigericin (final potassium concentration: 168 mM) for 2 hours provided optimal conditions, i.e. ensuring cell viability over the 2-hour period while effectively lowering intracellular pH to 6.9, as demonstrated in Supplementary Figure 1A-C.

(4) In vitro experiments should be performed on polarized cells with kAE1 properly inserted in the basolateral membrane. Experiments on subconfluent, non-polarized cells do not support the hypothesis that transport functions of AE1 initiate the cascade of events attributed to these SLC4A1 mutations.

To address this point, we have performed cell surface biotinylations on 70-80 % confluent mIMCD3 cells expressing kAE1 WT, S525F or R589H mutants and show that cell surface abundance of the mutants is not significantly different from the WT protein. This is now shown in Figure 3 A&B. As cell surface biotinylation provides a more quantitative assessment of protein cell surface abundance, we have removed the immunofluorescence images from non-polarised cells and replaced them with representative immunoblots from a cell surface biotinylation assay.

Concerns:

(1) No information about the B1 ATPase antibody used.

Now provided in Supplementary Material, ATP6V1B1 Antibody from Bicell cat#20901.

(2) No actin band in Figure 1E (as prepared).

Actin bands are provided for each blot in Figure 1D.

(3) Figures 1E and 1F are labelled wrong in the figure versus the results section. 

Thank you for letting us know, this is now corrected.

(4) The cortical sections shown in Figure 4 for the KI/KI do not appear to have the morphology of a CCD. The authors may want to consider including glomeruli to convince the reader of the localization of the tubules. Same concern with Figure 5G and I. The WT image in 5G does not have the morphology of a CCD. Principal cells should be predominant, and ICs should be dispersed.

Both figures 4 and 5 have been updated with images showing glomeruli (light blue “G” on figure) with neighbour and dispersed IC staining.

(5) The quantification of LAMP1 in Figure 4 is unclear. How did the authors determine the boundary of AICs, and how did they calculate the volume of lysosomes? If a zstack was used, how are the authors sure that their 10um section includes the entire AIC?

The quantification of LAMP1 is detailed under “Image analysis”, then “Volocity” sections in Supplementary Material. The boundary of A-IC was manually detected in Volocity based on the presence of the H⁺-ATPase before Volocity analysis for lysosomal volume as described in the Methods.

The 10 micron sections are expected to include full AIC as well as partial AIC, but the frequency of these events should be the same between WT and variants’ sections, therefore they were all included in the analysis if cells displayed H⁺-ATPase signal. 

(6) Figure 5: There is no description of how ATP rates are calculated from the provided traces.

We used Agilent Seahorse XF ATP rate assay kit for this experiment. In this assay, the total ATP rate is the sum of ATP production rate from both glycolysis and oxidative phosphorylation. Glycolysis releases protons in a 1:1 ratio with ATP hence the glycolytic ATP rate is calculated from the glycolytic proton efflux rate (glycoPER). GlycoPER is determined by subtracting respiration linked proton efflux from total proton efflux by inhibiting complex I and III. This information is now added to Supplementary Material, in the “Metabolic Flux analysis” section.

(7) Figure labels in Figure 5 are wrong. It seems 5H (as presented) should actually be labeled 5G. In 5H (G?), why did some cells not have any TOM20 pixel intensity for S525F and R589H variants?

Confocal image acquisition in this experiment was kept under the same settings to allow comparison between samples. Therefore, some cells show dimer fluorescence than others. From the figure 5 panels, all cells showed TOM 20 pixel intensity. Figure 5H panel has been relabelled Figure 5G.

(8) In Figure 2, the summary graphs show analysis of more samples than are visible on the included western blots. What is the rationale for this? Why does S525F have 9 samples in BafA1 while R295H only has 3 (2H)? Yet, R295H has 6 samples in 2I. In 2D, S525F has at least 9 samples. Explain.

Figure 2A-C shows representative immunoblots, among several ones independently conducted. Therefore, the final number of samples is higher than showed on Figure 2. This is now indicated in Figure 2 legend, line 603. It became clear quite early in our study that the recessive kAE1 R295H variant does not behave similarly to the other variants studied, maybe because it affects the cytosolic domain, so we did not perform as many replicates for this variant as we did for the others. However, we felt it was valuable to the research community to report the characterization of this variant and decided to keep it in our study. 

(9) In general, the actin loading does not appear to be equal between samples. And some figures show the same actin blot twice (2A, C) while some show independent actin bands for LC3B and p62. Equal loading seems a fairly significant control, considering the importance of quantification in the figures.

In addition to performing protein assays, we systematically conduct immunoblot with anti-b-actin antibody to control for loading variability. When possible, two or three proteins, including actin, are detected on the same blot, when molecular weight differ enough. This sometimes results in b-actin being used as a loading control for two different proteins, as seen on Figure 2A and 2C. This is now indicated on lines 605606.

(10) In the Supplemental Figure 2, which band is being quantified for mature CTSD at 33kDa? Same for intermediate CTSD. The quantification of V-ATPase seems questionable based on the actin variance shown in the blot. Surely the ratio of the fourth sample is greater than 1.

Supplementary Figure 2 has been updated to include arrows indicating which band was selected for the quantification. After verifying the measurements of band intensities from “Image Lab” quantification software, we confirm the results, including that fourth KI/KI sample has a ratio of 0.78 (Adj Total Band Vol (Int), lanes 10). Screen shots of quantifications are attached below.

Author response image 1.

Author response image 2.

(11) Why are the experiments performed on non-confluent IMCD cells? Figure 1D shows good basolateral localization of AE1, yet the other experiments in the manuscript appear to use IMCD cells in low confluent states, without proper localization of AE1. Figure 3A shows AE1 dispersed throughout the cytoplasm. Why have the authors decided to study the effects of an anion exchanger without it being properly localized to the basolateral membrane? Shouldn't all experiments be performed in polarized IMCDs? If AE1 isnt properly in the membrane, and the cells do not have defined apico-basolateral polarity, then what role can AE1-mediated intracellular pH change have on the results of the experiments? Were the pHi experiments in 3E performed on polarized cells? Or even 1F?

To address this point, we have performed cell surface biotinylations on 70-80 % confluent mIMCD3 cells expressing kAE1 WT, S525F or R589H mutants and show that cell surface abundance of the mutants is not significantly different from the WT protein. This is now shown in Figure 3A & B. As it provides a more quantitative assessment of protein cell surface abundance, we have removed the immunofluorescence images from non-polarised cells and replaced them with a representative immunoblot from a cell surface biotinylation assay.

(12) As mentioned in the public comments, how is the ratio A/(A+B) greater than 1? With A and B > 0. In Figure 3, the data is reasonable, but in Figure 2, the data is simply impossible. What is the explanation for this phenomenon? Why was this presentation of data approved? Is it supposedly a fold of WT, like 2K and 2L? Is the reader also to believe that total LC3B is 2-fold greater in KI/KI mice, as shown in 2K? My eyes, though not densitometry equipment, cannot confirm this. The actin bands are not equal. Yet again, there are 4 lanes of KI/KI mice, but the quantification shows 5 samples.

The ratios in figure 2D, 2F, 2H and 2L have been re-calculated and corrected. As indicated above, immunoblots are representative and quantification of additional blots has been included in the graphs.

(12) Spelling error Figure 4B: cels.

Corrected

References 

(1) Mumtaz, R. et al. Intercalated Cell Depletion and Vacuolar H+-ATPase Mistargeting in an Ae1 R607H Knockin Model. Journal of the American Society of Nephrology 28, 1507–1520 (2017).

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Lahtinen et al. evaluated the association between polygenic scores and mortality. This question has been intensely studied (Sakaue 2020 Nature Medicine, Jukarainen 2022 Nature Medicine, Argentieri 2025 Nature Medicine), where most studies use PRS as an instrument to attribute death to different causes. The presented study focuses on polygenic scores of non-fatal outcomes and separates the cause of death into "external" and "internal". The majority of the results are descriptive, and the data doesn't have the power to distinguish effect sizes of the interesting comparisons: (1) differences between external vs. internal (2) differences between PGI effect and measured phenotype. I have two main comments:

(1) The authors should clarify whether the p-value reported in the text will remain significant after multiple testing adjustment. Some of the large effects might be significant; for example, Figure 2C

We have now added Benjamini-Hochberg multiple-testing adjusted p-values in the text each time we present nominal p-values. Additionally, supplementary tables S5 and S6 provide multiple-adjusted p-values for all analysed PGIs.

Although this was not always the case, many comparisons remained significant after multiple testing adjustments, especially in Figure 2C that the reviewer commented on. In the revised version, we have placed more emphasis on describing these HRs that have low p-values after multiple-test adjustment. The revised text for Figure 2C in the Results section now reads:

Panel C analyses mortality in three age-specific follow-up periods. The PGIs were more predictive of death in younger age groups, although the difference between the 25–64 and 65–79 age groups was small, except for the PGI of ADHD (HR=1.14, 95% CI 1.08; 1.21 for 25–64-year-olds; HR=1.04, 95% CI 1.00; 1.08 for 65–79-year-olds; p=0.008 for difference, p=0.27 after multiple-testing adjustment). PGIs predicted death only negligibly among those aged 80+, and the largest differences between the age groups 25–64 and 80+ were for PGIs of self-rated health (HR 0.87, 95% CI 0.82; 0.93 for 25–64-year-olds, HR 1.00, 95% CI 0.94; 1.04 for 80+ year-olds, p=2*10^-4 for difference, p=0.006 after multiple-testing adjustment), ADHD (HR 1.14, 95% CI 1.08; 1.21 for 25–64-year-olds, HR 0.99, 95% CI 0.95; 1.03 for 80+ year-olds, p=7*10^-4 for difference, p=0.012 after multiple-testing adjustment) and depressive symptoms (HR 1.12, 95% CI 1.06; 1.18 for 25–64-year-olds, HR 1.00, 95% CI 0.96; 1.04 for 80+ year-olds, p=0.002 for difference, p=0.032 after multiple-testing adjustment). Additionally, the difference in HRs between these age groups achieved significance after multiple testing adjustment at the conventional 5% level for PGIs of cigarettes per day, educational attainment, and ever smoking.

We have also included the recent study by Argentieri et al. (2025) in the literature review, which was missing from our previous version. We appreciate the reference. Other references mentioned were already included in the previous version of the manuscript.

(note that the small prediction accuracy of PGI in older age groups has been extensively studied, see Jiang, Holmes, and McVean, 2021, PLoS Genetics).

We would like to thank the reviewer for suggesting the relevant reference by Jiang et al. We have now expanded on the discussion of age-specific differences in the discussion section and included this reference.

(2) The authors might check if PGI+Phenotype has improved performance over Phenotype only. This is similar to Model 2 in Table 1, but slightly different.

The reviewer raises an interesting angle to approach the analysis. We have now added an analysis assessing the information criteria and the significance of improvement between nested models in Supplementary table S8. All the tested PGI+phenotype models show improvement over the phenotype-only model that is statistically significant at all conventional levels when tested by likelihood-ratio tests between nested models . Additionally,  improvement was found when using Akaike and Bayesian (Schwarz) information criteria (albeit sometimes modest in size). We have added a passage in the results section briefly summarising this analysis:

Supplementary table S8 presents information criteria and significance tests on corresponding models. Models with PGI+phenotype (Models 2a–f) showed improvement over models with the phenotype only (Models 1a, 1c, 1e, 1g, 1i, 1k, with a p=0.0006 or lower) in terms of both Akaike information criterion (AIC) as well as Bayesian (Schwarz) information criterion (BIC) with a p=0.0006 or lower in all comparisons. The full Model 4 again showed improvement over the model with all PGIs jointly (Model 3b, with a p=0.0002 or p=0.00002, depending on continuous/categorical phenotype measurement), which had a lower AIC but not BIC.

Reviewer #2 (Public review): 

Summary:

This study provides a comprehensive evaluation of the association between polygenic indices (PGIs) for 35 lifestyle and behavioral traits and all-cause mortality, using data from Finnish population- and family-based cohorts. The analysis was stratified by sex, cause of death (natural vs. external), age at death, and participants' educational attainment. Additional analyses focused on the six most predictive PGIs, examining their independent associations after mutual adjustment and adjustment for corresponding directly measured baseline risk factors.

Strengths:

Large sample size with long-term follow-up.

Use of both population- and family-based analytical approaches to evaluate associations.

Weaknesses:

It is unclear whether the PGIs used for each trait represent the most current or optimal versions based on the latest GWAS data.

To our reading, this comment is closely related to the “recommendations for the author” number 3 by reviewer 2, and we thus address them together. 

If the Finnish data used in this study also contributed to the development of some of the PGIs, there is a risk of overestimating their associations with mortality due to overfitting or "double-dipping." Similar inflation of effect sizes has been observed in studies using the UK Biobank, which is widely used for PGI construction.

To our reading, this comment is closely related to the “recommendations for the author” 4 by reviewer 2, and we thus address them together.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Specific comments:

(1) Cited reference 1 also investigated the PRS association with life span; cited reference 8 explains PRS association with healthy lifespan. Can authors be clearer about what is new in the context of these references? Specifically, what are the PGIs studied here that were not analyzed in the cited analyses?

Although some previous studies on the topic do exist, our analysis arguably has novelty in touching upon several unstudied or scarcely studied themes. These include:

A set of PGIs focusing on social, psychological, and behavioural phenotypes or PGIs for typically non-fatal health conditions.

An assessment of direct genetic effects/ confounding with a within-sibship design.

An assessment of potential heterogeneous effects by several socio-demographic characteristics.

An analysis of external causes of deaths (which can be hypothesised to be particularly relevant here, given the choice of our PGIs not focusing directly on typical causes of death).

A detailed assessment of the interplay of the most predictive PGIs with their corresponding phenotypes.

We have substantially revised the Introduction section focusing on making these novel contributions more explicit.

(2) In the Methods section, it is not very clear why the authors specifically study the "within-sibship" samples. Is this for avoiding nurturing effects from parental genotypes or for controlling assortative mating? The authors should clarify the rationale behind the design.

The substance-related rationale behind this approach was briefly discussed in the Introduction section while in the Methods section, we focused more on the technical description of our analyses. However, it is certainly worthwhile to clarify to the reader why within-sibship methods have been used. The revised passage in the methods section now states:

“In addition to this population sample, we used a within-sibship analysis sample to assess the extent of direct and indirect genetic associations captured by the PGIs, as discussed in the introduction.”

(3) Residual correlations of PGIs were no more than 0.050..." As a minor comment, since PGIs is a noisy variable, the correlation would be low; however, I don't think there are better ways to evaluate Cox assumptions, and in many cases, this assumption is not correct for strong predictors.

Yes, these points are true. Overall, it is often implausible that empirical distributions exactly match distributional assumptions in statistical models. For example, it may not be realistic to expect that the mortality hazards across categories of independent variables stay exactly proportional during long mortality-follow-ups; some deviations from constant proportions are almost inevitable. However, there are reasonable grounds to argue that in case of moderate violations of the proportional hazards assumption, the estimates still remain interpretable for practical uses. They can be read as approximating average relative hazards over the study period (for discussion, see pages 42–47 in Allison P. 2014. Event history and survival analysis: Regression for longitudinal event data (second edition). Thousand Oaks: SAGE).

(4) "PGI of ADHD (HR=1.08 95%CI 1.04;1.11 among men; HR=1.01 95%CI 0.97;1.05 among women; p=0.012 for difference)." Is this difference significant after multiple testing correction?

We have presented multiple-testing adjusted p-values together with nominal ones in this and in all other instances where they are mentioned in the text. Additionally, Supplementary tables S5–S6 present multiple-adjusted p-values for each PGIs studied.

(5) "Panel D displays that most PGIs had stronger associations with external (accidents, violent, suicide, and alcohol related deaths) than natural causes of death." Similar to the comment above, are there any results that are significantly different between internal and external?

We have added the p-values of those variables that had larger differences in the revised text. Quoting from the revised article: “The HR differences between external and natural causes of death were nominally significant at the conventional 5% level for cannabis use (p=0.016), drinks per week (p=0.028), left out of social activity (p=0.029), ADHD (p=0.031), BMI (p=0.035) and height (p=0.049), but none of these differences remained significant after adjusting for 35 multiple tests. “

(6) Table 1: The effect of the phenotype is stronger than the PGI; this is expected as PGI is a weak predictor and can be considered as "noised" measurement of true genetic value (Becker 2021 Nature Human behavior). Is there a way to adjust for the impact of noise in PGI at tagging genetic value and compare if the PGI effect is different from the phenotype effect?

PGIs are certainly imperfect measures that contain a lot of noise. However, extracting new information from what is unknown is an extremely demanding exercise, and still further complicated for example, by that we do not know the exact benchmark of total genetic effect we should be aiming at. Different methods of heritability estimation, for instance, often give dramatically differing results – for reasons that are still up to scrutiny.

We are thus not familiar with a method that could achieve satisfactory answer for this challenging task.

Reviewer #2 (Recommendations for the authors):

(3) Justification and Selection of PGIs:

For several traits, such as BMI, multiple polygenic indices (PGIs) are currently available. The criteria used to select specific PGIs for this study are not clearly described. A more systematic and reproducible approach-for example, leveraging metadata from the PGS Catalog-could strengthen the justification for PGI selection and enhance the study's generalizability.

There are numerous PGIs developed in the extensive GWAS literature, but a finite set of PGIs always needs to be chosen for any analysis. The rationale behind our decision to include every PGI from the repository of Becker et al. 2021 (full reference in the manuscript, see also https://www.thessgac.org/pgi-repository) that was available for the Finnish data (including the possibility to exclude overlapping samples, see our response to the next comment for more discussion) was to provide rigorous analysis by limiting the researchers degrees of freedom in arbitrarily choosing PGIs. Although it would have been tempting to not use some PGIs that were not expected to substantially correlate with mortality, we believe that our conservative strategy increases the credibility of the reported p-values, particularly the multiple adjustment should now work as intended. 

We also mention now this rationale when discussing the chosen PGIs in the methods section: “As the independent variables of main interest, we used 35 different PGIs in the Polygenic Index repository by Becker et al., which were mainly based on GWASes using UK Biobank and 23andMe, Inc. data samples, but also other data collections. They were tailored for the Finnish data, i.e., excluding overlapping individuals between the original GWAS and our analysis and performing linkage-disequilibrium adjustment. We used every single-trait PGI defined in the repository (except for subjective well-being, for which we were unable to obtain a meta-analysis version that excluded the overlapping samples). By limiting the researchers’ freedom in selecting the measures, this conservative strategy should increase the validity of our estimates, particularly with regards to multiple-testing adjusted p-values.”

(4) Overlap Between PGI Training Data and Study Sample:

The authors should describe any overlap between the data used to develop the PGIs and the current study sample. If such overlap exists, it may lead to overestimation of effect sizes due to "double-dipping." A discussion of this issue and its potential implications is warranted, as similar concerns have been raised in studies using UK Biobank data.

This is, fortunately, not a concern of our analysis. Overlapping samples were excluded in creating the PGIs that we used. We have now described this more clearly in the revised methods section.

(1) Clarify the Methodology for Family-Based Cox Analysis:

It is unclear what specific method was used to perform Cox regression in the family-based analysis. Please provide additional methodological details. ”

We have described the method further and added an additional reference in the revision. The text now stands:

“We compared these models to the corresponding within-sibship models, using the sibship identifier as the strata variable. This method employs a sibship-specific (instead of a whole-sample-wide baseline hazard in the population models) baseline hazard, and corresponds to a fixed-effects model in some other regression frameworks (e.g., linear model with sibship-specific intercepts)”

(2) Clarify Timing of Measured Risk Factors Relative to Follow-Up:

The main text should provide more detailed information regarding the timing of data collection for directly measured risk factors. Specifically, it should be clarified whether the measurements used correspond to the first available data for each individual after the start of follow-up, or if a different criterion was applied.

BMI, self-rated health, alcohol consumption and smoking status were measured at the baseline survey of each dataset. Education was registered as the highest completed degree up to the end of 2019. Depression was a composite of survey self-report (at the time of the baseline survey), as well as depression-related medicine purchases and hospitalizations over a two-year period before the start of the individual’s follow-up.

We have added more comprehensive information on the measurement of the phenotypes of interest in Supplementary table 2, including the timing of the measurement.

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Author response:

Point-by-point description of the revisions:

Reviewer #1 (Evidence, reproducibility and clarity):

Summary

In this article, the authors used the synthetic TALE DNA binding proteins, tagged with YFP, which were designed to target five specific repeat elements in Trypanosoma brucei genome, including centromere and telomeres-associated repeats and those of a transposon element. This is in order to detect and identified, using YFP-pulldown, specific proteins that bind to these repetitive sequences in T. brucei chromatin. Validation of the approach was done using a TALE protein designed to target the telomere repeat (TelR-TALE) that detected many of the proteins that were previously implicated with telomeric functions. A TALE protein designed to target the 70 bp repeats that reside adjacent to the VSG genes (70R-TALE) detected proteins that function in DNA repair and the protein designed to target the 177 bp repeat arrays (177R-TALE) identified kinetochore proteins associated T. brucei mega base chromosomes, as well as in intermediate and mini-chromosomes, which imply that kinetochore assembly and segregation mechanisms are similar in all T. brucei chromosome.

Major comments:

Are the key conclusions convincing?

The authors reported that they have successfully used TALE-based affinity selection of proteinassociated with repetitive sequences in the T. brucei genome. They claimed that this study has provided new information regarding the relevance of the repetitive region in the genome to chromosome integrity, telomere biology, chromosomal segregation and immune evasion strategies. These conclusions are based on high-quality research, and it is, basically, merits publication, provided that some major concerns, raised below, will be addressed before acceptance for publication.

(1) The authors used TALE-YFP approach to examine the proteome associated with five different repetitive regions of the T. brucei genome and confirmed the binding of TALE-YFP with Chip-seq analyses. Ultimately, they got the list of proteins that bound to synthetic proteins, by affinity purification and LS-MS analysis and concluded that these proteins bind to different repetitive regions of the genome. There are two control proteins, one is TRF-YFP and the other KKT2-YFP, used to confirm the interactions. However, there are no experiment that confirms that the analysis gives some insight into the role of any putative or new protein in telomere biology, VSG gene regulation or chromosomal segregation. The proteins, which have already been reported by other studies, are mentioned. Although the author discovered many proteins in these repetitive regions, their role is yet unknown. It is recommended to take one or more of the new putative proteins from the repetitive elements and show whether or not they (1) bind directly to the specific repetitive sequence (e.g., by EMSA); (2) it is recommended that the authors will knockdown of one or a small sample of the new discovered proteins, which may shed light on their function at the repetitive region, as a proof of concept.

The main request from Referee 1 is for individual evaluation of protein-DNA interaction for a few candidates identified in our TALE-YFP affinity purifications, particularly using EMSA to identify binding to the DNA repeats used for the TALE selection. In our opinion, such an approach would not actually provide the validation anticipated by the reviewer. The power of TALE-YFP affinity selection is that it enriches for protein complexes that associate with the chromatin that coats the target DNA repetitive elements rather than only identifying individual proteins or components of a complex that directly bind to DNA assembled in chromatin.

The referee suggests we express recombinant proteins and perform EMSA for selected candidates, but many of the identified proteins are unlikely to directly bind to DNA – they are more likely to associate with a combination of features present in DNA and/or chromatin (e.g. specific histone variants or histone post-translational modifications). Of course, a positive result would provide some validation but only IF the tested protein can bind DNA in isolation – thus, a negative result would be uninformative.

In fact, our finding that KKT proteins are enriched using the 177R-TALE (minichromosome repeat sequence) identifies components of the trypanosome kinetochore known (KKT2) or predicted (KKT3) to directly bind DNA (Marciano et al., 2021; PMID: 34081090), and likewise the TelR-TALE identifies the TRF component that is known to directly associate with telomeric (TTAGGG)n repeats (Reis et al 2018; PMID: 29385523). This provides reassurance on the specificity of the selection, as does the lack of cross selectivity between different TALEs used (see later point 3 below). The enrichment of the respective DNA repeats quantitated in Figure 2B (originally Figure S1) also provides strong evidence for TALE selectivity.

It is very likely that most of the components enriched on the repetitive elements targeted by our TALE-YFP proteins do not bind repetitive DNA directly. The TRF telomere binding protein is an exception – but it is the only obvious DNA binding protein amongst the many proteins identified as being enriched in our TelR-TALE-YFP and TRF-YFP affinity selections.

The referee also suggests that follow up experiments using knockdown of the identified proteins found to be enriched on repetitive DNA elements would be informative. In our opinion, this manuscript presents the development of a new methodology previously not applied to trypanosomes, and referee 2 highlights the value of this methodological development which will be relevant for a large community of kinetoplastid researchers. In-depth follow-up analyses would be beyond the scope of this current study but of course will be pursued in future. To be meaningful such knockdown analyses would need to be comprehensive in terms of their phenotypic characterisation (e.g. quantitative effects on chromosome biology and cell cycle progression, rates and mechanism of recombination underlying antigenic variation, etc) – simple RNAi knockdowns would provide information on fitness but little more. This information is already publicly available from genome-wide RNAi screens (www.tritrypDB.org), with further information on protein location available from the genome-wide protein localisation resource (Tryptag.org). Hence basic information is available on all targets selected by the TALEs after RNAi knock down but in-depth follow-up functional analysis of several proteins would require specific targeted assays beyond the scope of this study.

(2) NonR-TALE-YFP does not have a binding site in the genome, but YFP protein should still be expressed by T. brucei clones with NLS. The authors have to explain why there is no signal detected in the nucleus, while a prominent signal was detected near kDNA (see Fig.2). Why is the expression of YFP in NonR-TALE almost not shown compared to other TALE clones?

The NonR-TALE-YFP immunolocalisation signal indeed is apparently located close to the kDNA and away from the nucleus. We are not sure why this is so, but the construct is sequence validated and correct. However, we note that artefactual localisation of proteins fused to a globular eGFP tag, compared to a short linear epitope V5 tag, near to the kinetoplast has been previously reported (Pyrih et al, 2023; PMID: 37669165).

The expression of NonR-TALE-YFP is shown in Supplementary Fig. S2 in comparison to other TALE proteins. Although it is evident that NonR-TALE-YFP is expressed at lower levels than other TALEs (the different TALEs have different expression levels), it is likely that in each case the TALE proteins would be in relative excess.

It is possible that the absence of a target sequence for the NonR-TALE-YFP in the nucleus affects its stability and cellular location. Understanding these differences is tangential to the aim of this study.

However, importantly, NonR-TALE-YFP is not the only control for used for specificity in our affinity purifications. Instead, the lack of cross-selection of the same proteins by different TALEs (e.g. TelR-TALE-YFP, 177R-TALE-YFP) and the lack of enrichment of any proteins of interest by the well expressed ingiR-TALE-YFP or 147R-TALE-YFP proteins each provide strong evidence for the specificity of the selection using TALEs, as does the enrichment of similar protein sets following affinity purification of the TelR-TALE-YFP and TRF-YFP proteins which both bind telomeric (TTAGGG)n repeats. Moreover, control affinity purifications to assess background were performed using cells that completely lack an expressed YFP protein which further support specificity (Figure 6).

We have added text to highlight these important points in the revised manuscript:

Page 8:

“However, the expression level of NonR-TALE-YFP was lower than other TALE-YFP proteins; this may relate to the lack of DNA binding sites for NonR-TALE-YFP in the nucleus.”

Page 8:

“NonR-TALE-YFP displayed a diffuse nuclear and cytoplasmic signal; unexpectedly the cytoplasmic signal appeared to be in the vicinity the kDNA of the kinetoplast (mitochrondria). We note that artefactual localisation of some proteins fused to an eGFP tag has previously been observed in T. brucei (Pyrih et al, 2023).”

Page 10:

Moreover, a similar set of enriched proteins was identified in TelR-TALE-YFP affinity purifications whether compared with cells expressing no YFP fusion protein (No-YFP), the NonR-TALE-YFP or the ingiR-TALE-YFP as controls (Fig. S7B, S8A; Tables S3, S4). Thus, the most enriched proteins are specific to TelR-TALE-YFP-associated chromatin rather than to the TALE-YFP synthetic protein module or other chromatin.

(3) As a proof of concept, the author showed that the TALE method determined the same interacting partners enrichment in TelR-TALE as compared to TRF-YFP. And they show the same interacting partners for other TALE proteins, whether compared with WT cells or with the NonR-TALE parasites. It may be because NonR-TALE parasites have almost no (or very little) YFP expression (see Fig. S3) as compared to other TALE clones and the TRF-YFP clone. To address this concern, there should be a control included, with proper YFP expression.

See response to point 2, but we reiterate that the ingi-TALE -YFP and 147R-TALE-YFP proteins are well expressed (western original Fig. S3 now Fig. S2) but few proteins are detected as being enriched or correspond to those enriched in TelR-TALE-YFP or TRF-YFP affinity purifications (see Fig. S9). Therefore, the ingi-TALE -YFP and 147R-TALE-YFP proteins provide good additional negative controls for specificity as requested. To further reassure the referee we have also included additional volcano plots which compare TelR-TALE-YFP, 70R-TALE-YFP or 177R-TALE-YFP to the ingiR-TALE-YFP affinity selection (new Figure S8). As with No-YFP or NonR-TALE-YFP controls, the use of ingiR-TALE-YFP as a negative control demonstrates that known telomere associated proteins are enriched in TelR-TALE-YFP affinity purification, RPA subunits enriched with 70R-TALE-YFP and Kinetochore KKT poroteins enriched with 177RTALE-YFP. These analyses demonstrate specificity in the proteins enriched following affinity purification of our different TALE-YFPs and provide support to strengthen our original findings.

We now refer to use of No-YFP, NonR-TALE-YFP, and ingiR-TALE -YFP as controls for comparison to TelR-TALE-YFP, 70R-TALE-YFP or 177R-TALE-YFP in several places:

Page10:

“Moreover, a similar set of enriched proteins was identified in TelR-TALE-YFP affinity purifications whether compared with cells expressing no YFP fusion protein (No-YFP), the NonR-TALE-YFP or the ingiR-TALE-YFP as controls (Fig. S7B, S8A; Tables S3, S4).”

Page 11:

“Thus, the nuclear ingiR-TALE-YFP provides an additional chromatin-associated negative control for affinity purifications with the TelR-TALE-YFP, 70R-TALE-YFP and 177R-TALE-YFP proteins (Fig. S8).”

“Proteins identified as being enriched with 70R-TALE-YFP (Figure 6D) were similar in comparisons with either the No-YFP, NonR-TALE-YFP or ingiR-TALE-YFP as negative controls.”

Top Page 12:

“The same kinetochore proteins were enriched regardless of whether the 177R-TALE proteomics data was compared with No-YFP, NonR-TALE or ingiR-TALE-YFP controls.”

Discussion Page 13:

“Regardless, the 147R-TALE and ingiR-TALE proteins were well expressed in T. brucei cells, but their affinity selection did not significantly enrich for any relevant proteins. Thus, 147R-TALE and ingiR-TALE provide reassurance for the overall specificity for proteins enriched TelR-TALE, 70R-TALE and 177R-TALE affinity purifications.”

(4) After the artificial expression of repetitive sequence binding five-TALE proteins, the question is if there is any competition for the TALE proteins with the corresponding endogenous proteins? Is there any effect on parasite survival or health, compared to the control after the expression of these five TALEs YFP protein? It is recommended to add parasite growth curves, for all the TALE proteins expressing cultures.

Growth curves for cells expressing TelR-TALE-YFP, 177R-TALE-YFP and ingiR-TALE-YFP are now included (New Fig S3A). No deficit in growth was evident while passaging 70R-TALE-YFP, 147R-TALE-YFP, NonR-TALE-YFP cell lines (indeed they grew slightly better than controls).

The following text has been added page 8:

“Cell lines expressing representative TALE-YFP proteins displayed no fitness deficit (Fig. S3A).”

(5) Since the experiments were performed using whole-cell extracts without prior nuclear fractionation, the authors should consider the possibility that some identified proteins may have originated from compartments other than the nucleus. Specifically, the detection of certain binding proteins might reflect sequence homology (or partial homology) between mitochondrial DNA (maxicircles and minicircles) and repetitive regions in the nuclear genome. Additionally, the lack of subcellular separation raises the concern that cytoplasmic proteins could have been co-purified due to whole cell lysis, making it challenging to discern whether the observed proteome truly represents the nuclear interactome.

In our experimental design, we confirmed bioinformatically that the repeat sequences targeted were not represented elsewhere in the nuclear or mitochondrial genome (kDNA). The absence of subcellular fractionation could result in some cytoplasmic protein selection, but this is unlikely since each TALE targets a specific DNA sequence but is otherwise identical such that cross-selection of the same contaminating protein set would be anticipated if there was significant non-specific binding. We have previously successfully affinity selected 15 chromatin modifiers and identified associated proteins without major issues concerning cytoplasmic protein contamination (Staneva et al 2021 and 2022; PMID: 34407985 and 36169304). Of course, the possibility that some proteins are contaminants will need to be borne in mind in any future follow-up analysis of proteins of interest that we identified as being enriched on specific types of repetitive element in T. brucei. Proteins that are also detected in negative control, or negative affinity selections such as No-YFP, NoR-YFP, IngiR-TALE or 147R-TALE must be disregarded.

(6) Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

As mentioned earlier, the author claimed that this study has provided new information concerning telomere biology, chromosomal segregation mechanisms, and immune evasion strategies. But there are no experiments that provides a role for any unknown or known protein in these processes. Thus, it is suggested to select one or two proteins of choice from the list and validate their direct binding to repetitive region(s), and their role in that region of interaction.

As highlighted in response to point 1 the suggested validation and follow up experiments may well not be informative and are beyond the scope of the methodological development presented in this manuscript. Referee 2 describes the study in its current form as “a significant conceptual and technical advancement” and “This approach enhances our understanding of chromatin organization in these regions and provides a foundation for investigating the functional roles of associated proteins in parasite biology.”

The Referee’s phrase ‘validate their direct binding to repetitive region(s)’ here may also mean to test if any of the additional proteins that we identified as being enriched with a specific TALE protein actually display enrichment over the repeat regions when examined by an orthogonal method. A key unexpected finding was that kinetochore proteins including KKT2 are enriched in our affinity purifications of the 177R-TALE-YFP that targets 177bp repeats (Figure 6F). By conducting ChIP-seq for the kinetochore specific protein KKT2 using YFP-KKT2 we confirmed that KKT2 is indeed enriched on 177bp repeat DNA but not flanking DNA (Figure 7). Moreover, several known telomere-associated proteins are detected in our affinity selections of TelRTALE-YFP (Figure 6B, FigS6; see also Reis et al, 2018 Nuc. Acids Res. PMID: 29385523; Weisert et al, 2024 Sci. Reports PMID: 39681615).

Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

The answer for this question depends on what the authors want to present as the achievements of the present study. If the achievement of the paper was is the creation of a new tool for discovering new proteins, associated with the repeat regions, I recommend that they add a proof for direct interactions between a sample the newly discovered proteins and the relevant repeats, as a proof of concept discussed above, However, if the authors like to claim that the study achieved new functional insights for these interactions they will have to expand the study, as mentioned above, to support the proof of concept.

See our response to point 1 and the point we labelled ‘6’ above.

Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

I think that they are realistic. If the authors decided to check the capacity of a small sample of proteins (which was unknown before as a repetitive region binding proteins) to interacts directly with the repeated sequence, it will substantially add of the study (e.g., by EMSA; estimated time: 1 months). If the authors will decide to check the also the function of one of at least one such a newly detected proteins (e.g., by KD), I estimate the will take 3-6 months.

As highlighted previously the proposed EMSA experiment may well be uninformative for protein complex components identified in our study or for isolated proteins that directly bind DNA in the context of a complex and chromatin. RNAi knockdown data and cell location data (as well as developmental expression and orthology data) is already available through tritrypDB.org and trtyptag.org

Are the data and the methods presented in such a way that they can be reproduced? Yes

Are the experiments adequately replicated, and statistical analysis adequate?

The authors did not mention replicates. There is no statistical analysis mentioned.

The figure legends indicate that all volcano plots of TALE affinity selections were derived from three biological replicates. Cutoffs used for significance: P < 0.05 (Student's t-test).

For ChiP-seq two biological replicates were analysed for each cell line expressing the specific YFP tagged protein of interest (TALE or KKT2). This is now stated in the relevant figure legends – apologies for this oversight. The resulting data are available for scrutiny at GEO: GSE295698.

Minor comments:

Specific experimental issues that are easily addressable.

The following suggestions can be incorporated:

(1) Page 18, in the material method section author mentioned four drugs: Blasticidine, Phleomycin and G418, and hygromycin. It is recommended to mention the purpose of using these selective drugs for the parasite. If clonal selection has been done, then it should also be mentioned.

We erroneously added information on several drugs used for selection in our labaoratory. In fact all TALE-YFP construct carry the Bleomycin resistance genes which we select for using Phleomycin. Also, clones were derived by limiting dilution immediately after transfection. We have amended the text accordingly:

Page 17/18:

“Cell cultures were maintained below 3 x 106 cells/ml. Pleomycin 2.5 µg/ml was used to select transformants containing the TALE construct BleoR gene.”

“Electroporated bloodstream cells were added to 30 ml HMI-9 medium and two 10-fold serial dilutions were performed in order to isolate clonal Pleomycin resistant populations from the transfection. 1 ml of transfected cells were plated per well on 24-well plates (1 plate per serial dilution) and incubated at 37°C and 5% CO2 for a minimum of 6 h before adding 1 ml media containing 2X concentration Pleomycin (5 µg/ml) per well.”

(2) In the method section the authors mentioned that there is only one site for binding of NonR-TALE in the parasite genome. But in Fig. 1C, the authors showed zero binding site. So, there is one binding site for NonR-TALE-YFP in the genome or zero?

We thank the reviewer for pointing out this discrepancy. We have checked the latest Tb427v12 genome assembly for predicted NonR-TALE binding sites and there are no exact matches. We have corrected the text accordingly.

Page 7:

“A control NonR-TALE protein was also designed which was predicted to have no target sequence in the T. brucei genome.”

Page 17:

“A control NonR-TALE predicted to have no recognised target in the T. brucei geneome was designed as follows: BLAST searches were used to identify exact matches in the TREU927 reference genome. Candidate sequences with one or more match were discarded.”

(3) The authors used two different anti-GFP antibodies, one from Roche and the other from Thermo Fisher. Why were two different antibodies used for the same protein?

We have found that only some anti-GFP antibodies are effective for affinity selection of associated proteins, whereas others are better suited for immunolocalisation. The respective suppliers’ antibodies were optimised for each application.

(4) Page 6: in the introduction, the authors give the number of total VSG genes as 2,634. Is it known how many of them are pseudogenes?

This value corresponds to the number reported by Consentino et al. 2021 (PMID: 34541528) for subtelomeric VSGs, which is similar to the value reported by Muller et al 2018 (PMID: 30333624) (2486), both in the same strain of trypanosomes as used by us. Based on the earlier analysis by Cross et al (PMID: 24992042), 80% of the identified VSGs in their study (2584) are pseudogenes. This approximates to the estimation by Consentino of 346/2634 (13%) being fully functional VSG genes at subtelomeres, or 17% when considering VSGs at all genomic locations (433/2872).

(5) I found several typos throughout the manuscript.

Thank you for raising this, we have read through the manuscipt several times and hopefully corrected all outstanding typos.

(6) Fig. 1C: Table: below TOTAL 2nd line: the number should be 1838 (rather than 1828)

Corrected- thank you.

- Are prior studies referenced appropriately? Yes

- Are the text and figures clear and accurate? Yes

- Do you have suggestions that would help the authors improve the presentation of their data and conclusions? Suggested above

Reviewer #1 (Significance):

Describe the nature and significance of the advance (e.g., conceptual, technical, clinical) for the field:

This study represents a significant conceptual and technical advancement by employing a synthetic TALE DNA-binding protein tagged with YFP to selectively identify proteins associated with five distinct repetitive regions of T. brucei chromatin. To the best of my knowledge, it is the first report to utilize TALE-YFP for affinity-based isolation of protein complexes bound to repetitive genomic sequences in T. brucei. This approach enhances our understanding of chromatin organization in these regions and provides a foundation for investigating the functional roles of associated proteins in parasite biology. Importantly, any essential or unique interacting partners identified could serve as potential targets for therapeutic intervention.

- Place the work in the context of the existing literature (provide references, where appropriate). I agree with the information that has already described in the submitted manuscript, regarding its potential addition of the data resulted and the technology established to the study of VSGs expression, kinetochore mechanism and telomere biology.

- State what audience might be interested in and influenced by the reported findings. These findings will be of particular interest to researchers studying the molecular biology of kinetoplastid parasites and other unicellular organisms, as well as scientists investigating chromatin structure and the functional roles of repetitive genomic elements in higher eukaryotes.

- (1) Define your field of expertise with a few keywords to help the authors contextualize your point of view. Protein-DNA interactions/ chromatin/ DNA replication/ Trypanosomes

- (2) Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. None

Reviewer #2 (Evidence, reproducibility and clarity):

Summary

Carloni et al. comprehensively analyze which proteins bind repetitive genomic elements in Trypanosoma brucei. For this, they perform mass spectrometry on custom-designed, tagged programmable DNA-binding proteins. After extensively verifying their programmable DNA-binding proteins (using bioinformatic analysis to infer target sites, microscopy to measure localization, ChIP-seq to identify binding sites), they present, among others, two major findings: 1) 14 of the 25 known T. brucei kinetochore proteins are enriched at 177bp repeats. As T. brucei's 177bp repeatcontaining intermediate-sized and mini-chromosomes lack centromere repeats but are stable over mitosis, Carloni et al. use their data to hypothesize that a 'rudimentary' kinetochore assembles at the 177bp repeats of these chromosomes to segregate them. 2) 70bp repeats are enriched with the Replication Protein A complex, which, notably, is required for homologous recombination. Homologous recombination is the pathway used for recombination-based antigenic variation of the 70bp-repeat-adjacent variant surface glycoproteins.

Major Comments

None. The experiments are well-controlled, claims well-supported, and methods clearly described. Conclusions are convincing.

Thank you for these positive comments.

Minor Comments

(1) Fig. 2 - I couldn't find an uncropped version showing multiple cells. If it exists, it should be linked in the legend or main text; Otherwise, this should be added to the supplement.

The images presented represent reproducible analyses, and independently verified by two of the authors. Although wider field of view images do not provide the resolution to be informative on cell location, as requested we have provided uncropped images in new Fig. S4 for all the cell lines shown in Figure 2A.

In addition, we have included as supplementary images (Fig. S3B) additional images of TelRTALE-YFP, 177R-TALE-YFP and ingiR-TALE YFP localisation to provide additional support their observed locations presented in Figure 1. The set of cells and images presented in Figure 2A and in Fig S3B were prepared and obtained by a different authors, independently and reproducibly validating the location of the tagged protein.

(2) I think Suppl. Fig. 1 is very valuable, as it is a quantification and summary of the ChIP-seq data. I think the authors could consider making this a panel of a main figure. For the main figure, I think the plot could be trimmed down to only show the background and the relevant repeat for each TALE protein, leaving out the non-target repeats. (This relates to minor comment 6.) Also, I believe, it was not explained how background enrichment was calculated.

We are grateful for the reviewer’s positive view of original Fig. S1 and appreciate the suggestion. We have now moved these analysis to part B of main Figure 2 in the revised manuscript – now Figure 2B. We have also provided additional details in the Methods section on the approaches used to assess background enrichment.

Page 19:

“Background enrichment calculation

The genome was divided into 50 bp sliding windows, and each window was annotated based on overlapping genomic features, including CIR147, 177 bp repeats, 70 bp repeats, and telomeric (TTAGGG)n repeats. Windows that did not overlap with any of these annotated repeat elements were defined as "background" regions and used to establish the baseline ChIP-seq signal. Enrichment for each window was calculated using bamCompare, as log₂(IP/Input). To adjust for background signal amongst all samples, enrichment values for each sample were further normalized against the corresponding No-YFP ChIP-seq dataset.”

Note: While revising the manuscript we also noticed that the script had a nomalization error. We have therefore included a corrected version of these analyses as Figure 2B (old Fig. S1)

(3) Generally, I would plot enrichment on a log2 axis. This concerns several figures with ChIP-seq data.

Our ChIP-seq enrichment is calculated by bamCompare. The resulting enrichment values are indeed log2 (IP/Input). We have made this clear in the updated figures/legends.

(4) Fig. 4C - The violin plots are very hard to interpret, as the plots are very narrow compared to the line thickness, making it hard to judge the actual volume. For example, in Centromere 5, YFP-KKT2 is less enriched than 147R-TALE over most of the centromere with some peaks of much higher enrichment (as visible in panel B), however, in panel C, it is very hard to see this same information. I'm sure there is some way to present this better, either using a different type of plot or by improving the spacing of the existing plot.

We thank the reviewer for this suggestion; we have elected to provide a Split-Violin plot instead. This improves the presentation of the data for each centromere. The original violin plot in Figure 4C has been replaced with this Split-Violin plot (still Figure 4C).

(5) Fig. 6 - The panels are missing an x-axis label (although it is obvious from the plot what is displayed).

Maybe the "WT NO-YFP vs" part that is repeated in all the plot titles could be removed from the title and only be part of the x-axis label?

In fact, to save space the X axis was labelled inside each volcano plot but we neglected to indicate that values are a log2 scale indicating enrichment. This has been rectified – see Figure 6, and Fig. S7, S8 and S9.

(6) Fig. 7 - I would like to have a quantification for the examples shown here. In fact, such a quantification already exists in Suppl. Figure 1. I think the relevant plots of that quantification (YFPKKT2 over 177bp-repeats and centromere-repeats) with some control could be included in Fig. 7 as panel C. This opportunity could be used to show enrichment separated out for intermediate-sized, mini-, and megabase-chromosomes. (relates to minor comment 2 & 8)

The CIR147 sequence is found exclusively on megabase-sized chromosomes, while the 177 bp repeats are located on intermediate- and mini-sized chromosomes. Due to limitations in the current genome assembly, it is not possible to reliably classify all chromosomes into intermediate- or mini- sized categories based on their length. Therefore, original Supplementary Fig. S1 presented the YFP-KKT2 enrichment over CIR147 and 177 bp repeats as a representative comparison between megabase chromosomes and the remaining chromosomes (corrected version now presented as main Figure 2B). Additionally, to allow direct comparison of YFP-KKT2 enrichment on CIR147 and 177 bp repeats we have included a new plot in Figure 7C which shows the relative enrichment of YFP-KKT2 on these two repeat types.

We have added the following text , page 12:

“Taking into account the relative to the number of CIR147 and 177 bp repeats in the current T.brucei genome (Cosentino et al., 2021; Rabuffo et al., 2024), comparative analyses demonstrated that YFP-KKT2 is enriched on both CIR147 and 177 bp repeats (Figure 7C).”

(7) Suppl. Fig. 8 A - I believe there is a mistake here: KKT5 occurs twice in the plot, the one in the overlap region should be KKT1-4 instead, correct?

Thanks for spotting this. It has been corrected

(8) The way that the authors mapped ChIP-seq data is potentially problematic when analyzing the same repeat type in different regions of the genome. The authors assigned reads that had multiple equally good mapping positions to one of these mapping positions, randomly.

This is perfectly fine when analysing repeats by their type, independent of their position on the genome, which is what the authors did for the main conclusions of the work.

However, several figures show the same type of repeat at different positions in the genome. Here, the authors risk that enrichment in one region of the genome 'spills' over to all other regions with the same sequence. Particularly, where they show YFP-KKT2 enrichment over intermediate- and mini-chromosomes (Fig. 7) due to the spillover, one cannot be sure to have found KKT2 in both regions.

Instead, the authors could analyze only uniquely mapping reads / read-pairs where at least one mate is uniquely mapping. I realize that with this strict filtering, data will be much more sparse. Hence, I would suggest keeping the original plots and adding one more quantification where the enrichment over the whole region (e.g., all 177bp repeats on intermediate-/mini-chromosomes) is plotted using the unique reads (this could even be supplementary). This also applies to Fig. 4 B & C.

We thank the reviewer for their thoughtful comments. Repetitive sequences are indeed challenging to analyze accurately, particularly in the context of short read ChIP-seq data. In our study, we aimed to address YFP-KKT2 enrichment not only over CIR147 repeats but also on 177 bp repeats, using both ChIP-seq and proteomics using synthetic TALE proteins targeted to the different repeat types. We appreciate the referees suggestion to consider uniquely mapped reads, however, in the updated genome assembly, the 177 bp repeats are frequently immediately followed by long stretches of 70 bp repeats which can span several kilobases. The size and repetitive nature of these regions exceeds the resolution limits of ChIP-seq. It is therefore difficult to precisely quantify enrichment across all chromosomes.

Additionally, the repeat sequences are highly similar, and relying solely on uniquely mapped reads would result in the exclusion of most reads originating from these regions, significantly underestimating the relative signals. To address this, we used Bowtie2 with settings that allow multi-mapping, assigning reads randomly among equivalent mapping positions, but ensuring each read is counted only once. This approach is designed to evenly distribute signal across all repetitive regions and preserve a meaningful average.

Single molecule methods such as DiMeLo (Altemose et al. 2022; PMID: 35396487) will need to be developed for T. brucei to allow more accurate and chromosome specific mapping of kinetochore or telomere protein occupancy at repeat-unique sequence boundaries on individual chromosomes.

Reviewer #2 (Significance):

This work is of high significance for chromosome/centromere biology, parasitology, and the study of antigenic variation. For chromosome/centromere biology, the conceptual advancement of different types of kinetochores for different chromosomes is a novelty, as far as I know. It would certainly be interesting to apply this study as a technical blueprint for other organisms with minichromosomes or chromosomes without known centromeric repeats. I can imagine a broad range of labs studying other organisms with comparable chromosomes to take note of and build on this study. For parasitology and the study of antigenic variation, it is crucial to know how intermediate- and mini-chromosomes are stable through cell division, as these chromosomes harbor a large portion of the antigenic repertoire. Moreover, this study also found a novel link between the homologous repair pathway and variant surface glycoproteins, via the 70bp repeats. How and at which stages during the process, 70bp repeats are involved in antigenic variation is an unresolved, and very actively studied, question in the field. Of course, apart from the basic biological research audience, insights into antigenic variation always have the potential for clinical implications, as T. brucei causes sleeping sickness in humans and nagana in cattle. Due to antigenic variation, T. brucei infections can be chronic.

Thank you for supporting the novelty and broad interest of our manuscript

My field of expertise / Point of view:

I'm a computer scientist by training and am now a postdoctoral bioinformatician in a molecular parasitology laboratory. The laboratory is working on antigenic variation in T. brucei. The focus of my work is on analyzing sequencing data (such as ChIP-seq data) and algorithmically improving bioinformatic tools.

Unlike Mitskell's article, Croll highlights how industrial elements could become a key part of the leisure industry through the advertisement of industrial ports as a tourist attraction, leisure and industrialisation having a strong and positive relationship in this example.

like mitskell, highlights the importance of the railway in the influx of tourism. Both authors draw a clear link between this industrial development and the development of tourism. This concept of the connecting power of railway is greater developed by (blah) as he notes how this then connected the industrial workers of the rhondda valleys to Barry. This further suggests a strong relationship between industry and leisure. Industrialisation had facilitated a new working (), which saw disposable income increase for many, while industrial action encouraged parliamentary acts such as the (factory act and bank holiday acts with dates) which increased the free-time that workers could use to engage in the leisure industry, resorts like swansea and Barry, as both authors note, becoming places to do it (altho swansea's clientele was a little more posh

Reviewer #1 (Public review):

Summary:

The authors assess the role of map3k1 in adult Planaria through whole body RNAi for various periods of time. The authors' prior work has shown that neoblasts (stem cells that can regenerate the entire body) for various tissues are intermingled in the body. Neoblasts divide to produce progenitors that migrate within a "target zone" to the "differentiated target tissues" where they differentiate into a specific cell type. Here the authors show that map3k1-i animals have ectopic eyes that form along the "normal" migration path of eye progenitors, ectopic neurons and glands along the AP axis and pharynx in ectopic anterior positions. The rest of the study shows that positional information is largely unaffected by loss of map3k1. However, loss of map3k1 leads to premature differentiated of progenitors along their normal migratory route. They also show that "long-term" whole body depletion of map3k1 results in mis-specified organs and teratomas. In short, this study convincingly demonstrates that in planaria, map3k1 maintains progenitor cells in an undifferentiated state, preventing premature fate commitment until they encounter the appropriate signals, either positional cues within a designated region or contact-dependent inputs from surrounding tissues.

Strengths:

(1) The study has appropriate controls, sample sizes and statistics.

(2) The work is high-quality.

(3) The conclusions are supported by the data.

(4) Planaria is a good system to analyze the function of map3k1, which exists in mammals but not other invertebrates.

Weaknesses:

None noted.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors assess the role of map3k1 in adult Planaria through whole body RNAi for various periods of time. The authors' prior work has shown that neoblasts (stem cells that can regenerate the entire body) for various tissues are intermingled in the body. Neoblasts divide to produce progenitors that migrate within a "target zone" to the "differentiated target tissues" where they differentiate into a specific cell type. Here the authors show that map3k1-i animals have ectopic eyes that form along the "normal" migration path of eye progenitors (Fig. 1), ectopic neurons and glands along the AP axis (Fig. 2) and pharynx in ectopic anterior positions (Fig. 3). The rest of the study show that positional information is largely unaffected by loss of map3k1 (Fig. 4,5). However, loss of map3k1 leads to premature differentiated of progenitors along their normal migratory route (Fig. 6). They also show that an ill-defined "long-term" whole body depletion of map3k1 results in mis-specified organs and teratomas.

Strengths:

(1) The study has appropriate controls, sample sizes and statistics.

(2) The work appears to be high-quality.

(3) The conclusions are supported by the data.

(4) Planaria is a good system to analyze the function of map3k1, which exists in mammals but not in other invertebrates.

Weaknesses:

(1) The paper is largely descriptive with no mechanistic insights. 

The mechanistic insights we aim to address are primarily at the cellular systems level – how adult progenitor cells produce pattern. Specifically, we uncovered strong evidence that regulation of differentiation is an active process occurring in migratory progenitors and that this regulation is a major component of pattern formation during the adult processes of tissue turnover and regeneration. The map3k1 phenotype provided a tool used to reveal the existence of this regulation, and to understand the patterning abnormalities prevented by this regulatory mechanism. We updated the text in several places to make clearer some of this emphasis. For example, in the Discussion: "We suggest that differentiation is restricted during migratory targeting as an essential component of pattern formation, with the map3k1 RNAi phenotype indicating the existence and purpose of this element of patterning." 

Naturally, identifying a particular molecule involved in this process is of interest for understanding molecular mechanism; this would allow for comparison to other cellular systems in other organisms and would focus future molecular inquiry. Future molecular studies into the mechanism of Map3k1 regulation and its downstream signaling will be fascinating as next steps towards understanding the process at the molecular level more deeply. We also added some discussion considering the types of upstream activation cues that could potentially be associated with Map3k1 regulation to suppress differentiation. 

(2) Given the severe phenotypes of long-term depletion of map3k1, it is important that this exact timepoint is provided in the methods, figures, figure legends and results. 

We removed the use of the term “long-term” and instead added timepoints used to all figure legends. We also added a summary of timepoints used in the methods section and included RNAi timepoint labels in figures where a phenotype progression over time is relevant to interpretation. For timecourses, we also added suitable time information to text in the results. 

(3) Figure 1C, the ectopic eyes are difficult to see, please add arrows. 

To improve visualization, we replaced the example animal in the original Figure 1C with one that has a stronger phenotype, including arrows pointing to every ectopic event. Additionally, we included magnified images of optic cup cells and photoreceptor neurons in the trunk and tail region. This is now Figure 1B.

(4) line 217 - why does the n=2/12 animals not match the values in Figure 3B, which is 11/12 and 12/12. The numbers don't add up. Please correct/explain. 

In Figure 3B in the submitted version (3/18 had cells in the tail) had more animals scored (6 animals from a replicate experiment where 1/6 showed the cells in the tail) than the total scored (2/12 had cells in the tail) in the text, which did not have the animals from the replicate added during writing. The results are the same, just different sample sizes were noted in those locations and we fixed this issue. In the updated Figure 3, the order of presentation has shifted (e.g., prior 3B is now in 3C and Figure 3_figure supplement 1). We made sure to include numbers to all figure panels. 

(5) Figure panels do not match what is written in the results section. There is no Figure 6E. Please correct.

Thank you for catching this. We have gone through figures and text after editing to make sure that text callouts are appropriately matched to the figures. 

Reviewer #2 (Public review):

Summary:

The flatworm planarian Schmidtea mediterranea is an excellent model for understanding cell fate specification during tissue regeneration and adult tissue maintenance. Planarian stem cells, known as neoblasts, are continuously deployed to support cellular turnover and repair tissues damaged or lost due to injury. This reparative process requires great precision to recognize the location, timing, and cellular fate of a defined number of neoblast progeny. Understanding the molecular mechanisms driving this process could have important implications for regenerative medicine and enhance our understanding of how form and function are maintained in long-lived organisms such as humans. Unfortunately, the molecular basis guiding cell fate and differentiation remains poorly understood.

In this manuscript, Canales et al. identified the role of the map3k1 gene in mediating the differentiation of progenitor cells at the proper target tissue. The map3k1 function in planarians appears evolutionarily conserved as it has been implicated in regulating cell proliferation, differentiation, and cell death in mammals. The results show that the downregulation of map3k1 with RNAi leads to spatial patterning defects in different tissue types, including the eye, pharynx, and the nervous system. Intriguingly, long-term map3k1-RNAi resulted in ectopic outgrowths consistent with teratomas in planarians. The findings suggest that map3k1 mediates signaling, regulating the timing and location of cellular progenitors to maintain correct patterning during adult tissue maintenance.

Strengths:

The authors provide an entry point to understanding molecular mechanisms regulating progenitor cell differentiation and patterning during adult tissue maintenance.

The diverse set of approaches and methods applied to characterize map3k1 function strengthens the case for conserved evolutionary mechanisms in a selected number of tissue types. The creativity using transplantation experiments is commendable, and the findings with the teratoma phenotype are intriguing and worth characterizing.

Thank you to the reviewer for the positive feedback

Weaknesses:

The article presents a provocative idea related to the importance of positional control for organs and cells, which is at least in part regulated by map3k1. Nonetheless, the role of map3k1 or its potential interaction with regulators of the anterior-posterior, mediolateral axes, and PCGs is somewhat superficial. The authors could elaborate or even speculate more in the discussion section and the different scenarios incorporating these axial modulators into the map3k1 model presented in Figure 8 

First, to strengthen the support for our finding that positional information is largely unaffected in map3k1 RNAi animals, we added data regarding the expression of additional relevant position control genes (PCGs) –ndl-4, ptk7, sp5, and wnt11-1 – to the PCG panel in Figure 5. The expression domain of ndl-4, an FGF receptor-like protein family member that contributes to head patterning and anterior pole maintenance, was normal in map3k1 RNAi. wnt11-1, a PCG with expression concentrated in the posterior end of the animal and with expression dependent on general Wnt activity, was also normal in map3k1 RNAi animals. ptk7, RNAi of which can result in supernumerary pharynges, also showed normal expression in map3k1 RNAi animals. Finally, sp5, a Wnt-activated gene with expression in the tail, also showed normal expression in map3k1 RNAi animals. 

Second, to further support the conclusion that cells are not suitably responding to positional information after map3k1 RNAi, which we argue normally dictates where differentiation should occur, we added examples of differentiated cell types that are ectopically positioned within an atypical PCG expression domain for that cell type (Figure 5C). This underscores that following map3k1 RNAi the PCG expression domains do not change, but the pattern of differentiated cell types relative to these domains does shift. We also added data showing that regenerating tails had a proper wntP-2 gradient, but an anterior regenerating pharynx appeared outside of this wntP-2⁺ zone and inside of an ndl-5⁺ zone (Figure 5- figure supplement 1E). We added some discussion of these new data in the Figure 5 results section. We also noted, regarding independent recent map3k1 work (Lo, 2025), some evidence exists that a minor posterior shift in ndl-5 expression can occur after map3k1 RNAi.

Next, we added a new element to the model figure (Figure 8B) depicting that PCG expression domains remain normal after map3k1 RNAi, with ectopic differentiation occurring in an incorrect positional information environment. We refer to this new panel in the discussion: "We suggest that map3k1 is not required for the spatial distribution of progenitor-extrinsic differentiation-promoting cues themselves, but for progenitors to be restricted from differentiating until these cues are received (Figure 8B)."; we then follow this statement with a summary in the Discussion of six pieces of evidence that support this model.

Finally, we added some additional text to the discussion section about candidate mechanisms by which extrinsic cues could potentially regulate Map3k1, pointing to potential future inquiry directions. We suggest that map3k1 is not involved in regulating PCG activity domains themselves, but instead acts as a brake on differentiation within migratory progenitors through active signaling. This brake is then lifted when the progenitors hit their correct PCG-based migratory target, or when they hit their target tissue. How that occurs mechanistically is unknown. One scenario is that each progenitor is tuned to respond to a particular PCG-regulated environment (such as a particular ECM or signaling environment) to generate a molecular change that inactivates Map3K1 signaling, such as by inactivating or disengaging an RTK signal. Alternatively, the migratory process in progenitors could engage the Map3K1 signal, enabling signal cessation with arrival at a target location. When Map3K1 is active it could result in a transcriptional state that prevents full expression of differentiated factors required for maturation, tissue incorporation, and cessation of migration. These considerations are now added to the discussion.

The article can be improved by addressing inconsistencies and adding details to the results, including the main figures and supplements. This represents one of the most significant weaknesses of this otherwise intriguing manuscript. Below are some examples of a few figures, but the authors are expected to pay close attention to the remaining figures in the paper.

Details associated with the number of animals per experiment, statistical methods used, and detailed descriptions of figures appear inconsistent or lacking in almost all figures. In some instances, the percentage of animals affected by the phenotype is shown without detailing the number of animals in the experiment or the number of repeats. Figures and their legends throughout the paper lack details on what is represented and sometimes are mislabeled or unrelated. 

We endeavored to ensure that these noted details are present throughout the legends and figures for all figure panels.

Specifically, the arrows in Figure 1A are different colors. Still, no reasoning is given for this, and in the exact figure, the top side (1A) shows the percentages and the number of animals below. 

The only reason for the different colored arrows was for visibility purposes. To avoid confusion, we now use white arrows for all FISH images in figure 1, and where ever else possible. We also replaced the percentages with n numbers in the bottom left corner of the live images in Figure 1A. 

Conversely, in Figures 1B, C, and D, no details on the number of animals or percentages are shown, nor an explanation of why opsin was used in Figure 1A but not 1B. 

The original Figure 1B represented a few different examples of ectopic eye/eye cell patterns in the map3k1 RNAi animals to demonstrate the variable and disorganized nature of the phenotype. To address this, we added further explanation in the legend. We also merged 1A and 1B for simplicity of interpretation. opsin was used in Figure 1A to label cell bodies of photoreceptors. anti-Arrestin was used in the example FISH images to see if these cells were interconnected via projections, which we now clarify in the legend and in the text. 

Is Figure 1B missing an image for the respective control? Figure 1C needs details regarding what the two smaller boxes underneath are. 

The control for Figure 1B was in Figure 1A; the merger of Figures 1A/B should address this. Boxes in Figure 1C were labelled with numbers corresponding to the image above them.

Figure 1C could use an AP labeling map in 10 days (e.g., AP6 has one optic cup present). Figure 1C and F counts do not match. 

We added a cartoon to 1C to show the region imaged. Note that the 36d trunk image (now Fig. 1B) has now been replaced with a full animal image and magnified boxes that show locations of example ectopic cells. That cell in 1C was categorized as in AP5. Note that additional animals were analyzed and data added to the distribution (now Fig. 1D). 

In Figure 1C, we do not know the number of animals tested, controls used, the scale bar sizes in the first two images, nor the degree of magnification used despite the pharynx region appearing magnified in the second image.  Figure 1C is also shown out of chronological order; 36 days post RNAi is shown before 10 days post RNAi. Moreover, the legends for Figures 1C and 1D are swapped.

We have endeavored to ensure sample numbers, control images, and appropriate scale bar notation in legends are present for all images. Figure 1C has now been split into two panels: Figure 1B and Figure 1C. It does not follow a chronological order in presentation for the following logic flow: we uncover and describe the phenotype; then, with knowledge of the defect, we walk back to see how early the phenotype starts after RNAi and what the pattern of ectopic cell distribution is when the phenotype starts to emerge (using the knowledge of which cells are affected from the overt phenotype described in 1A/B). 

Additionally, Figure 1F and many other figures throughout the paper lack overall statistical considerations. Furthermore, Figure 1F has three components, but only one is labeled. Labeling each of them individually and describing them in the corresponding figure legend may be more appropriate.

The main point of the graphs in 1F (now 1D) was the overt overall pattern difference with the wild-type, which never has ectopic eye cells in the midbody or tail, and that the ectopic eye cells progress throughout the entire AP axis. However, we concur that a statistical test is a reasonable thing to show here and that is now included in the legend. The 3 components (in Figure 1F, now Figure 1D) where kept together with one figure label (D) for simplicity, but were rearranged (top and bottom) with a cartoon to the side and with modified labeling for extra clarity. 

Figure 2C shows images of gene expression for two genes, but the counts are shown for only one in Figure 2D. It is challenging to follow the author's conclusions without apparent reasoning and by only displaying quantitative considerations for one case but not the other. These inconsistencies are also observed in different figures. 

In Figure 2C, FISH images of cintillo+ and dd_17258+ neurons are shown to display the specificity of this effect to some neurons and not others. Because cintillo+ cells did not expand at all (n=24/24 animals), the counts for them would all be zero values. We only counted data for dd_17258 cells because it was the neuron that expanded compared to the control animals. We have now added a note in the legend explaining this.

In Figure 2D, 24/24 animals were reported to show the phenotype, but only eight were counted (is there a reason for this?).

8 animals were used to quantitatively characterize the spread of cells along the AP axis, as it was deemed an adequate sample size to capture the change in distribution of 17258+ cells from being head restricted to being present throughout the body. Through multiple cohorts of animals in replicates, a total of 24/24 examined animals showed this expansion phenotype. Double FISH experiments were additionally carried out using dd_17258 and various PCGs; these data are now included in Figure 5C, and these animals were added to the total counts regarding quantitative analysis of the phenotype in Figure 2D. 

In Figure 2E, the expression for three genes is shown, with some displaying anterior and posterior regions while others only show the anterior picture. Is there a particular reason for this? 

The original first panel in Figure 2E showed an example of a non-expanding gland cell type, dd_9223, which is very restricted to the head in both control and map3k1 RNAi animals. Because we did not observe a phenotype for this cell type (no cells in all control and map3k1 RNAi animal tails), we only included tail images of cell types that showed an abnormal phenotype with clear expanded to the posterior (dd_8476 and dd_7131). However, we have now included tail images of dd_9223 cells and added data for dd_9223 to the graph in Figure 2E. 

Also, in Figure 2F, the counts are shown for only the posterior region of two genes out of the three displayed in Figure 2E. It is unclear why the authors do not show counts for the anterior areas considered in Figure 2E. Furthermore, the legend for Figure 2D is missing, and the legend for 2F is mislabeled as a description for Figure 2D.

We now include tail images for dd_9223 in Figure 2E to show that there are no ectopic cells in tails. We did not originally include counts of dd_9223 because there was no phenotype observed. dd_7131 and dd_8476 cell types appeared in the posterior of even control animals at a low frequency, unlike dd_9223 cells. However, we did now add counts for dd_9223 tail regions in the graph. We did not count the anterior regions of the animal because our goal was to show data for the visible phenotype (ectopic cells in the tail) not only with an example image, but also by showing the number of cells in the tail with a graph and statistical test. Legends have been updated with correct details.

Supplement Figure 1 B reports data up to 6 weeks, but no text in the manuscript or supplement mentions any experiment going up to 6 weeks. There are no statistics for data in Supplement Figure 1E. Any significance between groups is unclear.

More details about the RNAi feeding schedules have been added in the methods section. All RNAi timepoints are now specified specifically in the legends. The Figure 1F and Figure 1- figure supplement 1E (additional data: ovo⁺; smedwi-1^- cell counts) and legends now mention the statistical tests performed and annotations (not significant *ns) or p values have been added to the graphs. For simplicity, we decided to include all smedwi-1+ counts together rather than splitting them into low and high smedwi-1+ cells, because we weren't really making any claims about low and high cells. 

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

It would be good to acknowledge in the discussion the recent paper from the Petersen lab on map3k1, published in PLoS Genet 2025, especially if the results differ between the two labs.

We added reference/discussion regarding the recent PLoS Genetics Lo, 2025 map3k1 paper at several suitable points in the manuscript.

Reviewer #2 (Recommendations for the authors):

Please pay close attention to the description of experimental details and the consistency throughout the paper. It seems like the reader has to assume or come across information that is not readily available from the text or the legends in the paper. This is an interesting paper with intriguing findings. However, the version presented here appears rushed or put together on the flight.

Thank you for your thorough feedback. We have endeavored to ensure all appropriate details are present in figures and/or figure legends.

Simplifying data may frequently be convenient when creating a widely-applicable program, but it involves leaving at least one group or perspective out. Because of this, simplification of data often contains inherent bias and developers should be aware of this.

This definitely opened my perspective on data constraints. In the reflection before, I figured that the best way to store information for social media would be through pre-set categories (for things like relationship status, address, etc), but there are definitely important details that can be hard to simplify and cut out (though, I'm sure no one needs additional details on someone's relationship status). I guess that's why there are some instances where you're able to put down a permanent address and a temporary address for those who are only residing somewhere for a short-term opportunity.

Reviewer #1 (Public review):

Summary:

This manuscript by Joshi and colleagues demonstrates that the precise theta-phase timing of spikes is causal for CA1 hippocampal theta sequences during locomotion on a linear track and is necessary for learning the cognitively demanding outbound component of a hippocampus-dependent alternation task (W-maze), independently of replay during immobility. To reach these conclusions, the authors developed a theta-phase-specific, closed-loop manipulation that used optogenetic activation of medial septal parvalbumin (PV) interneurons at the ascending phase of theta during locomotion. This protocol preserved immobility periods, allowing a clean and elegant dissociation from SWR-associated replay.

The manuscript is well written and was a pleasure to read. The work described is of high quality and introduces several notable advances to the field:

(a) It extends prior studies that manipulated theta oscillations by examining precise temporal structure (specifically theta sequences) rather than only LFP features.

(b) The closed-loop manipulation enabled dissociation between deficits in theta sequences during a behavioural task and SWR-associated replay activity.

(c) As controls, the authors included rats with suboptimal viral transduction or optic-fibre placement, and, within subjects, both stimulation-on (stim-on) and stimulation-off (stim-off) trials. Notably, sequence disruption persisted into stim-off periods within the same session.

Overall, this is a strong manuscript that will provide valuable insights to the field. I have only minor comments:

(1) As the authors note, it is striking that both behavioural performance and spike patterns are altered during stim-off trials. They propose that "disruption of theta sequences during the initial experience in an environment is sufficient to have lasting effects," implying that rapid, experience-dependent plasticity is driven by sequential firing. Does this imply that if rats were previously trained on the task, subsequent stim-on and stim-off trials would yield different outcomes, with stim-off trials showing improved performance and intact theta sequences? For example, if the sequence of one-third stim-on, one-third stim-off, one-third stim-on were inverted to off-on-off, would theta sequences be expected to emerge, disappear, and potentially re-emerge? While I am not asking for additional experiments, I think the discussion could be extended in this aspect.

Alternatively, could the number of stim-off trials (one third of the total) be insufficient to support learning/induce plasticity? In the controls, ~50-100 trials appear necessary to achieve high performance.

(2) In line with the point above, the authors characterise the behavioural changes induced by MS optogenetic stimulation specifically as a "learning deficit," as rats failed to improve across 300 trials in an initially novel environment (W-maze). While they present this as complementary to prior demonstrations of impaired performance on previously learned tasks (Zutshi et al., 2018; Quirk et al., 2021; Etter et al., 2023; Petersen et al., 2020), an alternative interpretation is a working-memory deficit. This would produce the same behavioural pattern, with reference memory (the less cognitively demanding trials) remaining intact despite stimulation and concomitant changes in theta sequences. This interpretation would also be consistent with work in certain disease models, where reduced synaptic plasticity and working-memory deficits co-occur with preserved place coding despite impaired theta sequences (e.g., Viana da Silva et al., 2024; Donahue et al., 2025).

(3) It was not immediately clear whether SWR-associated activity was derived from the interleaved ~15-min rest sessions in a rest box, or from periods of immobility or reward consumption in the maze (aSWR, as in Jadhav et al 2012). Regardless, it would be informative to compare aSWR events within the maze to rest-box SWRs that may occur during more prolonged slow-wave episodes (even if not full sleep). This contrasts with Liu et al. (2024), who analysed replay during ~1.5-h sleep sessions.

Reviewer #2 (Public review):

Summary:

The authors of this study developed a closed-loop optogenetic stimulation system with high temporal precision in rats to examine the effect of medial septum (MS) stimulation on the disruption of hippocampal activity at both behavioral and compressed time scales. They found that this manipulation preserved hippocampus single-cell-level spatial coding but affected theta sequences and performance during a spatial alternation task. The performance deficits were observed during the more cognitively demanding component of the task and even persisted after the stimulation was turned off. However, the effects of this disruption were confined to locomotor periods and did not impact waking rest replay, even during the early phase of stimulation-on. Their conclusion is consistent with previous findings from the Pastalkova lab, where MS disruption (using different methods) affected theta sequences and task performance but spared replay (Wang et al., 2015; Wang et al., 2016). However, it differs from a recent study in which optogenetic disruption of EC inputs during running affected both theta sequences and replay (Liu et al., 2023).

Strengths:

The experiments were well designed and controlled, and the results were generally well presented.

Weaknesses:

Major concerns are primarily technical but also conceptual. To further increase the impact of this study by contrasting findings from different disruptions, it is necessary to better align the analysis and detection methods.

Major concerns:

(1) To show that MS disruption does not affect spatial tuning, the authors computed the KL divergence of tuning curves between stimulation-on and stimulation-off conditions. I have two main questions about this analysis:

(1.1) The authors seem to impose stringent inclusion criteria requiring a large number of spikes and a strong concentration of tuning curves. These criteria may have selected strongly spatially tuned cells, which are typically more stable and potentially less vulnerable to perturbations. Based on the Figure 2 caption, it seems that fewer than 10% of cells were included in the KL divergence analysis, which is lower than the usual proportion of place cells reported in the literature. What is the rationale for using such strict inclusion criteria? What happens to the cells that are not as strongly tuned but are still identified as significant place cells?

(1.2) The KL divergence was computed between stimulation-on and stimulation-off conditions within the same animal group. However, the authors also showed that MS stimulation had lasting effects on theta sequences and performance even during stimulation-off periods. Would that lasting effect also influence spatial tuning? Based on these questions, the authors should perform additional analyses that directly measure spatial tuning quality and compare results across control and experimental groups - for example, spatial information of spikes (Skaggs et al., 1996), tuning stability, field length, and decoding error during running.

(2) The authors compared their results with those from Liu et al. (2023) and proposed that the different outcomes could be explained by different sites of disruption. However, the detection and quantification methods for theta sequences and replay differ substantially between the two studies, emphasizing different aspects of the phenomenon. I am not suggesting that either method is superior, but providing additional analyses using aligned detection methods would better support the authors' interpretations and benefit the field by enabling clearer comparisons across studies. In the current analysis, the power spectrum of the decoded ahead/behind distance only indicates that there is a rhythmic pattern, without specifying the decoding features at different theta phases. Moreover, the continuous non-local representations during ripples could include stationary representations of a location or zigzag representations that do not exhibit a linear sequential trace. Given that, the authors should show averaged decoding results corrected by the animal's actual position within theta cycles and compute a quadrant ratio. For replay analysis, they could use a linear fit (as in Liu et al., 2023) and report the proportion of significant replay events.

(3) The finding that theta sequences and performance were impaired even during stimulation-off periods is particularly interesting and warrants deeper exploration. In the Discussion, the authors claim that this may arise from "the rapid plasticity engaged during early learning." However, this explanation does not fully account for the observation. Previous studies have shown that theta sequences can develop very rapidly (Feng et al., Foster lab, 2015; Zhou et al., Dragoi lab, 2025). If the authors hypothesize that rapid plasticity during early stimulation-on disrupts the theta sequence, then the plasticity window must also be short and terminate during the subsequent stimulation-off period. Otherwise, why can't animals redevelop theta sequences during stimulation-off? The authors should conduct additional analyses during the stimulation-off periods of the W-maze task. For example:

(3.1) What is the spike-theta phase relationship? Do the phases return to normal or remain altered as during stimulation-on?

(3.2) Is there a significant place-field remapping from stimulation-on to stimulation-off? (Supplementary Figure 3F includes only a small subset of cells; what if population vector correlations are computed across all cells, or Bayesian decoding of stimulation-on spikes is performed using stimulation-off tuning curves?)

(3.3) The authors should also discuss why the stimulation-off epochs were not sufficient to support learning, and if the stimulation-off place cell sequences could have supported replay.

(4) Citations and/or discussion of key studies relevant to the current work are missing: Wang et al. in Pastalkova lab 2015-2016 studies for disruption of theta sequence (but not place cell sequence) disrupting learning but not replay, Drieu et al. in Zugaro lab 2018 study on disruption of theta sequence affecting sleep replay, Farooq and Dragoi 2019 for association between a lack of theta sequence and presence of waking rest replay during postnatal development, etc. The authors should discuss what the conceptually new findings in the current study are, given the findings of the previous literature above.

(5) The assessment of theta sequence is not state-of-the-art:

(5.1) Detecting the peak of cross-correlograms between neurons (CCG) relates to behavioral timescale CCG, not the theta sequence one; for the theta sequence, the closest to zero local peak should be used instead.

(5.2) How were other methods of detecting theta sequences performing on the stimulation-on/stimulation-off data: Bayesian decoding, firing sequences?

(5.3) How was phase precession during stimulation-on/stimulation-off?

(6) It would be important to calculate additional variables in the replay part of the study to compare the quality of replay across the 2 groups:

(6.1) Proportion of significant replay events out of the detected multiunit events.

(6.2) The average extent of trajectory depicted by the significant replay events in the targeted compared to the control, stimulation-on/stimulation-off.

Reviewer #3 (Public review):

Joshi et al. present an elegant and technically rigorous study examining how the temporal structure of hippocampal spiking during locomotion contributes to spatial learning. Using a closed-loop, theta phase-specific optogenetic manipulation of medial septal parvalbumin-expressing neurons in rats, the authors demonstrate that disrupting theta-timescale coordination impairs performance on the cognitively demanding component outbound trajectory of a spatial alternation task, while sparing hippocampal replay, place coding, and the simpler inbound learning. The work aims to dissociate the role of theta-associated temporal organization during navigation from sharp-wave ripple-associated replay during subsequent rest periods, providing a mechanistic link between theta sequences and learning. The findings have important implications for models of septo-hippocampal coordination and the functional segregation between online (theta) and offline (SWR) network states. That said, there are a few conceptual and methodological issues that need to be addressed.

One concern is the overall novelty of this work; the dissociation between online temporal sequence and offline replay events following memory deficits has previously been shown by Wang et al., 2016 elife. While the authors discuss Lui et al., 2023, which demonstrates MEC activation of inhibitory neurons at gamma frequencies during locomotion disrupts theta sequences, subsequent replay and learning (line 65-66), they do not reference Wang et al., 2016 who performed a very similar study with MS pharmacological inactivation, and report large decreases in theta power, attenuated theta frequencies together with behavioural deficits but SWR replay persisted. Given strong similarities in the manipulation and findings, this study should be discussed.

Along the same lines, it should be noted that Brandon et al. (2014, Neuron) demonstrated that hippocampal place codes can still form in novel environments despite MS inactivation and loss of theta, indicating that spatial representations can emerge without intact septal drive. Referencing this study would strengthen the discussion of how temporal coordination, rather than spatial coding per se, underlies the learning deficits observed here.

The conclusion that disrupting "theta microstructure" impairs learning relies on the assumption that the observed behavioral deficits arise from altered temporal coding from within hippocampal CA1 only. However, optogenetic modulation of medial septal PV neurons influences multiple downstream regions (entorhinal cortex, retrosplenial cortex) via widespread GABAergic projections. While the authors do touch on this, their discussion should expand to include the network-level consequences of entorhinal grid-cell disruption and how this could affect temporal coding both online and offline.

The finding that replay content, rate, and duration are unchanged is critical to the paper's claim of dissociation. However, the analysis is restricted to immobility on the track. Given evidence for distinct awake vs. sleep replay, confirming that off-track rest and post-session sleep replays are similarly unaffected would confirm the conclusions of the paper. If these data are unavailable, the limitation should be acknowledged explicitly. Moreover, statistical power for detecting subtle differences in replay organization or spatial bias should be added to the supplement (n of events per animal, variability across sessions).

The exact protocol for optogenetic stimulation is a bit confusing. For the task, the first and final third (66%) of trials were disrupted and were only stimulated when away from the reward well and only when the animal was moving. What proportion of time within "stimulated" trials remained unstimulated? Why were only 66% of trials stimulated?

Author response:

We thank all reviewers for their overall assessment, thoughtful comments, and suggestions. We are working to address each reviewer’s comment in detail. In this provisional response, we provide clarifications regarding our experimental approach and the novelty of our work, and include additional analyses that we have performed since the submission of the manuscript. We are also happy to report that we have now shared the raw data, intermediate analysis files, and the complete repository to facilitate replication of the analysis and figures.

Code repo: github.com/LorenFrankLab/ms_stim_analysis

Data repo: dandiarchive.org/dandiset/001634

Docker containers (see GitHub repo for use instructions):

Database: https://hub.docker.com/r/samuelbray32/spyglass-db-ms_stim_analysis

Python notebooks: https://hub.docker.com/r/samuelbray32/spyglass-hub-ms_stim_analysis

(1) Novelty and contrast with earlier manipulations:

We thank the reviewers for suggesting that we explicitly contrast our results with prior pharmacological (Wang et al., 2016; Wang et al., 2015; Koenig et al., 2011; Brandon et al., 2014), systemic (Robbe & Buzsaki 2009; Petersen and Buzsáki 2020), and behavioral (Drieu et al., 2018) manipulations that also assessed some of the physiological features we evaluated. We will add a discussion of these studies, which will help us emphasize both the insights and discrepancies observed using these prior approaches. We will also more clearly explain the the novelty and importance of our specific approach for temporally and physiologically precise manipulation. Specifically, our approach (closed-loop theta-phase stimulation during locomotion) provides a level of physiological specificity that made it possible to dissociate theta-state dynamics from other hippocampal processes. This in turn allowed us to address a question that has remained unresolved across prior studies: Are hippocampal spatial sequences during locomotion (i.e., theta sequences) necessary to learn a novel hippocampal-dependent task?

(2) Additional analysis on SWRs during rest:

since submitting the manuscript, we have conducted additional analysis on the rate and length of SWRs in the rest box and found that their rate and length are also indistinguishable between targeted and control animals (effect of manipulation between control and targeted animals; rSWR rate: p=0.45; rSWR length: p=0.94, mixed effect model). We also find evidence for sequential neural representations in the rest box, when the encoding was performed in the behavioral arena. Example trajectories are shown below. These results are consistent with our observations on SWRs rate, length, and content in the behavioral arena. Additionally, we are in the process of evaluating and quantifying the results of decoding the rSWRs and will include those in the next version of the manuscript.

Author response image 1.

Sequential replay events observed in the rest box

(3) Theta sequence measurement in the absence of theta:

In the next version of the manuscript, we will explicitly explain why our manipulation makes it is more appropriate to measure sequential hippocampal representations during locomotion (i.e., theta sequences) without using theta oscillation or an epoch-averaged relatively large sliding window as a reference. The key insight here is that our manipulation suppresses theta and thus makes it difficult or impossible to accurately identify theta phase. We understand that theta-phase based approaches were used in prior work; however, these prior analyses may have confounded the absence of hippocampal theta sequences during locomotion by the inability to detect theta oscillatory phase reliably. We will show that our method of using clusterless Bayesian decoding in which we estimate the decoded position at every 2ms timestep is indeed able to capture endogenous hippocampal sequences even without imposing any requirements of aligning to theta oscillations, thus providing an unbiased estimate of the rhythmicity of hippocampal spatial representations.

(4) Additional analysis on place cell stability and tuning:

We thank the reviewer for this question. For the KL divergence analysis, we have imposed a spike-count criterion (100 spikes for each interval type —stimulation-off, stimulation-on, and the stimulus sub-interval) and a coverage criterion (50% HPD of the units’ spatial firing distribution was contained within 40cm on the linear track and 100cm on the w-track). These criteria were chosen to ensure that spatial tuning curves were sufficiently well sampled and localized to allow reliable estimation of KL divergence, which is particularly sensitive to noise arising from low spike counts or diffuse firing. Based on the reviewer’s suggestion, we have relaxed the unit inclusion criteria for KL divergence by relaxing the criteria for number of spikes and spatial coverage criterion to include more weakly tuned place cells and replicated our results (p=.146). Further, we have also evaluated the stability of place field order between stimulation-on and stimulation-off conditions using more standard methods (as in Wang et. al., 2015; spearman correlation of place field order, control vs targeted, p = .920, t-test). These results are consistent with our observations about place field stability during stimulation-off and stimulation-on conditions (Fig. 2F).

Author response image 2.

Spearman correlation of place field order during stimulation-on and stimulation-off conditions.

Synthèse : Enfants Violents à l'École - Entre Aide et Répression

Résumé Exécutif

Ce document de synthèse analyse les tensions et les débats entourant la gestion de la violence chez les jeunes enfants au sein du système scolaire et de la société française.

Il ressort que l'école se trouve démunie face à des comportements extrêmes, conduisant à la création de structures expérimentales comme "r'école" pour éviter la déscolarisation.

Parallèlement, une tendance croissante à la médicalisation des troubles du comportement, incarnée par le diagnostic d'hyperactivité et la prescription de Ritaline, suscite une vive controverse.

Des experts dénoncent l'influence du lobbying pharmaceutique et une simplification qui ignore les causes profondes de la souffrance de l'enfant.

Cette approche s'inscrit dans un contexte de "psychose médiatique" qui exagère le phénomène de la violence infantile, contredit par la réalité judiciaire qui atteste de la rareté des cas criminels chez les très jeunes.

L'analyse des cas individuels révèle que la violence est souvent le symptôme d'une souffrance psychique profonde, liée à des contextes familiaux difficiles (ruptures, violence parentale) et socio-économiques précaires.

Face à des réponses répressives ou médicamenteuses, des initiatives de prévention de proximité, comme l'association "Mission Possible", démontrent qu'un accompagnement axé sur l'écoute et le soutien aux familles est non seulement plus humain, mais aussi considérablement moins coûteux et plus efficace à long terme pour la société.

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1. Le Défi de l'École Face aux Comportements Extrêmes

Le système éducatif est confronté à une difficulté croissante pour gérer les comportements violents et ingérables de certains très jeunes élèves.

Les enseignants et les directions d'école expriment un sentiment d'impuissance et de manque de formation, menant à des situations d'échec et à l'exclusion des enfants concernés comme ultime recours.

• Le Cas d'Ethan Paul et Hamadi (6 ans) : Ces deux élèves de CP ont été exclus de leur école pour "comportement violent et ingérable".

Ethan Paul aurait tenté d'étrangler un camarade, conduisant des parents à porter plainte. Sa maîtresse reconnaît son échec :

"Je n'ai pas réussi à ce qu'ils puissent être intégrés en classe et faire les apprentissages de la place de façon satisfaisante." Elle décrit un enfant en "très très très très grande souffrance".

• La Structure "r'école" : Mise en place en janvier 2009 par le rectorat de Paris, cette structure unique en France accueille pour trois mois des enfants exclus.

◦ Objectif : Éviter la déscolarisation en offrant une "surveillance éducative renforcée" et en réapprenant aux enfants les règles de la vie en groupe.  

◦ Fonctionnement : Les enfants sont pris en charge par un personnel mixte (enseignante, auxiliaire de vie scolaire), mais seule l'enseignante est spécifiquement formée. Les crises de violence y sont fréquentes et difficiles à gérer pour le personnel.   

◦ Limites : Bien que présentée comme le "seul espoir", cette solution est coûteuse et soulève le risque de créer des filières alternatives pour enfants "difficiles", comme le souligne Jean-Louis Barateau, initiateur du projet : "Ça pourrait être dangereux si on multipliait des r'écoles au point d'avoir finalement des écoles alternatives."

2. La Médicalisation de la Violence Infantile : Une Solution Controversée

Face à l'inquiétude grandissante, une approche tendant à considérer les troubles du comportement comme des pathologies médicales à traiter a émergé, non sans susciter de vives critiques.

2.1. Le Rapport de l'INSERM et la Récupération Politique

• Le Rapport (2006) : Consacré aux "troubles des conduites", ce rapport d'experts visait à dépister les "facteurs de risque" et de "vulnérabilité" chez l'enfant.

• La Controverse : Le rapport a servi de caution scientifique à un projet de loi sur la délinquance des mineurs, porté par Nicolas Sarkozy, alors ministre de l'Intérieur.

Celui-ci affirmait : "Plutôt on n'intervient plus mieux on a de chances d'éviter le drame d'un enfant qui évolue vers la délinquance."

• La Réponse de la Société Civile : Des experts ayant participé au rapport ont précisé n'avoir "jamais écrit un rapport sur la prévention de la délinquance".

En réaction, la pétition "Pas de zéro de conduite pour les enfants de 3 ans" a recueilli près de 200 000 signatures, dénonçant le risque de transformer des comportements normaux (morsures, colères) en symptômes d'un trouble mental à rééduquer.

2.2. L'Hyperactivité et la Ritaline : Remède ou Simplification ?

Le diagnostic de "l'hyperactivité avec déficit de l'attention" (TDAH) et son traitement par la Ritaline, une amphétamine, sont au cœur du débat.

• La Défense du Traitement : La pédopsychiatre Marie-France Le Heuzey justifie son usage pour "améliorer la qualité de vie et d'améliorer le quotidien de ces enfants", soulignant la souffrance liée au rejet social et familial.

Pour elle, si le médicament permet à l'enfant de ne plus être puni et aux parents de moins se disputer, "on a aussi fait du bien largement à l'enfant".

• La Critique de la Sur-médicalisation :

◦ Rareté de la pathologie : Selon le Pr Bernard Golse (Hôpital Necker), l'hyperactivité "vraie" est très rare (1 à 2 cas pour 1000), loin des 5 à 10% avancés par certains. Il dénonce "l'effet direct du lobbying pharmaceutique qui veut élargir coûte que coûte la prescription de médicaments".   

◦ Création de la demande : Philippe Pignarre, ancien cadre de l'industrie pharmaceutique, explique la stratégie marketing : "L'industrie pharmaceutique travaille à créer à la fois l'offre et la demande... On va la créer la demande en disant aux gens... ce que vous saviez pas, c'est qu'il a un trouble mental et qu'on peut soigner ce trouble mental."   

◦ Traitement des symptômes, pas des causes : La Ritaline, surnommée "pilule de l'obéissance", agit sur les symptômes mais ne traite pas les causes sous-jacentes de la souffrance.

2.3. L'Expérience Vécue : Le Cas d'Aymeric

Aymeric, 16 ans, a été traité à la Ritaline pendant des années.

Son témoignage illustre l'ambivalence du traitement : "C'était bien mais c'est pas bien. Pourquoi c'était bien ? Parce que ça me calmait d'un côté. Mais... c'était bien pour eux, mais pour moi c'était pas bien parce que là je mangeais plus... j'étais tout le temps fatigué."

3. Psychose Médiatique et Réalité Judiciaire

La perception publique de la violence infantile est fortement influencée par un traitement médiatique qui tend à se focaliser sur des faits divers extrêmes, créant une forme de psychose collective.

• L'Affaire d'Uckange (2009) : Un Emballement Révélateur :

◦ Un garçon de 5 ans est accusé d'avoir poignardé sa sœur de 8 ans, prétendument sous l'influence de jeux vidéo.

L'affaire est largement médiatisée, et la thèse de l'enfant coupable est acceptée par les médias, la police et la justice.   

◦ Quelques jours plus tard, la mère avoue être l'auteure du coup de couteau. L'affaire démontre la rapidité avec laquelle "les médias ont véhiculé un peu trop vite le scénario de l'enfant criminel".

• La Perspective des Magistrats : La juge pour enfants Marie-Pierre Hourcade affirme que les affaires de violence au pénal impliquant de très jeunes enfants sont très rares.

Le critère de la responsabilité pénale est le discernement, qui apparaît vers 7-8 ans. "En aucune façon le parquet ne nous saisit pour des situations de violences commises par des très jeunes enfants."

4. Derrière la Violence : Souffrance Psychique et Contexte Familial

L'analyse approfondie des cas révèle que les comportements violents sont presque toujours l'expression d'une souffrance profonde, souvent enracinée dans des histoires familiales et sociales complexes.

• L'Expression de la Souffrance : Le Cas de Sami (13 ans) :

◦ Retiré à 8 ans d'un contexte familial violent, Sami a été ballotté de foyer en foyer. Sa violence est une manifestation de sa tristesse face aux ruptures affectives répétées.  

◦ Le Dr Roger Teboul, psychiatre, explique : "Bien souvent, quand vous parlez de la violence, vous parlez de la tristesse... Le seul truc qui permet de tenir, c'est d'être en colère."

L'objectif de son service est de permettre à ces jeunes d'exprimer leur tristesse pour ne plus avoir à l'agir par la violence.

• L'Impact du Contexte Socio-économique : Le Cas de Florian (7 ans) :

◦ Florian vit dans un quartier précaire d'Amiens. Sa mère élève seule 5 enfants avec le RMI. L'État s'est largement désengagé du quartier.  

◦ Cet environnement de précarité rend l'éducation extrêmement difficile. La mère de Florian exprime sa peur que son fils devienne délinquant si elle n'est pas soutenue.

5. Stratégies d'Intervention : La Prévention comme Alternative

Face aux approches répressives ou médicales, les initiatives de prévention axées sur l'accompagnement et le soutien des familles démontrent leur pertinence humaine et économique.

• "Mission Possible" : Un Modèle de Prévention de Proximité :

◦ Créée par le juge des enfants Claude Baud, cette association à Amiens accueille librement des familles sans obligation judiciaire.  

◦ Elle offre un soutien aux parents, souvent démunis et en rupture avec les services sociaux, sans les juger ni les culpabiliser.   

◦ Elle apprend aux enfants les règles de vie en société par le dialogue et un cadre clair, en cherchant à comprendre le sens de leurs comportements plutôt qu'à les étiqueter.

• L'Analyse d'un Juge : Coût et Efficacité : Claude Baud souligne les avantages de la prévention :

◦ Moins Stigmatisant : "Un parcours judiciaire pour un enfant est dix fois plus stigmatisant qu'un parcours de prévention."   

◦ Moins Coûteux : Il établit une comparaison financière éloquente :      

▪ Prévention (Mission Possible) : 8 € par jour et par enfant.     

▪ Placement Éducatif : 200 à 400 € par jour.    

▪ Détention en section mineurs : 700 à 1000 € par jour.

La conclusion est claire : investir dans des moyens humains, de l'écoute et du personnel bien formé pour aider les familles et les enfants en souffrance est infiniment moins coûteux que de devoir gérer, quelques années plus tard, les conséquences de cette souffrance non traitée.

Reviewer #1 (Public review):

Summary:

This study examines the role of the long non-coding RNA Dreg1 in regulating Gata3 expression and ILC2 development. Using Dreg1-deficient mice, the authors show a selective loss of ILC2s but not T or NK cells, suggesting a lineage-specific requirement for Dreg1. By integrating public chromatin and TF-binding datasets, they propose a Tcf1-Dreg1-Gata3 regulatory axis. The topic is relevant for understanding epigenetic regulation of ILC differentiation.

Strengths:

(1) Clear in vivo evidence for a lineage-specific role of Dreg1.

(2) Comprehensive integration of genomic datasets.

(3) Cross-species comparison linking mouse and human regulatory regions.

Weaknesses:

(1) Mechanistic conclusions remain correlative, relying on public data.

(2) Lack of direct chromatin or transcriptional validation of Tcf1-mediated regulation.

(3) Human enhancer function is not experimentally confirmed.

(4) Insufficient methodological detail and limited mechanistic discussion.

Reviewer #2 (Public review):

The authors investigate the role of the long non-coding RNA Dreg1 for the development, differentiation, or maintenance of group 2 ILC (ILC2). Dreg1 is encoded close to the Gata3 locus, a transcription factor implicated in the differentiation of T cells and ILC, and in particular of type 2 immune cells (i.e., Th2 cells and ILC2). The center of the paper is the generation of a Dreg1-deficient mouse. While Dreg1-/- mice did not show any profound ab T or gd T cell, ILC1, ILC3, and NK cell phenotypes, ILC2 frequencies were reduced in various organs tested (small intestine, lung, visceral adipose tissue). In the bone marrow, immature ILC2 or ILC2 progenitors were reduced, whereas a common ILC progenitor was overrepresented, suggesting a differentiation block. Using ATAC-seq, the authors find that the promoter of Dreg1 is open in early lymphoid progenitors, and the acquisition of chromatin accessibility downstream correlates with increased Dreg1 expression in ILC2 progenitors. Examining publicly available Tcf1 CUT&Run data, they find that Tcf1 was specifically bound to the accessible sites of the Dreg1 locus in early innate lymphoid progenitors. Finally, the syntenic region in the human genome contains two non-coding RNA genes with an expression pattern resembling mouse Dreg1.

The topic of the manuscript is interesting. However, there are various limitations that are summarized below.

(1) The authors generated a new mouse model. The strategy should be better described, including the genetic background of the initially microinjected material. How many generations was the targeted offspring backcrossed to C57BL/6J?

(2) The data is obtained from mice in which the Dreg1 gene is deleted in all cells. A cell-intrinsic role of Dreg1 in ILC2 has not been demonstrated. It should be shown that Dreg1 is required in ILC2 and their progenitors.

(3) The data on how Dreg1 contributes to the differentiation and or maintenance of ILC2 is not addressed at a very definitive level. Does Dreg1 affect Gata3 expression, mRNA stability, or turnover in ILC2? Previous work of the authors indicated that knockdown of Dreg1 does not affect Gata3 expression (PMID: 32970351).

(4) How Dreg1 exactly affects ILC2 differentiation remains unclear.

Synthèse sur la Maltraitance Infantile : Thèmes, Intervenants et Cas d'Étude

Synthèse Exécutive

Ce document de synthèse analyse les thèmes centraux, les dynamiques et les conséquences de la maltraitance infantile, en se basant sur une série d'études de cas et d'interventions d'experts.

L'analyse révèle que la maltraitance est un phénomène polymorphe, incluant la violence physique extrême, le syndrome du bébé secoué, les abus sexuels et les négligences graves.

Une conclusion alarmante émerge : dans la majorité des cas (neuf sur dix), les sévices sont infligés au sein même de la cellule familiale, transformant le lieu de sécurité supposé en principal foyer de danger.

Le silence des victimes, la complicité passive ou active de certains membres de la famille et l'aveuglement de l'entourage constituent des obstacles majeurs à la protection des enfants.

La chaîne d'intervention, bien que complexe, est clairement définie : elle commence par une alerte (via le 119 ou un signalement médical), se poursuit par une enquête policière (Brigade des Mineurs), aboutit à une réponse judiciaire (Procureur, Juge des enfants) et se conclut par une prise en charge spécialisée (placement, suivi psychologique).

Les séquelles de la maltraitance sont profondes et durables, affectant les victimes sur les plans physique, psychologique et comportemental.

Néanmoins, les témoignages de résilience, illustrés par des parcours de reconstruction personnelle et la recréation de liens affectifs, soulignent que la guérison, bien que longue et ardue, reste possible grâce à un soutien adéquat et continu.

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1. Les Multiples Visages de la Maltraitance

La maltraitance infantile se manifeste sous diverses formes, souvent cumulatives, allant des sévices physiques aux abus psychologiques et sexuels.

Violences Physiques et Sévices Graves

La violence physique est la forme la plus visible de la maltraitance. Les statistiques présentées sont alarmantes : chaque semaine en France, trois enfants meurent des suites de mauvais traitements.

• Le cas de Gaël : Adolescent de 15 ans, il a été victime de violences extrêmes de la part de son père pendant 21 mois, à l'âge de 8 ans. Ses témoignages décrivent un calvaire :

◦ Brûlures de cigarettes.  

◦ Coups portés avec divers objets : manche à balai, fourchette à poulet, bouteille de verre, assiette, pare-chocs de voiture.  

◦ Tentative de noyade dans la baignoire.  

◦ Humiliations extrêmes, comme être forcé à manger des excréments de chien.

Son père a été condamné à 14 ans de prison ferme.

Gaël utilise aujourd'hui la boxe à haut niveau pour "dégager la haine" et se reconstruire.

• Le cas de Dylan : Enfant de 4 ans décédé en 2003, son corps présentait d'innombrables traces de coups, de morsures et de brûlures de cigarettes, infligées par son beau-père.

Il était devenu son "souffre-douleur" depuis plusieurs mois.

Le Syndrome du Bébé Secoué

Une forme de violence spécifique aux nourrissons est mise en évidence : le syndrome du bébé secoué.

• Mécanisme : Le Dr Philippe Meyer explique qu'il ne s'agit pas d'un simple jeu, mais de "mouvements très répétitifs" et "extrêmement violents".

La tête du bébé, très lourde et mal soutenue par les muscles du cou, subit des accélérations et décélérations qui provoquent des hémorragies cérébrales (hématome sous-dural).

• Prévalence : L'hôpital Necker reçoit plus de 50 bébés par an présentant ces symptômes.

• Conséquences : Les séquelles peuvent être irréversibles, et un bébé secoué sur dix en meurt.

• Cas étudiés :

◦ Louis (6 jours) : Arrivé pour un hématome sous-dural, son cas est d'autant plus suspect que son frère est décédé cinq ans plus tôt dans des circonstances similaires, conduisant les médecins à faire un signalement au procureur.  

◦ Willy (3,5 mois) : Admis pour le même symptôme, son père avoue lui avoir porté un coup lors d'une dispute.

Il reconnaît son geste : "J'ai craqué [...] j'ai fait ces gestes là j'ai regretté".

Abus Sexuels

Les abus sexuels, souvent perpétrés par des proches, sont une autre facette de la maltraitance.

• Le cas d'Elena (7 ans) : La fillette se plaint d'avoir été touchée par Yvon, l'ami de sa grand-mère.

L'enquête de la Brigade des Mineurs révèle que l'agresseur présumé a déjà des antécédents pour "agression sexuelle sur mineur" en 1998. Confronté, il avoue les faits.

• Le cas d'Estelle : Violée de 2 à 12 ans par son grand-père maternel, elle n'a osé en parler que dix ans plus tard.

Son parcours illustre la difficulté de la révélation et le poids de la culpabilité et de la honte, qui se sont traduits par des conduites à risque (tentatives de suicide, drogue) à l'adolescence.

Négligences Graves et Violences Psychologiques

La maltraitance ne se limite pas aux actes de commission.

• Le placement d'un enfant de 8 ans : La Brigade des Mineurs intervient pour retirer un enfant de sa famille suite à des "graves négligences". L'enfant n'est pas scolarisé et les services sociaux n'ont plus accès à la famille.

• Le cas de Marie : Placée à 15 ans, elle a fui une famille où, au-delà des violences physiques, régnait une "violence psychologique permanente". Elle témoigne : "chaque fois je faisais quelque chose ma mère me disait que ça allait pas tout le temps tout le temps". Cette emprise psychologique l'a conduite à des pensées suicidaires.

2. L'Environnement Familial : Principal Foyer de Danger

Le documentaire souligne de manière récurrente que le danger provient le plus souvent de l'entourage immédiat de l'enfant.

La Responsabilité des Auteurs et le Silence Complice

Les auteurs des violences sont les parents, beaux-parents ou des proches. Le silence d'un des parents peut être assimilé à une forme de complicité.

• Le cas d'Adeline, mère de Dylan : Elle est jugée pour ne pas avoir dénoncé les violences infligées par son compagnon à son fils.

Elle a retiré Dylan de l'école pour cacher ses blessures. Son procès en appel aboutit à une peine alourdie à 20 ans de réclusion criminelle.

Pour l'avocat du père de Dylan, son comportement n'était pas un simple silence mais une "dissimulation" active des faits, court-circuitant toute aide possible.

L'Aveuglement et la Culpabilité de l'Entourage

L'entourage élargi peine souvent à percevoir ou à admettre la réalité de la maltraitance, ce qui engendre une profonde culpabilité a posteriori.

• L'entourage de Gaël : La mère de Gaël, Carole, a lutté seule pendant deux ans pour récupérer son fils, séquestré par son ex-mari.

Les grands-parents expriment leur regret : "Carole disait toujours mon enfant est en danger et nous autour d'elle, on le croyait pas [...] on ne peut pas imaginer qu'on s'est rendu compte de rien." Ils avouent même avoir pensé qu'elle "amplifiait la chose".

• L'indifférence du voisinage : Gaël raconte avoir dormi en slip sur le toit du garage, visible par des centaines de personnes, y compris les parents et enfants de l'école voisine. "Personne a jugé bon de signaler qu'il y avait un souci, c'est inadmissible."

3. La Chaîne d'Intervention : Du Signalement à la Protection

Le processus de prise en charge d'un enfant en danger implique une succession d'acteurs institutionnels.

| Étape | Acteurs Clés | Actions et Observations | | --- | --- | --- | | L'Alerte | Ligne 119, entourage, écoles, médecins | Le service du 119 reçoit plus de 4000 appels par jour. L'alerte est le point de départ crucial qui déclenche l'intervention. | | Le Diagnostic Médical | Médecins hospitaliers (pédiatres, réanimateurs) | Ils sont en première ligne pour détecter les signes physiques (hématomes, fractures). Leur rôle est de soigner mais aussi de signaler les suspicions aux autorités judiciaires, comme dans le cas de Louis. | | L'Enquête Policière | Brigade de Protection des Mineurs | Les policiers mènent des auditions et des interrogatoires pour établir les faits. Leur travail consiste à démêler le vrai du faux face aux dénégations initiales des parents (cas du bébé secoué) ou à obtenir les aveux (cas d'Elena). | | La Réponse Judiciaire | Procureur de la République, Juge des enfants | Le procureur décide des suites à donner (mise en examen, contrôle judiciaire, procès). Le juge des enfants prend les mesures de protection nécessaires (enquête sociale, placement) et évalue la sécurité de l'enfant dans son milieu familial (cas d'Elena et de Marie). | | Le Placement et le Soin | Foyers, pouponnières, éducateurs spécialisés, pédopsychiatres | Lorsque le danger est avéré, les enfants sont retirés de leur famille et placés dans des structures spécialisées. Le placement est souvent un traumatisme, comme le montre l'intervention forcée pour l'enfant de 8 ans. Le soin vise à "réparer" les traumatismes (cas de Roxane et Charlotte à la pouponnière). |

4. Les Séquelles et le Chemin de la Reconstruction

Les conséquences de la maltraitance sont profondes et nécessitent un travail de reconstruction de longue haleine.

Traumatismes Physiques et Psychologiques

• Séquelles physiques : Gaël conserve de multiples cicatrices de ses blessures.

• Séquelles psychologiques : Me Brun Meyrin, l'avocate de Gaël, souligne : "Il a surtout des séquelles morales dont on se demande bien comment elles pourraient ne pas avoir de conséquences dans son futur."

La psychologue Martine Nisse explique que la communication paradoxale dans les familles maltraitantes ("c'est pour ton bien que je te frappe") rend les enfants "difficiles à comprendre".

• Comportements post-traumatiques : Les enfants placés en pouponnière manifestent des troubles du comportement :

Roxane, exposée à la violence, développe de l'agressivité et des difficultés relationnelles ; Charlotte, bébé secoué, a appris à "éviter la relation" en se protégeant du contact physique.

La Thérapie comme Voie de Guérison

Le suivi psychologique est essentiel pour surmonter le traumatisme.

• Le cas d'Estelle : Après quatre ans de thérapie, elle a pu mettre des mots sur l'inceste subi et déconstruire le sentiment de culpabilité. "Là j'ai compris vraiment que j'y étais pour rien [...] la honte elle reste mais elle s'estompe."

• L'importance de la parole : L'éducateur de Marie souligne que "le fait qu'il y ait une intervention du commissariat [...] n'a pas réglé les problèmes". Il a fallu deux ans et demi pour qu'elle arrive progressivement à "prendre en main sa vie".

La Résilience et la Reconstruction des Liens

Malgré la gravité des faits, des parcours de résilience sont possibles.

• Gaël : La boxe lui sert d'exutoire et il retisse un lien fort avec sa mère, Carole. Il parvient à formuler : "Grâce à ma mère, je suis là."

• Marie : Bien qu'inquiète de sa majorité, elle demande à la juge de continuer à la protéger, montrant sa volonté de se construire un avenir stable.

• Cindy, mère de Roxane : En désintoxication et séparée de son conjoint violent, elle s'engage dans un processus pour recréer un lien avec ses enfants et espère pouvoir un jour les récupérer.

5. Citations Clés

• Gaël, victime de violences paternelles : "C'est pour pouvoir me défendre, c'est pour pouvoir dégager la haine que j'ai sur lui."

• Père de Willy, auteur de secouement : "J'ai craqué [...] je pardonne pas parce qu'on fait pas ça à un bébé mais je sais que ça peut arriver à n'importe qui."

• Grand-père de Gaël, sur sa culpabilité : "Carole disait toujours mon enfant est en danger et nous autour d'elle, on le croyait pas [...] je m'imaginais jamais ce qui se passe."

• Maître Bejo, avocat du père de Dylan : "On n'est pas dans le silence, on est dans un comportement actif de dissimulation des faits et c'est ce comportement actif qui a court-circuité toutes les velléités d'intervention."

• Dr Renier, pionnier sur le syndrome du bébé secoué : "Ce qui fait la différence entre un bien-traitant pour un bébé et un non bien-traitant [...] c'est la maîtrise et la maîtrise elle est indispensable en toutes circonstances."

• Françoise Achard, médecin scolaire, aux enseignants : "On sait que tout le monde peut être maltraitant, c'est-à-dire que ces parents qui avaient l'air bien sympathiques, et ben ça veut pas dire pour autant qu'ils soient pas maltraitants dans l'intimité de leur maison."

• Martine Nisse, psychologue : "Je crois que les principaux sévices c'est la famille, c'est le principal danger pour l'enfant."

• Carole, mère de Gaël : "On essaie de récupérer mais on récupérera jamais ces années, c'est des années qui vont nous manquer toujours."

Note d'information : La Stratégie d'Expansion du Groupe Emeis (ex-Orpea) dans le Secteur de la Psychiatrie en France

Synthèse Exécutive

Cette note d'information analyse la stratégie d'expansion du groupe privé lucratif Emeis (anciennement Orpea) dans le secteur de la psychiatrie en France.

Elle s'appuie sur une enquête journalistique qui met en lumière comment le groupe, marqué par le scandale de ses EHPAD, capitalise sur la crise profonde de la psychiatrie publique pour s'implanter sur ce marché jugé très rentable.

L'analyse révèle une situation de crise systémique dans le secteur public : un sous-financement chronique, un manque criant de personnel (seulement 600 pédopsychiatres en France), des infrastructures vétustes et une explosion de la demande de soins, notamment chez les jeunes depuis la crise du Covid (+77 % d'épisodes dépressifs chez les 18-24 ans).

Dans ce contexte, Emeis déploie une stratégie agressive pour s'imposer, illustrée par un projet de clinique de 80 lits près de Strasbourg.

Cette implantation, menée via sa filiale Clinea, s'est initialement appuyée sur une alliance "étonnante" avec un concurrent, Clinipsy.

L'enquête suggère que cette alliance aurait pu servir de "cheval de Troie" pour Emeis, lui permettant d'obtenir des autorisations administratives que le groupe, sous le nom d'Orpea, s'était vu refuser à plusieurs reprises depuis 2007.

Les principales préoccupations soulevées sont le risque d'affaiblissement de l'hôpital public par le débauchage de son personnel, une complémentarité illusoire où le privé se concentrerait sur les cas les plus rentables en laissant les plus complexes au public, et un modèle économique basé sur la rentabilité qui pourrait se faire au détriment de la qualité des soins par la réduction des effectifs.

Enfin, le document souligne l'opacité de l'Agence Régionale de Santé (ARS) Grand Est, qui a refusé de communiquer des documents essentiels sur ce projet malgré les importants financements publics engagés.

1. Le Contexte : Une Psychiatrie Publique en Crise Profonde

La psychiatrie en France est décrite comme étant "malade" et "abandonnée par les pouvoirs publics".

Ce secteur est devenu le "parent pauvre de la santé", confronté à un manque critique de moyens alors que les besoins de soins explosent.

• Explosion de la demande : La crise du Covid et les confinements ont provoqué une forte augmentation des pathologies mentales.

◦ +77 % d'épisodes dépressifs chez les 18-24 ans.    ◦ +133 % d'hospitalisations pour tentative de suicide ou automutilation.

• Manque de moyens structurel : Le secteur public souffre d'un sous-investissement chronique.

◦ Une politique ambulatoire non financée : Depuis les années 1980, une politique de fermeture de lits a été menée au profit de soins ambulatoires (hors de l'hôpital).

Cependant, les moyens financiers n'ont pas suivi pour développer ces structures alternatives comme les Centres Médico-Psychologiques (CMP).  

◦ Pénurie de personnel : La France compte environ 600 pédopsychiatres, laissant des départements entiers sans spécialiste.  

◦ Diminution des capacités : L'hôpital public a perdu près de 7 000 places de prise en charge psychiatrique à temps complet en 15 ans.

• Vétusté des infrastructures : L'état des bâtiments publics est alarmant.

À Strasbourg, le secteur de la pédopsychiatrie des Hôpitaux Universitaires est logé dans des "bâtiments complètement vétustes" et des "préfabriqués".

L'Inspection générale des affaires sociales (Igas) a signalé des risques d'incendie et demandé un déménagement en urgence, qui n'a été annoncé que 15 ans plus tard.

"On a alerté qu'on allait droit dans le mur et le mur aujourd'hui on se le prend en pleine face." - Un soignant, cité dans le documentaire de Laurence Deur.

2. L'Émergence d'un Nouvel Eldorado : Le Secteur Privé Lucratif

La défaillance du système public crée une opportunité majeure pour les groupes privés à but lucratif, qui considèrent la psychiatrie comme un "marché très rentable".

• Un secteur profitable : Selon un rapport récent du Sénat, la psychiatrie est l'un des secteurs de la santé les plus rentables, avec des marges estimées entre 5 % et 8 %.

L'investissement principal étant l'humain, la réduction du personnel est le principal levier pour augmenter les profits.

• Une croissance rapide : La part du secteur privé lucratif dans l'offre de soins psychiatriques a considérablement augmenté :

◦ 1975 : 11 % des lits.    ◦ Aujourd'hui : Plus de 30 % des lits.

• Un parallèle avec les EHPAD : La situation actuelle en psychiatrie est comparée à la privatisation du secteur des EHPAD dans les années 1980.

Face à des établissements publics vieillissants et coûteux à rénover, l'État avait ouvert la porte au privé qui promettait de "faire moins cher, plus vite".

• Le rôle des ARS : Les Agences Régionales de Santé, autrefois réticentes à ouvrir la psychiatrie au privé, sont aujourd'hui plus enclines à le faire.

Face à l'incapacité du public à répondre à la demande immense, elles autorisent l'ouverture de cliniques et d'hôpitaux de jour privés.

3. Étude de Cas : La Stratégie d'Implantation d'Emeis à Strasbourg

L'enquête se concentre sur un projet de clinique psychiatrique privée de 80 lits à Schiltigheim, près de Strasbourg, porté par le groupe Emeis (ex-Orpea), rebaptisé pour faire oublier le scandale révélé par le livre Les Fossoyeurs. Ce projet est jugé "démesuré" et "anachronique" par les acteurs locaux.

Une Alliance Stratégique Inédite

Le projet est né d'une alliance "étonnante" entre deux concurrents :

1. Clinea : La filiale sanitaire d'Emeis/Orpea.

2. Clinipsy : Un acteur plus petit, déjà connu pour une enquête du Parquet National Financier (PNF) concernant des autorisations obtenues en région Rhône-Alpes par d'anciens fonctionnaires de l'ARS locale, ensuite embauchés par des filiales du groupe.

Cette collaboration entre concurrents directs est jugée inhabituelle, comparable à "si Intermarché et Leclerc montaient un supermarché ensemble".

L'Hypothèse du "Cheval de Troie"

L'enquête soulève l'hypothèse que cette alliance aurait servi de stratégie à Emeis pour contourner des obstacles réglementaires.

• Dissimulation : Emeis se serait "dissimulé un petit peu" derrière le nom de Clinipsy, un groupe plus petit avec une "moins mauvaise image" auprès des ARS, pour obtenir plus facilement les autorisations.

• Historique des refus : Des documents montrent qu'Orpea tentait d'ouvrir une clinique psychiatrique dans la région depuis au moins 2007 et avait essuyé au moins deux refus de la part de l'agence régionale (alors ARH).

Depuis, Clinipsy s'est désengagé du projet de clinique de 80 lits pour se concentrer sur des hôpitaux de jour, des structures moins coûteuses et "extrêmement rentables", laissant le champ libre à Emeis pour le projet principal.

"La question [...] se pose de savoir si Clinipsy a été un petit peu le cheval de Troie d'Orpea dans cette affaire." - Laurence Deur, journaliste.

4. Les Risques Systémiques de la Privatisation

L'arrivée massive d'acteurs privés lucratifs comme Emeis dans la psychiatrie fait peser plusieurs risques majeurs sur l'équilibre global du système de santé mentale.

Le "Pillage" des Ressources Humaines du Public

La principale inquiétude est que les nouvelles cliniques privées, en offrant de meilleures conditions de travail ou de rémunération, ne débauchent le personnel médical et soignant déjà en sous-effectif dans le secteur public.

• Un exemple concret : Un courrier de 2022 révèle qu'une clinique privée près de Nancy a débauché cinq médecins de l'hôpital public local, fragilisant ce dernier.

• L'inquiétude de la Mairie de Strasbourg : La maire, Jeanne Barségan, craint que le projet de 80 lits n'aggrave la pénurie de psychiatres et ne "vide" l'hôpital public de ses forces.

Une Complémentarité Illusoire : Le "Triage" des Patients

L'offre privée est souvent présentée comme "complémentaire" du public. Cependant, l'analyse montre qu'elle ne remplit pas les mêmes missions.

• Évitement des cas complexes : Le privé évite généralement les missions les plus lourdes et les moins rentables, comme l'hospitalisation sous contrainte, qui nécessite plus de personnel et de temps.

• Gestion des urgences "à la carte" : Dans le projet d'Emeis, la prise en charge des urgences se ferait "de gré à gré", sans obligation contraignante. Le médecin du privé peut accepter ou refuser un patient envoyé par le public.

• La conclusion : "Tout ce qui est complexe reste dans l'hôpital public", tandis que le privé se positionne sur des missions "plus faciles à assurer".

Le Modèle Économique : Profit vs. Qualité des Soins

Emeis est une entreprise cotée en bourse qui doit générer du profit pour ses actionnaires.

• Le levier du personnel : En psychiatrie, où "l'investissement, c'est l'humain", la principale méthode pour augmenter la rentabilité est de réduire les effectifs.

• Conflits sociaux : Plusieurs conflits sociaux ont éclaté dans des cliniques psychiatriques d'Emeis (Thionville, Nord, Isère) où le personnel dénonçait un manque d'effectifs et une réorganisation du travail impactant la qualité des soins.

Une grève de trois semaines a eu lieu à Seyssins, un événement "extrêmement rare dans le privé".

"Une entreprise est là pour faire du profit alors que l'hôpital public on lui demande pas d'être profitable, on lui demande d'être à l'équilibre." - Laurence Deur, journaliste.

5. Le Rôle et l'Opacité des Autorités de Régulation

L'enquête met en cause le manque de transparence de l'Agence Régionale de Santé (ARS) Grand Est.

• Rétention d'information : La journaliste a été "baladée pendant un mois et demi" sans obtenir de réponse ni les documents demandés concernant le projet de clinique Emeis.

L'ARS a fini par envoyer un document public générique qui ne correspondait pas à la demande.

• Recours à la CADA : Il a fallu saisir la Commission d'Accès aux Documents Administratifs (CADA) pour obtenir une partie des informations.

• Enjeux financiers publics : Cette opacité est jugée problématique car le projet engage d'importants fonds publics.

Les autorisations délivrées "valent des millions d'euros" et le groupe peut prétendre à une "dotation d'amorçage" de l'État pour financer son démarrage.

Cette situation soulève des questions sur le contrôle et la régulation de l'expansion du secteur privé lucratif, financée en partie par de l'argent public, dans un domaine aussi sensible que la santé mentale.

Synthèse du rapport : Protection de l’enfance et maltraitances — État des lieux 2025

Résumé Exécutif

Ce document de synthèse présente les principales conclusions du rapport "Protection de l’enfance et maltraitances — État des lieux 2025", publié par l’Observatoire national de la protection de l’enfance (ONPE).

L'analyse des données, arrêtées au 31 décembre 2023, révèle plusieurs tendances structurelles profondes qui redéfinissent le paysage de la protection de l'enfance en France.

Au 31 décembre 2023, 364 200 prestations et mesures étaient en cours pour les mineurs et 33 400 pour les jeunes majeurs, des chiffres en augmentation significative sur la dernière décennie.

Les dynamiques clés sont les suivantes :

1. Une croissance globale et continue : Le nombre total d'interventions pour les mineurs a augmenté de 22 % entre 2013 et 2023.

Le taux de prise en charge pour 1 000 mineurs a quant à lui progressé de 27 % sur la même période, passant de 20,3 ‰ à 25,8 ‰, une hausse accentuée par la baisse démographique de cette tranche d'âge.

2. Un basculement structurel vers l'accueil : Pour la première fois depuis le début du suivi, l'accueil (placement hors du domicile familial) est devenu majoritaire, représentant 52,2 % des interventions pour les mineurs.

Cette inversion de tendance, amorcée en 2018, marque un changement profond par rapport au suivi en milieu ouvert (à domicile).

3. La prédominance de l'hébergement en établissement : Une seconde inversion de tendance est observée dans les modalités d'accueil.

L'hébergement en établissement (41 %) dépasse désormais l'accueil familial traditionnel chez les assistants familiaux (36 %), qui voit sa part diminuer de manière continue.

4. Une judiciarisation accrue des mesures : La part des interventions décidées par un juge ne cesse de croître, atteignant 82,4 % de l'ensemble des mesures pour mineurs en 2023, contre 78,6 % en 2013.

Cette tendance est particulièrement marquée pour les mesures d'accueil (92,1 %).

5. L'impact majeur des mineurs non accompagnés (MNA) : La forte augmentation du nombre de MNA pris en charge (46 200 mineurs et jeunes majeurs fin 2023) influence profondément les statistiques globales, notamment la hausse des accueils, la prédominance masculine chez les adolescents et l'augmentation des saisines judiciaires.

6. Des disparités territoriales persistantes et croissantes : Des écarts considérables subsistent entre les départements, que ce soit pour les taux de prise en charge globaux, les taux de judiciarisation ou les modalités d'intervention. Ces disparités tendent à se creuser au fil du temps.

7. Une attention renforcée aux jeunes majeurs : Bien qu'en légère baisse depuis un pic en 2021, le soutien aux 18-20 ans a fortement augmenté sur dix ans (+53 %).

Le taux de poursuite de l'accompagnement après la majorité a atteint 52 % en 2023, retrouvant son niveau d'avant 2013, signe d'une politique active contre les "sorties sèches".

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1. Vue d'ensemble de la prise en charge en protection de l'enfance

1.1. Augmentation continue des interventions

Le nombre d'interventions en protection de l'enfance pour les mineurs (0-17 ans) a connu une croissance soutenue sur la dernière décennie.

• Nombre total d'interventions : Au 31 décembre 2023, 364 200 prestations administratives et mesures judiciaires étaient en cours, soit une augmentation de 22 % par rapport à 2013 (297 500).

• Taux de prise en charge : Le taux d'intervention pour 1 000 mineurs est passé de 20,3 ‰ en 2013 à 25,8 ‰ en 2023, une augmentation de 27 %.

Cette hausse est plus rapide que celle des effectifs en raison d'une diminution de 4 % de la population des moins de 18 ans sur la même période.

• Estimation du nombre de mineurs : En croisant diverses sources (DREES, Olinpe), le nombre de mineurs uniques suivis est estimé à environ 351 500 au 31 décembre 2023.

| Année | Nombre de prestations et mesures | Taux pour 1 000 mineurs | | --- | --- | --- | | 2013 | 297 500 | 20 ‰ | | 2017 | 342 900 | 23 ‰ | | 2020 | 338 600 | 24 ‰ | | 2022 | 347 100 | 24 ‰ | | 2023 | 364 200 | 26 ‰ |

Source : DREES, DPJJ, Insee, calculs ONPE

La croissance a été particulièrement marquée entre 2022 et 2023, avec une hausse de 5 %.

Cette dynamique fait écho à l'augmentation des saisines des juges des enfants observée après la crise sanitaire.

1.2. Disparités territoriales croissantes

Les écarts de prise en charge entre les départements non seulement persistent mais se sont accentués entre 2013 et 2023.

• Écart des taux : Au 31 décembre 2023, le taux de prise en charge des mineurs variait de 13,5 ‰ (Yvelines) à 48,1 ‰ (Nièvre). En 2013, l'écart était moins prononcé, allant de 10,9 ‰ à 37 ‰.

• Tendances géographiques :

◦ Les taux les plus faibles se concentrent majoritairement en Île-de-France et en Auvergne-Rhône-Alpes.  

◦ Les taux les plus élevés sont observés dans des territoires souvent moins densément peuplés.

• Évolutions hétérogènes : Entre 2013 et 2023, le taux de prise en charge a augmenté dans 98 départements, mais avec des variations extrêmes, allant de -7 % (Hauts-de-Seine, Loiret) à +101 % (Lozère).

2. La dynamique des types d'intervention

2.1. Une judiciarisation accrue

La prise en charge des mineurs est majoritairement décidée par l'autorité judiciaire, et cette tendance se renforce.

• Part des mesures judiciaires : Au 31 décembre 2023, 82,4 % des 364 200 interventions résultaient d'une décision judiciaire, contre 78,6 % en 2013.

• Répartition par type d'intervention :

◦ Accueil : 92,1 % des mesures sont judiciaires.    ◦ Milieu ouvert : 71,7 % des mesures sont judiciaires.

• Disparités départementales : Le "taux de judiciarisation" varie fortement, de 66,9 % (Morbihan) à 94,9 % (Seine-Saint-Denis).

Les départements des Hauts-de-France et du Grand-Est affichent des taux particulièrement élevés.

2.2. Le basculement vers l'accueil au détriment du milieu ouvert

Un changement structurel majeur s'est opéré : le nombre de placements d'enfants (accueil) dépasse désormais le nombre d'interventions à domicile (milieu ouvert).

• Inversion de la tendance : En 2023, les mesures d'accueil s'élèvent à près de 190 300 (52,2 % du total), tandis que les mesures en milieu ouvert sont de 174 000 (47,8 %). Le point de bascule s'est produit en 2018.

• Croissance différentielle (2013-2023) :

◦ Le taux de mineurs accueillis a augmenté de 40 % (passant de 9,7 ‰ à 13,5 ‰).    ◦ Le taux de mineurs suivis en milieu ouvert a augmenté de 16 % (passant de 10,7 ‰ à 12,3 ‰).

• Facteurs explicatifs : Cette évolution est notamment liée à la forte augmentation des accueils de mineurs non accompagnés (MNA) et au développement de nouvelles mesures comme le "placement à domicile", comptabilisées comme de l'accueil.

3. L'accueil des mineurs : modalités et profils

3.1. Évolution des modes d'hébergement : l'établissement devance la famille d'accueil

Pour la deuxième année consécutive, l'accueil en établissement est la modalité la plus fréquente, devant l'accueil familial traditionnel.

| Mode d'hébergement | Part en 2013 | Part en 2023 | Évolution en effectifs (2013-2023) | | --- | --- | --- | --- | | Établissement | 38 % | 41 % | +50 % | | Famille d'accueil | 52 % | 36 % | \-4 % (depuis 2019) | | Hébergement autonome | 4 % | 6 % | +143 % | | Autres modes d'hébergement | 6 % | 17 % | +321 % |

Source : DREES, calculs ONPE. Champ : Mineurs et jeunes majeurs confiés à l'ASE.

• La catégorie "Autres modes d'hébergement" inclut les placements chez un tiers digne de confiance, en internat, l'accueil durable et bénévole, etc. Son explosion est un facteur clé de la restructuration du secteur.

• Cette tendance coïncide avec une baisse de 11 % du nombre d'assistants familiaux employés par les départements entre 2016 et 2023.

3.2. Le placement direct : le recours croissant au "tiers digne de confiance"

Le placement direct, décidé par un juge sans passer par une mesure de confiement à l'ASE, évolue également.

• Au 31 décembre 2023, 17 100 enfants bénéficiaient d'un placement direct.

• La part des placements chez un "tiers digne de confiance" a fortement augmenté, passant de 69 % en 2013 à 86 % en 2023.

• Cette évolution est directement liée à la loi du 7 février 2022, qui systématise la recherche d'un membre de la famille ou d'un proche pour accueillir l'enfant.

3.3. Profils démographiques des enfants accueillis

• Prédominance masculine : Les garçons représentent 59 % des mineurs accueillis (hors placement direct).

Ce déséquilibre s'accentue avec l'âge, atteignant 69 % chez les 16-17 ans, principalement en raison de la population de MNA (à plus de 90 % masculine).

• Répartition par âge : Entre 2015 et 2023, la croissance des accueils a été la plus forte aux âges extrêmes : +38 % pour les moins de 6 ans et +50 % pour les 16-17 ans.

• Profil en placement direct : La population en placement direct est très différente, avec un équilibre quasi parfait entre les sexes (50,2 % de filles) et une part plus importante de 11-15 ans (39 % contre 34 %).

4. La situation spécifique des jeunes majeurs (18-20 ans)

4.1. Tendances générales et disparités

L'accompagnement des jeunes majeurs a connu une croissance massive, bien qu'en léger recul depuis 2021.

• Effectifs : 33 400 jeunes majeurs étaient pris en charge fin 2023, soit une augmentation de 53 % depuis 2013. Le pic a été atteint en 2021 avec 35 100 jeunes.

• Nature de l'intervention : La prise en charge est quasi exclusivement administrative (99,8 %) et consiste très majoritairement en un accueil (92,2 %).

• Taux de prise en charge : Le taux national est de 13,6 ‰, mais les disparités départementales sont extrêmes, allant de 1,6 ‰ (Hautes-Alpes) à 28,5 ‰ (Allier).

4.2. La poursuite de l'accompagnement après 18 ans

Un nouvel indicateur, le "taux de poursuite en Accueil Provisoire Jeunes Majeurs (APJM)", mesure la probabilité pour un jeune confié à 17 ans de continuer à être hébergé après sa majorité.

• Après une chute à un niveau plancher de 37 % en 2018, ce taux a connu une remontée spectaculaire pour atteindre 52 % en 2023.

• Cette hausse s'explique par les mesures liées à la crise sanitaire puis par la Stratégie nationale de prévention et de protection de l’enfance, qui a fait de la lutte contre les "sorties sèches" un objectif prioritaire.

5. Facteurs d'influence et dynamiques transversales

5.1. L'impact des mineurs non accompagnés (MNA)

Les MNA constituent une part croissante et influente de la population protégée.

• Effectifs : Au 31 décembre 2023, 46 200 mineurs et jeunes majeurs MNA étaient pris en charge, une hausse de 17 % en un an.

• Répartition : 65 % sont mineurs et 35 % sont de jeunes majeurs. Cette proportion de jeunes majeurs a diminué depuis son pic à 50 % en 2021.

• Influence statistique : Les MNA contribuent significativement à la hausse du nombre d'accueils, à la surreprésentation des garçons de 16-17 ans et à l'augmentation des saisines judiciaires.

5.2. L'augmentation des saisines des juges des enfants

L'activité judiciaire en assistance éducative est en forte croissance.

• En 2023, les juges des enfants ont été saisis pour 124 117 nouveaux mineurs, un chiffre en hausse de 10 % par rapport à 2022 et de 50 % depuis 2013.

• Le rapport note une corrélation entre la courbe des saisines judiciaires et celle des évaluations de minorité pour les MNA, suggérant un lien de cause à effet partiel.

Analyse de l'Avis du CESE sur les Temps de Vie de l'Enfant

Résumé Exécutif

Cet avis du Conseil économique, social et environnemental (CESE), intitulé « Satisfaire les besoins fondamentaux des enfants et garantir leurs droits », dresse un constat critique de la situation des enfants en France, dont les temps de vie sont davantage structurés par les contraintes des adultes que par leurs propres besoins fondamentaux.

Fruit d'une saisine gouvernementale faisant suite à une Convention citoyenne, le rapport souligne un décalage majeur entre les droits constitutionnels et internationaux de l'enfant et leur application effective, particulièrement pour les plus vulnérables.

Les principales conclusions révèlent des inégalités sociales, territoriales et économiques profondes qui entravent le développement, la santé et le bien-être des enfants.

L'avis pointe du doigt des rythmes scolaires inadaptés, une sédentarité croissante, un manque de sommeil chronique, une surexposition aux écrans, et une déconnexion préoccupante de la nature.

La pression sur les familles, notamment monoparentales, et le manque de coordination entre les acteurs éducatifs aggravent ces constats.

Pour y remédier, le CESE formule 19 préconisations interdépendantes visant une transformation systémique. Celles-ci incluent des mesures politiques fortes comme l'instauration d'une « clause impact enfance » dans chaque projet de loi, une réforme ambitieuse des rythmes scolaires sur la base des besoins physiologiques, et la création d'un Service Public de la Continuité Éducative (SPCE) pour assurer une meilleure coordination des acteurs.

L'avis appelle également à renforcer le soutien à la parentalité, à garantir l'accès de tous les enfants aux loisirs, à la culture et aux activités de plein air, et à allouer des financements publics pérennes pour faire de l'enfance un véritable investissement d'avenir.

Introduction et Contexte

En réponse à une saisine du Premier ministre de mai 2025, le CESE a élaboré cet avis suite aux travaux d'une Convention citoyenne dédiée aux temps de vie des enfants. Cent trente-trois citoyens et un panel de vingt enfants et adolescents ont été invités à répondre à la question :

« Comment mieux structurer les différents temps de la vie quotidienne des enfants afin qu’ils soient plus favorables à leurs apprentissages, à leur développement et à leur santé ? ».

Le constat principal de la Convention citoyenne, repris par le CESE, est que les enfants subissent les rythmes effrénés d'une société qui construit leurs temps autour des contraintes des adultes plutôt qu'en réponse à leurs besoins biologiques et de développement.

Le rapport du CESE, s'appuyant sur les 20 propositions citoyennes, formule 19 préconisations qui constituent une position commune de la société civile organisée.

Cet avis s'inscrit dans la continuité de travaux antérieurs du CESE sur l'éducation, la protection de l'enfance et la santé mentale, et vise à proposer des réponses globales et articulées.

Partie 1 : Droits et Besoins Fondamentaux de l'Enfant : Un Constat Alarmant

A. L'Écart entre Droits Reconnus et Réalité Vécue

La France a consacré les droits de l'enfant dans sa Constitution et a ratifié la Convention Internationale des Droits de l'Enfant (CIDE) en 1990, s'engageant sur quatre principes fondamentaux : le droit à la vie, l'intérêt supérieur de l'enfant, la non-discrimination et le respect de son opinion.

Cependant, l'avis du CESE met en lumière une ineffectivité préoccupante de ces droits pour une part significative des enfants.

• Pauvreté et Précarité : En 2023, 21,9 % des enfants de moins de 18 ans vivent sous le seuil de pauvreté monétaire.

À la rentrée 2025, au moins 2 159 enfants se sont retrouvés sans solution d'hébergement.

Ces réalités percutent violemment la capacité de la société à répondre à leurs besoins fondamentaux.

• Critiques Internationales : Le Comité des droits de l'enfant de l'ONU a enjoint la France en 2023 à prendre des mesures urgentes concernant la violence, la protection de l'enfance, la détention d'enfants étrangers, la pauvreté et l'inclusion des enfants en situation de handicap.

• L'« Infantisme » : Le rapport dénonce la persistance de l'« infantisme », un concept désignant les préjugés et la discrimination fondée sur l'âge, qui considère les enfants comme des êtres inférieurs et moins dignes de respect.

Cette culture conduit à ignorer leur parole et leur capacité à être des acteurs sociaux. Pour le combattre, le CESE réaffirme la nécessité d'un débat de société et la création d'un Code de l'enfance.

• Clause « Impact Enfance » : S'inspirant de la « clause impact jeunesse », le CESE préconise (Préconisation #1) d'intégrer un volet enfance dans chaque étude d'impact des projets de loi afin de s'assurer que toute politique publique soit fondée sur le respect des droits de l'enfant.

B. Le Rôle de la Famille et les Obstacles Socio-économiques

La famille est le premier lieu de développement de l'enfant, mais elle fait face à de nombreux obstacles.

• Soutien à la Parentalité : Face à la diversité des modèles familiaux (nucléaire, monoparentale, recomposée...), un soutien renforcé à la parentalité est jugé nécessaire pour aider les parents à répondre aux besoins de leurs enfants (Préconisation #7).

• Inégalités de Genre : Les femmes continuent d'assumer l'essentiel des responsabilités familiales et de la charge mentale, ce qui impacte leur santé et leur carrière.

Le rapport souligne la nécessité d'une répartition équitable des tâches.

• Conciliation Vie Professionnelle/Familiale : Les contraintes professionnelles empiètent sur le temps familial.

Le CESE préconise (Préconisation #2) la transposition complète de la directive européenne sur l'équilibre vie professionnelle-vie personnelle, en créant un droit à des « formules souples de travail » (aménagement du temps, télétravail) négocié dans les branches et la fonction publique.

• Enfants Séparés de leur Famille :

◦ Parents séparés : Il est crucial de soutenir les dispositifs comme les Espaces de rencontre pour préserver la relation parent-enfant tout en prenant en compte le point de vue de l'enfant (Préconisation #3).   

◦ Aide Sociale à l'Enfance (ASE) : L'avis dénonce une crise systémique de la protection de l'enfance, où les droits des enfants confiés, notamment l'accès aux loisirs et à la culture, sont négligés.

Il est préconisé (Préconisation #4) que le Projet Pour l'Enfant (PPE) soit co-construit avec les parents et l'enfant, et qu'il intègre l'ensemble de ses besoins.

Partie 2 : Les Enjeux des Temps et des Espaces de Vie

L'avis analyse en profondeur la manière dont les temps et les espaces de l'enfant sont organisés, révélant de multiples fractures et inadéquations.

A. Les Temps de Vie : Entre Contraintes et Qualité

La vie de l'enfant est rythmée par trois grands temps : familial, scolaire, et les "tiers temps" (périscolaire, extrascolaire).

• Qualité des Temps : Le rapport insiste sur la nécessité d'un équilibre entre temps contraints et temps libre, temps individuel et collectif, activité et repos.

La qualité des interactions avec les adultes et un environnement sécurisant sont déterminants.

Le CESE préconise (Préconisation #6) d'intégrer des temps libres de qualité dans toutes les activités d'apprentissage.

• Le Temps Scolaire : La France se distingue par des journées scolaires longues et un temps d'instruction élevé, sans que cela se traduise par de meilleurs résultats.

Le rythme de la semaine de quatre jours est jugé contraire aux besoins des enfants. Le CESE estime que le statu quo n'est plus tenable et appelle (Préconisation #8) à une évolution des rythmes scolaires :

◦ Premier degré : Réorganiser la journée et la semaine scolaire après concertation.   

◦ Second degré : Adapter les amplitudes horaires aux besoins physiologiques des jeunes (ex: commencer plus tard).   

◦ Calendrier scolaire : Organiser le calendrier hexagonal autour de deux zones de vacances, avec une alternance de 7 semaines de cours et 2 semaines de vacances.

• Les Tiers Temps et le Droit aux Loisirs : Les activités périscolaires et extrascolaires, portées par les associations et les collectivités, sont essentielles mais menacées par le désengagement de l'État et la marchandisation.

L'accès à ces activités, ainsi qu'aux vacances, est fortement marqué par les inégalités sociales.

Un enfant sur deux ne part pas en vacances. Le CESE réaffirme (Préconisation #9) que chaque enfant a droit aux vacances et aux loisirs, et appelle à renforcer le financement des accueils collectifs de mineurs et l'information sur les aides existantes.

B. Les Espaces de Vie : De l'« Enfant d'Intérieur » à la Reconnexion au Dehors

L'environnement physique joue un rôle crucial dans le développement de l'enfant.

• L'« Enfant d'Intérieur » : Le rapport alerte sur le phénomène des « enfants d'intérieur », qui passent de moins en moins de temps à l'extérieur et en contact avec la nature, en raison de la peur du risque, de l'urbanisation centrée sur la voiture et de l'attrait des écrans.

• Repenser l'Aménagement : Il est impératif de repenser l'aménagement des territoires « à hauteur d'enfant », en créant des espaces publics (rues, places) sécurisés, propices au jeu, à la socialisation et aux mobilités douces.

Le CESE préconise (Préconisation #11) d'associer les enfants à l'élaboration des projets d'urbanisme.

• Le Bâti et le Cadre de Vie : Les bâtiments accueillant des enfants (écoles, centres de loisirs) sont souvent inadaptés, notamment face aux enjeux climatiques (vagues de chaleur).

Leur rénovation écologique et leur accessibilité sont des priorités. Toute rénovation doit faire l'objet d'une concertation incluant les enfants et les jeunes (Préconisation #12).

Partie 3 : Leviers d'Action pour la Santé et le Bien-être

L'avis identifie quatre domaines d'action prioritaires pour améliorer la santé physique et mentale des enfants.

• Reconnecter à la Nature : Le contact avec la nature est fondamental pour la santé.

Le CESE appelle à valoriser et accompagner l'éducation au dehors (Préconisation #10) et à garantir que chaque enfant bénéficie d'un accès à des espaces naturels, de sorties régulières et d'au moins un séjour en classe de découverte par cycle scolaire (Préconisation #13).

• Lutter contre le Manque de Sommeil : Le déficit de sommeil touche plus de 30 % des enfants et 70 % des adolescents, avec des conséquences graves sur l'apprentissage et la santé.

Le CESE demande une campagne nationale de sensibilisation (Préconisation #14) et la garantie de temps de repos et de sieste dans toutes les structures, notamment en maternelle (Préconisation #15).

• Favoriser l'Activité Physique : Face à une sédentarité alarmante, il est crucial de faciliter l'accès au sport pour tous. Le CESE préconise (Préconisation #16) une tarification sociale et l'élargissement du dispositif Pass'Sport, récemment restreint.

• Mieux Réguler les Écrans : L'omniprésence des écrans a des effets néfastes documentés (sommeil, sédentarité, exposition à des contenus inappropriés). L'avis souligne la nécessité d'une meilleure régulation et d'un accompagnement à la parentalité numérique.

Partie 4 : Gouvernance, Coordination et Financement

Pour que ces changements soient effectifs, une transformation de la gouvernance des politiques de l'enfance est indispensable.

• Coordination des Acteurs : L'action publique est jugée trop fragmentée. Le CESE préconise (Préconisation #17) de réhabiliter le Projet Éducatif Territorial (PEDT) et d'en faire le volet éducation des Conventions Territoriales Globales (CTG) pour assurer une coordination efficace au niveau local.

• Un Service Public de la Continuité Éducative (SPCE) : Pour garantir une offre éducative cohérente sur tous les temps de l'enfant, l'avis propose la création d'un SPCE (Préconisation #18).

Ce service, confié aux collectivités locales, serait chargé de diagnostiquer les besoins et de planifier les actions en associant tous les acteurs.

• Formation et Financement : La revalorisation des métiers éducatifs et le développement d'une culture commune des droits de l'enfant sont essentiels.

Enfin, le CESE alerte sur l'insuffisance des budgets alloués aux politiques de l'enfance et appelle (Préconisation #19) à un effort budgétaire conséquent et pérenne de l'État et de la Sécurité sociale, considérant ces dépenses comme un investissement fondamental pour l'avenir.

Synthèse des 19 Préconisations du CESE

| Numéro | Thème Principal | Résumé de la Préconisation | | --- | --- | --- | | #1 | Droits de l'enfant | Mettre en œuvre une « clause impact enfance » dans chaque étude d'impact de projet de loi ou de texte réglementaire pour garantir que les politiques publiques respectent les droits de l'enfant. | | #2 | Parentalité & Travail | Créer un droit aux « formules souples de travail » (aménagement du temps, télétravail) pour les parents, par la négociation dans les branches et la fonction publique. | | #3 | Séparation parentale | Développer et soutenir financièrement les Espaces de rencontre pour aider les parents séparés à assumer leurs responsabilités parentales en prenant en compte le point de vue de l'enfant. | | #4 | Protection de l'enfance (ASE) | Rendre le Projet pour l'enfant (PPE) systématiquement co-construit avec les parents et l'enfant, et y intégrer tous les besoins, y compris les loisirs et la culture. Simplifier la gestion des actes usuels. | | #5 | Accès à la culture | Soutenir financièrement et développer tous les dispositifs culturels et artistiques pour les enfants (scolaires, ACM), via des contrats multipartites (État, collectivités, réseau culturel). | | #6 | Qualité des temps | Intégrer des temps libres de qualité dans les activités d'apprentissage, ce qui implique de former les adultes et personnels encadrants. | | #7 | Soutien à la parentalité | Mieux faire connaître, rendre accessibles et valoriser financièrement les lieux et actions d'aide aux parents (maisons des familles, groupes de parole, LAEP, PMI...). | | #8 | Rythmes scolaires | Faire évoluer les rythmes scolaires : réorganiser la journée et la semaine au primaire ; adapter les horaires aux besoins physiologiques au secondaire ; organiser un calendrier national à 2 zones (7 semaines de cours / 2 de vacances). | | #9 | Droit aux vacances et loisirs | Mobiliser les pouvoirs publics pour rendre effectif le droit aux vacances. Renforcer l'information sur les aides et financer davantage les accueils collectifs de mineurs (ACM). | | #10 | Éducation à la nature | Valoriser et accompagner l'éducation au dehors et en lien avec la nature (formation des acteurs, verdissement des espaces, aires éducatives, terrains d'aventure...). | | #11 | Aménagement du territoire | Aménager les territoires « à hauteur d'enfant » dans une démarche participative, en repensant les espaces publics comme lieux de sociabilité, de mixité et de jeu. | | #12 | Bâti et cadre de vie | Rendre obligatoire la concertation avec les enfants et les jeunes pour tout projet d'aménagement ou de rénovation de bâtiments (écoles, centres de loisirs, gymnases...). | | #13 | Lien à la nature | Garantir que chaque enfant bénéficie d'un accès à des espaces naturels, de sorties régulières, et d'au moins un séjour en classe de découverte par cycle de scolarité. | | #14 | Sommeil | Organiser une campagne nationale d'information et de sensibilisation sur le rôle fondamental du sommeil et les facteurs qui lui nuisent. | | #15 | Temps de repos | Prévoir des temps de repos, de calme et de sieste (préservée en maternelle) dans toutes les structures accueillant des enfants, et repenser les locaux pour créer une atmosphère paisible. | | #16 | Activité physique et sportive | Soutenir une tarification sociale pour l'accès au sport. Étendre et revaloriser le Pass'Sport, en y incluant les associations sportives scolaires. | | #17 | Coordination locale | Réhabiliter le Projet Éducatif Territorial (PEDT) et en faire le volet "éducation" des Conventions Territoriales Globales (CTG) pour une coordination globale des acteurs. | | #18 | Gouvernance | Créer un Service Public de la Continuité Éducative (SPCE), confié aux collectivités, pour diagnostiquer les besoins et planifier les actions éducatives sur le territoire. | | #19 | Financement | Assurer un effort budgétaire conséquent et pérenne de l'État et de la Sécurité sociale pour financer les politiques publiques en faveur de l'enfance. |

Synthèse de l'Étude sur la Protection des Mineurs en Ligne

Synthèse Exécutive

Cette étude, menée par l'Arcom en septembre 2025, révèle que les plateformes numériques sont devenues un pilier central et inévitable de la vie des adolescents de 11 à 17 ans, avec des implications majeures en matière d'exposition aux risques et d'efficacité des mesures de protection.

L'accès à ces services est quasi universel, de plus en plus précoce, et se fait souvent en contournant les restrictions d'âge conçues pour protéger les plus jeunes.

Les principaux points à retenir sont les suivants :

• Usage quasi universel et intensif : 99 % des 11-17 ans utilisent au moins une plateforme en ligne, et 83 % fréquentent quotidiennement une très grande plateforme (VLOP).

En moyenne, les adolescents utilisent 3,6 plateformes différentes chaque jour, motivés principalement par le besoin de lien social, de divertissement et d'accès à l'information.

• Contournement systématique des restrictions d'âge : L'âge moyen de la première utilisation des réseaux sociaux est de 12,3 ans, bien en deçà du seuil légal de 13 ans.

Une part significative (62 %) des adolescents reconnaît avoir menti sur son âge lors de l'inscription, principalement pour accéder à des services pour lesquels ils n'avaient pas l'âge requis (65 %).

Cette tendance à une inscription précoce s'accentue chez les plus jeunes générations.

• Faiblesse des mécanismes de vérification : Les systèmes de vérification d'âge des plateformes s'avèrent largement inefficaces.

Seulement 18 % des mineurs déclarent avoir déjà dû prouver leur âge ou avoir vu leur compte bloqué.

Les observations techniques montrent que le contournement des blocages à l'inscription est souvent simple, notamment sur des plateformes majeures comme Instagram, Snapchat et Facebook.

• Encadrement parental ambivalent et contourné : Bien que 94 % des foyers instaurent des règles sur l'usage du numérique, près de la moitié des adolescents (45 %) admettent les contourner régulièrement.

Il existe une perception partagée des risques entre parents et enfants, mais les parents se montrent nettement plus inquiets et moins convaincus des bénéfices des plateformes.

• Perception dichotomique : Les adolescents et leurs parents entretiennent un rapport ambivalent aux plateformes, les considérant à la fois comme des outils d'intégration sociale et de divertissement indispensables, mais aussi comme des sources d'inquiétude et d'exposition à des risques graves.

1. Contexte et Méthodologie de l'Étude

Objectifs de l'Étude

L'étude menée pour l'Arcom vise à dresser un état des lieux complet de la protection des mineurs dans l'univers numérique. Elle s'articule autour de trois axes principaux d'investigation :

1. L'Exposition : Mesurer le degré de conscience des mineurs face aux risques en ligne et leur exposition réelle.

2. La Protection : Analyser les moyens de prévention mis en place par les mineurs et leur entourage, ainsi que leurs réactions post-exposition.

3. Les Attentes : Recueillir les attentes des mineurs, des parents et des professionnels pour une meilleure protection.

L'objectif est de comprendre les compétences que les adolescents mobilisent pour naviguer en ligne, dans un contexte oscillant entre la conscience des dangers et la prise de risques.

Approche Méthodologique

Pour garantir une vision exhaustive, l'étude a été réalisée en quatre volets complémentaires entre novembre 2024 et avril 2025, en partenariat avec Ipsos BVA et OpinionWay.

| Volet | Type d'étude | Période | Participants et Méthodes | | --- | --- | --- | --- | | 1 | Entretiens préparatoires | Nov - Déc 2024 | Entretiens avec des experts, des représentants de plateformes. | | 2 | Étude qualitative | Fév - Mars 2025 | Entretiens avec des experts (associations, psychologue, pédiatre), 16 entretiens individuels et 4 triades avec des mineurs (11-17 ans). | | 3 | Étude sémiologique et observations | Avril 2025 | Analyse des outils et CGU des plateformes ; simulation de parcours utilisateurs avec 8 profils fictifs ; focus sur les thèmes de la maigreur et du masculinisme. | | 4 | Étude quantitative | Avril 2025 | Questionnaire en ligne auprès de 2 000 mineurs (11-17 ans) et de leurs parents. |

Le périmètre de l'étude couvre les réseaux sociaux (Snapchat, TikTok, Facebook, Instagram, etc.), les plateformes de partage de vidéos (YouTube, Twitch, etc.) et les messageries instantanées (WhatsApp, Discord, etc.).

2. L'Usage Incontournable des Plateformes par les Mineurs

Omniprésence et Intensité d'Usage

Les plateformes en ligne sont omniprésentes dans la vie des 11-17 ans. L'étude révèle des chiffres qui témoignent d'une adoption quasi totale et d'un usage quotidien intensif.

• 99 % des 11-17 ans utilisent au moins une plateforme en ligne.

• 83 % utilisent au moins une Très Grande Plateforme en Ligne (VLOP) chaque jour.

• En moyenne, les adolescents utilisent 3,6 plateformes différentes quotidiennement.

La ventilation par catégorie de services montre une forte pénétration de tous les types de plateformes.

| Catégorie de Service | Taux d'Utilisation (11-17 ans) | | --- | --- | | Plateformes de vidéos en ligne | 98 % | | Messageries instantanées | 91 % | | Réseaux sociaux | 88 % | | Jeux en ligne | 87 % | | Sites de rencontres | 15 % |

YouTube, Snapchat, TikTok et WhatsApp sont les plateformes les plus utilisées au quotidien par plus de la moitié des 11-17 ans. L'usage quotidien des VLOP augmente de manière significative avec l'âge, passant de 62 % chez les 11 ans à 96 % chez les 17 ans.

Motivations Principales des Adolescents

Trois motivations majeures expliquent pourquoi les plateformes sont devenues incontournables pour les adolescents.

1. Le besoin d'appartenance et de lien social : Les plateformes sont perçues comme un vecteur essentiel d'intégration sociale et de communication avec les pairs.

2. La recherche de divertissement et d'évasion : Les contenus ludiques et humoristiques sont massivement plébiscités pour se détendre et s'évader du quotidien.

3. L'accès à l'information : Les plateformes servent également de canal d'information pour se tenir au courant de l'actualité et des sujets d'intérêt.

3. Le Contournement Systématique des Restrictions d'Âge

Malgré les dispositifs de restriction, l'accès des mineurs aux plateformes est de plus en plus précoce, grâce à des stratégies de contournement généralisées et à une faible application des règles par les services en ligne.

Précocité de l'Accès

L'âge de la première utilisation des plateformes se situe bien en dessous des seuils réglementaires.

• Âge moyen déclaré de la 1ère utilisation :

◦ 11,2 ans pour les plateformes vidéos.    ◦ 12,3 ans pour les réseaux sociaux.

L'étude met en évidence une tendance à un accès toujours plus précoce : 22 % des jeunes de 11 ans actuels déclarent avoir utilisé les réseaux sociaux pour la première fois à 10 ans ou moins, contre seulement 4 % des jeunes de 17 ans.

Déclaration d'Âge et Manquements à la Vérification

Le contournement de l'âge minimum requis est une pratique massive et assumée par les adolescents.

• 62 % des adolescents reconnaissent ne pas avoir mis leur vraie date de naissance sur au moins une de leurs inscriptions.

• 17 % l'ont fait sur toutes leurs inscriptions.

La principale raison invoquée est l'impossibilité de s'inscrire autrement :

• 65 % n'avaient pas l'âge minimum requis.

• 31 % ne voulaient pas donner leurs données personnelles.

• 12 % voulaient passer pour plus âgés.

"Tout le monde peut y aller, parce que quand tu t'inscris, tu as juste à mettre une fausse date de naissance, ils ne la vérifient pas." - Garçon, 15 ans.

Face à cette pratique, les mesures de contrôle des plateformes apparaissent très limitées :

• Seulement 18 % des 11-17 ans ont déjà dû prouver leur âge ou ont vu leur compte bloqué.

• Facebook est la plateforme où les contrôles sont les plus fréquents (12 % des utilisateurs concernés), suivie par TikTok (10 %) et Instagram (7 %).

Failles Techniques et Contournement à l'Inscription

Les observations de parcours utilisateurs confirment la facilité avec laquelle les restrictions peuvent être contournées.

• L'interdiction d'inscription pour les moins de 13 ans n'est pas clairement explicitée lors du processus.

• Sur Instagram, Snapchat et Facebook, il est possible de contourner un premier refus en modifiant simplement sa date de naissance lors d'une nouvelle tentative.

• Le contournement est plus complexe sur d'autres plateformes comme TikTok, YouTube ou X, nécessitant des manipulations comme la réinitialisation de l'application ou la création d'une nouvelle adresse mail.

4. Perceptions Ambivalentes et Encadrement Familial

Une Perception Dichotomique des Risques et Bénéfices

Les adolescents et leurs parents partagent une vision ambivalente des plateformes, oscillant entre l'attrait des bénéfices et l'inquiétude face aux risques. Cependant, les parents se montrent systématiquement plus préoccupés et moins convaincus des avantages.

| Perception des plateformes (% d'accord) | Mineurs | Parents | | --- | --- | --- | | Permettent d’avoir une vie sociale riche | 80 % | 37 % | | Permettent d’accéder à des contenus éducatifs | 76 % | 56 % | | Exposent les mineurs à des risques graves | 77 % | 89 % | | Inquiètent quant à leur impact sur moi / votre enfant | 83 % | 86 % |

L'Encadrement Parental : Règles et Contournement

L'encadrement familial est une réalité dans la quasi-totalité des foyers, mais son efficacité est relative.

• 94 % des familles ont instauré au moins une règle concernant l'usage du numérique, avec une moyenne de 3,5 règles par foyer.

• Les règles les plus fréquentes sont l'interdiction du téléphone pendant les repas (63 %) et au coucher (55 %).

Malgré ce cadre, 45 % des mineurs admettent contourner ces règles régulièrement (8 % "souvent" et 37 % "de temps en temps"). Les adolescents reconnaissent la finalité protectrice de ces règles mais développent des stratégies pour s'y soustraire.

Utilisation des Comptes Supervisés

Une majorité de jeunes déclarent utiliser des dispositifs de protection intégrés aux plateformes, mais une part non négligeable ignore leur statut.

• 71 % des 11-17 ans déclarent utiliser au moins un compte paramétré pour un adolescent ou supervisé par un adulte.

• Le taux d'utilisation de ces comptes varie selon les plateformes : 63 % sur Snapchat, 60 % sur TikTok, 58 % sur Instagram et 49 % sur YouTube.

• Cependant, une part importante des jeunes (par exemple, 26 % sur Instagram) ne savent pas si leur compte est un compte "adulte" ou un compte "ado/supervisé", ce qui questionne la clarté et l'efficacité de ces dispositifs.

Rapport sur l’Éducation aux Médias, à l’Information et à la Citoyenneté Numérique 2024-2025

Résumé Exécutif

Ce rapport de l'Arcom pour l'année 2024-2025 analyse les initiatives en matière d’éducation aux médias, à l’information et à la citoyenneté numérique (EMI&CN) menées par les acteurs de l'audiovisuel et du numérique.

L'engagement global est en nette progression, avec une augmentation de 35 % des actions déclarées par les chaînes de télévision et de radio.

La croissance la plus spectaculaire concerne les actions de terrain, qui ont bondi de 75 %, témoignant d'une volonté d'aller à la rencontre des publics.

Cette dynamique s'accompagne d'une diversification des publics cibles, touchant non seulement le public scolaire mais aussi de plus en plus les étudiants, le grand public, les seniors et même le public carcéral.

Cependant, si les thématiques de la lutte contre la désinformation et la découverte du journalisme dominent, un effort reste à fournir pour diversifier les sujets abordés.

De plus, la proportion de programmes spécifiquement dédiés au décryptage des médias reste faible sur les antennes (12 %) et les plateformes numériques (27 %).

Les plateformes en ligne concentrent leurs efforts sur des campagnes de sensibilisation à la désinformation et à la détection des contenus générés par l'IA, en s'appuyant sur des partenariats stratégiques.

De son côté, l'Arcom a intensifié ses propres actions, sensibilisant plus de 13 000 personnes sur tout le territoire, développant de nouvelles ressources pédagogiques et renforçant ses collaborations institutionnelles.

Les préconisations pour l'avenir incluent le renforcement des actions de proximité, l'élargissement des publics cibles (notamment les parents et seniors), la diversification des thématiques traitées et la mise en place systématique de dispositifs d'évaluation de l'impact des actions menées.

Contexte et Enjeux de l'EMI&CN

L'intégration des médias audiovisuels et numériques dans la vie quotidienne des Français s'intensifie.

Le Baromètre du numérique 2025 révèle que 94 % des 12 ans et plus utilisent Internet, dont 82 % quotidiennement, et 91 % de la population possède un smartphone.

Cette omniprésence numérique transforme les usages : bien que la télévision demeure un média majeur, les jeunes générations se tournent massivement vers les écrans numériques pour consommer des contenus.

Dans ce contexte, une étude de l'Arcom sur la protection des mineurs en ligne (septembre 2025) souligne que 53 % des mineurs souhaitent être mieux accompagnés face aux risques en ligne.

Ces évolutions confirment l'impératif de renforcer les initiatives d'EMI&CN pour outiller l'ensemble des citoyens.

L'objectif est de développer un usage critique et responsable des médias, en s'adressant tant aux publics scolaires qu'aux responsables éducatifs comme les enseignants et les parents.

L'Arcom appelle à un engagement collectif, coordonné et durable pour répondre à ces enjeux démocratiques fondamentaux.

Cadre Réglementaire et Rôle des Acteurs

L'implication des différents acteurs dans l'EMI&CN est encadrée par des obligations légales et réglementaires précises, sous la supervision de l'Arcom.

1. Les Chaînes de Télévision et de Radio

• Secteur public : Les groupes France Télévisions, Radio France et France Médias Monde sont soumis à des obligations légales issues de la loi du 30 septembre 1986. L'article 43-11 stipule qu'ils doivent :

• Secteur privé : Depuis 2020, l'Arcom intègre une stipulation relative à l'EMI&CN dans les conventions signées avec les chaînes privées. Celles-ci s'engagent à mener des actions dédiées et à en rendre compte annuellement à l'Autorité.

2. Les Plateformes en Ligne

Le cadre réglementaire européen et français impose des responsabilités spécifiques aux plateformes :

• Règlement sur les Services Numériques (RSN) : Ce règlement européen du 19 octobre 2022 impose aux très grandes plateformes (VLOPSEs) de lutter contre les risques systémiques, notamment la désinformation. La participation à des campagnes d'éducation aux médias est une des mesures d'atténuation prévues.

• Loi SREN : La loi du 21 mai 2024 visant à sécuriser et réguler l'espace numérique conforte les missions de l'Arcom dans la lutte contre la manipulation de l'information.

3. Le Rôle de l'Arcom

En tant que garante des libertés de communication, l'Arcom considère l'EMI&CN comme un volet essentiel de sa mission. Consciente que la seule régulation normative ne suffit plus, elle s'investit dans une démarche pédagogique pour donner à tous les publics les clés de compréhension des écosystèmes médiatiques. L'Arcom incite les chaînes et les plateformes à contribuer à cet effort, valorise leurs actions et présente dans ce rapport annuel une analyse des déclarations reçues.

Analyse des Actions d'EMI&CN en 2024-2025

Une Hausse Globale de 35 % des Actions des Médias Audiovisuels

En 2024-2025, les chaînes de télévision et de radio ont déclaré 267 initiatives de plus que l'exercice précédent, soit une hausse de 35 %. Cette augmentation concerne tous les types d'actions : +125 sur les antennes, +45 sur le numérique et +97 sur le terrain. L'Arcom salue cet engagement constant, notamment celui des médias locaux qui jouent un rôle de relais de confiance essentiel.

Forte Progression des Actions de Terrain (+75 %) et Diversification des Publics

La hausse la plus significative concerne les actions de terrain, avec une progression de 75 % (près de 100 actions supplémentaires). Ces actions de contact direct gagnent en importance par rapport aux diffusions sur les antennes.

| Type d'action | Part en 2024 | Part en 2025 | | --- | --- | --- | | Programmes diffusés sur les antennes | 70% | 64% | | Contenus sur les prolongements numériques | 13% | 14% | | Actions de terrain | 17% | 22% |

Cette progression s'accompagne d'une diversification notable des publics ciblés. La part du public scolaire, bien que majoritaire, est passée de 58 % à 51 %, au profit des étudiants (13 %, soit +4 points) et du "tout public" (30 %, soit +8 points). Cette évolution est portée par des projets innovants touchant des publics spécifiques (seniors, public carcéral).

Les thématiques abordées sur le terrain restent cependant concentrées sur :

• La lutte contre la désinformation (56 %)

• La découverte du métier de journaliste (30 %)

• L'éducation au numérique (5 %)

Exemples d'Actions de Terrain Inspirantes

• Actions itinérantes : Le « Camion de l’info TropMytho » (Lumières sur l’info, TF1, M6, FTV, etc.) et le « Tour de France académique de l’EMI » (France Télévisions, CLEMI) vont à la rencontre des publics sur tout le territoire.

• Apprentissage par la pratique : « L’Ecole des Odyssées » (Radio France) initie 33 600 élèves de CM2 à la création de podcasts, tandis que le « Prix de la Jeune Création » (Groupe M6) encourage les talents de 18-30 ans.

• Actions auprès de publics isolés : Des ateliers en centre pénitentiaire (TF1, Ministère de la Justice) ont été organisés pour 130 détenus. Radio France a participé au festival « En Quête d’info » avec des ateliers pour seniors sur l'information via les réseaux sociaux.

• Nouvelles thématiques : France Médias Monde a animé des tables rondes sur l'intelligence artificielle lors du Sommet de la Francophonie.

Contenus sur les Antennes et le Numérique : un Potentiel à Mieux Exploiter

Si la diffusion de contenus éducatifs au sens large a augmenté (+23 % sur les antennes, +45 % sur le numérique), la part des programmes spécifiquement dédiés à l'EMI&CN reste faible : 12 % sur les antennes et 27 % sur le numérique. Les thèmes principaux restent la lutte contre la désinformation et l'éducation au numérique. L'Arcom encourage un traitement plus approfondi de sujets comme la distinction entre faits et opinions, la reconnaissance des ingérences étrangères ou la lutte contre la haine en ligne.

Initiatives des Plateformes en Ligne et Réseaux Sociaux

Les plateformes en ligne mènent des actions diversifiées, souvent en partenariat avec des acteurs de référence (associations, agences de presse, etc.). Leurs principales initiatives incluent :

• Campagnes d'EMI&CN sous forme de vidéos ou de messages d'intérêt général sur la désinformation et la détection de contenus générés par l'IA.

• Intégration de fonctionnalités pédagogiques pour expliquer le fonctionnement des services (ex: systèmes de recommandation).

• Création de ressources et d'initiatives de qualité en collaboration avec des experts de l'EMI&CN.

L'Action de l'Arcom sur le Terrain

L'Arcom s'investit directement sur le terrain pour sensibiliser aux enjeux de ses missions.

• Publics touchés : En 2024-2025, plus de 13 000 personnes ont été sensibilisées (enseignants, élèves, parents, conseillers numériques, bibliothécaires) sur tout le territoire, grâce aux Arcom locales et au prestataire Génération Numérique.

• Création de ressources : De nouvelles ressources ont été créées sur la haine en ligne (avec Pharos, CNCDH), l'impact de l'IA (avec "Café IA") et la découverte du numérique (avec le ministère de l'Éducation nationale, la CNIL, etc.).

• Partenariats : Un partenariat a été signé avec l'Institut Français de Presse de l'Université Paris-Panthéon-Assas.

• Actions emblématiques : Participation à la « Semaine de la presse et des médias dans l’école », au forum « Numérique en commun[s] », au Forum de la parentalité numérique, et création du Réseau francophone en EMI (REFEMI).

Préconisations et Perspectives

Préconisations Clés

Pour renforcer l'efficacité des actions d'EMI&CN, l'Arcom formule quatre préconisations majeures :

1. Multiplier les actions de proximité pour toucher les publics éloignés des écosystèmes audiovisuels et numériques.

2. Élargir les publics cibles, en s'adressant notamment aux parents et aux seniors, qui jouent un rôle clé dans l'accompagnement et peuvent être sensibles à la désinformation.

3. Diversifier les thématiques abordées pour permettre au public de différencier connaissances et opinions, d'identifier les ingérences numériques étrangères et de prévenir les discours de haine.

4. Évaluer la pertinence des actions mises en place pour mesurer leur impact réel.

Prochaines Actions de l'Arcom

L'Arcom poursuivra son engagement à travers un programme d'actions dense début 2026 :

| Date | Action | | --- | --- | | Janvier 2026 | Renouvellement de la convention avec le ministère de l’Éducation nationale et ses opérateurs (Réseau Canopé, CLEMI). | | Janvier 2026 | Signature d’une convention de partenariat avec Pix. | | Fin janvier 2026 | Publication d’une ressource pédagogique sur la transition écologique, en partenariat avec ARTE Education. | | À partir de jan. 2026 | Organisation par les Arcom locales de rencontres entre acteurs de l’éducation, des médias et associatifs. | | Mars 2026 | Participation à la « Semaine de la presse et des médias dans l’école » et organisation d'une table ronde sur la citoyenneté numérique. | | Courant mars 2026 | Signature d’une convention de partenariat avec l’INSPE de Lille. | | Avril 2026 | Mise en place d’un partenariat avec la Ville de Marseille pour des interventions dans les écoles primaires. |

Note de synthèse : Usages des réseaux sociaux et santé des adolescents - Analyse et recommandations de l'Anses

1. Introduction : Contexte et portée de l'expertise de l'Anses

Face à l'expansion massive des réseaux sociaux numériques et aux préoccupations croissantes concernant leur impact sur la santé, l'Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (Anses) s'est autosaisie en septembre 2019 pour évaluer les risques sanitaires encourus par les adolescents.

Cette démarche répond à un besoin d'expertise scientifique indépendante sur un phénomène socio-culturel majeur qui reconfigure en profondeur les modes de vie et de socialisation des plus jeunes.

L'expertise, menée par un groupe de travail pluridisciplinaire, s'appuie sur une analyse rigoureuse de plus d'un millier d'études scientifiques internationales.

Elle se concentre sur la population des 11-17 ans, une période charnière du développement marquée par une vulnérabilité particulière.

L'objectif est de caractériser les usages, d'identifier les risques avérés et de formuler des recommandations pour protéger la santé de cette population.

La conclusion centrale de l'Agence est sans équivoque : l'usage des réseaux sociaux numériques a des effets négatifs documentés sur la santé physique et mentale des adolescents.

Ces effets ne sont pas le fruit du hasard mais découlent en grande partie de la conception même des plateformes. Ils nécessitent une réponse coordonnée et systémique impliquant les pouvoirs publics, les plateformes elles-mêmes, ainsi que les acteurs du monde éducatif et de la santé.

2. Le Modèle Économique des Plateformes : Un Facteur de Risque Systémique

Pour évaluer les risques sanitaires des réseaux sociaux, il est indispensable de comprendre leur fonctionnement. Leur conception n'est pas neutre mais répond à des impératifs économiques précis qui constituent le cœur du problème.

Le modèle économique dominant des grandes plateformes repose sur la monétisation de l'attention et des données des utilisateurs.

En offrant un accès gratuit à leurs services, ces entreprises transforment l'usager en une source de profit, principalement par la vente d'espaces publicitaires ciblés et l'exploitation de ses données comportementales.

Ce modèle induit une course à l'engagement maximal. Pour y parvenir, les plateformes intègrent délibérément des mécanismes de captation de l'attention conçus pour influencer le comportement de l'utilisateur, maximiser le temps passé sur le service et, in fine, induire une perte de contrôle. Parmi ces techniques figurent notamment les interfaces persuasives ou trompeuses, qui exploitent des biais cognitifs pour inciter les utilisateurs à réaliser des actions qu'ils ne feraient pas autrement, et le défilement infini, qui élimine les points d'arrêt naturels pour favoriser une consultation prolongée et passive.

Les adolescents sont particulièrement vulnérables à ces stratégies.

Leurs capacités de régulation émotionnelle et comportementale étant encore en développement, ils peinent davantage à maîtriser leur temps de connexion.

De plus, la conception de ces plateformes entre en résonance directe avec leurs aspirations fondamentales : besoin d'interactions sociales avec les pairs, recherche de sensations et construction de l'identité.

Les réseaux sociaux exploitent ainsi une vulnérabilité psychologique et développementale inhérente à cette période de la vie. Ces mécanismes de conception sont à l'origine de risques sanitaires spécifiques et documentés.

3. Principaux Risques Sanitaires Identifiés et Populations Vulnérables

L'expertise de l'Anses établit des corrélations claires et préoccupantes entre l'usage des réseaux sociaux et la détérioration de la santé des jeunes.

Ces risques ne sont pas des externalités malheureuses, mais des conséquences directes des stratégies de captation de l'attention et d'exploitation des vulnérabilités développementales décrites précédemment. Un constat transversal émerge de l'analyse : les filles constituent une population particulièrement à risque.

Cette vulnérabilité accrue n'est pas monolithique ; elle résulte d'une confluence de facteurs : un temps d'usage quantitativement supérieur, une orientation vers des plateformes hautement visuelles qui exacerbent la pression sur l'apparence, et une plus grande exposition aux dynamiques de cyberviolence genrée.

D'autres populations, comme les jeunes LGBTQIA+ ou ceux présentant des troubles préexistants (anxiodépressifs, TDAH), sont également surexposées.

3.1. Dégradation de la Santé Mentale et de l'Image de Soi

L'expertise de l'Anses établit que l'usage des réseaux sociaux constitue un facteur contributif aux troubles anxiodépressifs.

Cette relation est médiée par plusieurs mécanismes psychologiques délétères, tels que la comparaison sociale ascendante, qui génère un sentiment d'insatisfaction ; le FoMO (Fear of Missing Out), qui nourrit une connexion anxiogène ; et le cyberharcèlement.

L'expertise met en lumière un cercle vicieux : un mal-être initial peut conduire un adolescent à se réfugier dans les réseaux sociaux dans une stratégie d'« escapisme », ce qui renforce paradoxalement ses difficultés psychologiques.

L'impact sur l'image corporelle est particulièrement prononcé. L'exposition continue à des corps idéalisés, souvent modifiés par des filtres et des retouches, favorise l'insatisfaction corporelle.

L'expertise identifie cette exposition comme étant corrélée à l’intériorisation des idéaux corporels, l’auto-objectification et la comparaison sociale ascendante, autant de facteurs intermédiaires des troubles des conduites alimentaires.

L'effet est amplifié par les algorithmes de personnalisation qui créent un effet « silo », enfermant les jeunes vulnérables dans des boucles de contenus délétères (valorisation de la maigreur, automutilation, suicide), banalisant ces comportements et augmentant le risque d'imitation.

3.2. Altération du Sommeil : Un Médiateur Clé des Troubles de Santé

L'Anses identifie la perturbation du sommeil comme un effet sanitaire majeur et un médiateur central entre l'usage des réseaux sociaux et la dégradation de la santé mentale.

L'impact négatif sur le sommeil s'opère via trois mécanismes principaux :

• Réduction de la durée du sommeil : l'augmentation du temps d'écran retarde systématiquement l'heure du coucher.

• Altération de la qualité du sommeil : les contenus et interactions en ligne provoquent une stimulation cognitive et émotionnelle qui entrave l'endormissement et fragmente le sommeil.

• Perturbation du rythme circadien : l'exposition à la lumière bleue des écrans en soirée inhibe la sécrétion de mélatonine, l'hormone de l'endormissement.

Or, une perturbation chronique du sommeil est elle-même un facteur de risque majeur pour le développement de troubles de santé mentale et de maladies chroniques.

3.3. Exposition aux Conduites à Risques et aux Cyberviolences

Les réseaux sociaux agissent comme de puissants vecteurs de conduites à risques.

Ils contribuent à la normalisation de la consommation de substances psychoactives (alcool, tabac, cannabis) et assurent la propagation virale de défis dangereux (challenges), dont l'attrait repose sur la quête de reconnaissance sociale.

Le cyberharcèlement est une autre menace centrale. Il prolonge les dynamiques de harcèlement hors ligne, mais son impact est amplifié par des facteurs spécifiques au numérique : l'anonymat (réel ou perçu), la persistance des contenus et l'ampleur de leur diffusion.

L'expertise souligne que le fait d’appartenir à une communauté LGBTQIA+ est associé à une probabilité plus élevée d’être cybervictime.

Les conséquences documentées par l'Anses sont graves :

• Augmentation des symptômes dépressifs

• Risque accru d'idées suicidaires et de tentatives de suicide

• Comportements d'automutilation

• Augmentation de l'usage problématique des réseaux sociaux

Enfin, l'expertise alerte sur les cyberviolences à caractère sexuel, comme le sexting non consenti ou la coercition numérique. Ces pratiques constituent une nouvelle expression du sexisme, particulièrement risquée pour les filles.

Ce tableau de risques multifactoriels, systémiquement liés à la conception des plateformes, appelle une réponse stratégique et coordonnée, que l'Anses articule en quatre axes d'intervention.

4. Axes d'Intervention Stratégiques : Les Recommandations de l'Anses

L'Anses préconise une approche systémique et coordonnée qui ne fait pas reposer la charge uniquement sur les individus. Les recommandations visent à la fois les plateformes, les pouvoirs publics, les acteurs de l'éducation et la communauté scientifique. L'Agence insiste sur la nécessité d'impliquer les adolescents dans l'élaboration de ces mesures pour garantir leur pertinence et faciliter leur adhésion.

4.1. Axe 1 : Réguler et Sécuriser l'Environnement Numérique

Cet axe vise directement les plateformes et les pouvoirs publics, considérant que la responsabilité première incombe aux concepteurs des services. Les recommandations phares incluent :

• Instaurer un cahier des charges technique pour les réseaux sociaux accessibles aux mineurs, afin de garantir un design protecteur.

• Appliquer des mécanismes fiables de vérification de l'âge et du consentement parental.

• Encadrer légalement les interfaces persuasives ou trompeuses et les algorithmes de personnalisation, en s'appuyant sur les dispositions du Digital Services Act (DSA) européen pour interdire les techniques d'influence trompeuse et la diffusion de contenus délétères.

• Imposer un paramétrage par défaut protecteur pour les comptes des mineurs (limitation des notifications, suppression des indicateurs d'activité en ligne).

• Mettre en place des procédures de signalement simples et efficaces pour les contenus problématiques.

4.2. Axe 2 : Développer une Éducation aux Médias Numériques

L'éducation est un levier complémentaire indispensable. Pour les parents et adolescents, il s'agit de co-construire des repères de bonnes pratiques et d'alerter sur les pressions sociales spécifiques (stéréotypes de genre, harcèlement).

Pour le milieu scolaire, l'Anses préconise de renforcer les programmes d'éducation au numérique, de développer l'esprit critique et les compétences socio-émotionnelles, et de promouvoir des espaces de parole entre pairs.

4.3. Axe 3 : Renforcer la Prévention des Effets sur la Santé

Une approche de santé publique globale est nécessaire. L'Anses préconise de :

• Mener des campagnes de sensibilisation sur l'hygiène de vie (sommeil, sédentarité) et l'hygiène numérique (risques liés à l'image de soi, aux images intimes).

• Renforcer la prévention en santé mentale, par la formation des professionnels et l'augmentation des moyens du système de santé et du personnel médical scolaire.

• Lutter activement contre les cyberviolences et toutes les formes de discrimination.

• Développer des alternatives attractives à la socialisation en ligne (infrastructures sportives, culturelles, associatives).

4.4. Axe 4 : Soutenir la Recherche Scientifique

Pour combler les lacunes de la recherche, l'Anses recommande de garantir l'accès des chercheurs aux données des plateformes, comme le prévoit le Digital Services Act (DSA) européen, et d'améliorer la méthodologie des études pour mieux objectiver les usages et les effets sanitaires.

Enfin, l'Agence appelle la communauté scientifique à étudier la pertinence de qualifier l'« usage problématique » des réseaux sociaux comme une addiction comportementale, au même titre que les jeux d’argent et de hasard.

5. Conclusion Générale

L'expertise de l'Anses dresse un constat sévère : les effets négatifs documentés des réseaux sociaux sur la santé des adolescents sont étroitement liés aux caractéristiques de conception et au modèle économique des plateformes.

Le problème n'est donc pas réductible à une simple question de responsabilité individuelle.

Les stratégies de captation de l'attention sont systémiques et exploitent des vulnérabilités psychologiques propres à l'adolescence.

Ces constats invalident l'approche de l'autorégulation et démontrent l'urgence d'adopter un cadre de gouvernance robuste pour les réseaux sociaux, à la hauteur des enjeux de santé publique.

Si l'éducation au numérique et l'accompagnement parental sont des piliers nécessaires, ils demeurent insuffisants face à un problème d'une telle ampleur structurelle.

L'expertise de l'Anses fournit la base factuelle pour une politique publique plus musclée, engageant la responsabilité des plateformes pour imposer des modifications profondes de leurs services.

Une vigilance continue s'impose face aux évolutions technologiques rapides, notamment l'intégration de l'intelligence artificielle, qui pourrait démultiplier les risques identifiés.

Synthèse du rapport de l'Anses sur les usages des réseaux sociaux et la santé des adolescents

Résumé Exécutif

Ce document synthétise l'avis et le rapport d'expertise collective de l'Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (Anses), publiés en décembre 2025, concernant les effets de l'usage des réseaux sociaux numériques sur la santé des adolescents de 11 à 17 ans.

S'appuyant sur l'analyse de plus d'un millier d'études scientifiques, l'expertise établit un lien clair entre l'utilisation des réseaux sociaux et une augmentation des risques pour la santé mentale et le bien-être des jeunes.

Les conclusions principales indiquent que le modèle économique des plateformes, fondé sur une "économie de l'attention", induit des conceptions (interfaces persuasives, défilement infini, algorithmes de personnalisation) qui exploitent les vulnérabilités propres à l'adolescence.

Ces mécanismes favorisent un usage excessif et une perte de contrôle, entraînant des conséquences sanitaires multifactorielles.

Les principaux effets négatifs identifiés sont :

• Perturbation du sommeil : Réduction de la durée et de la qualité du sommeil, agissant comme un médiateur clé pour d'autres troubles de santé mentale.

• Troubles anxiodépressifs : L'usage des réseaux sociaux est un facteur contributif, notamment via la comparaison sociale, le cyberharcèlement et la "peur de manquer" (FoMO).

• Image corporelle et troubles des conduites alimentaires : L'exposition à des contenus idéalisés renforce l'insatisfaction corporelle, particulièrement chez les filles.

• Conduites à risques : Les plateformes agissent comme des vecteurs pour la normalisation de la consommation de substances, la participation à des défis dangereux et l'exposition aux cyberviolences.

L'expertise souligne que les filles constituent une population particulièrement à risque, étant plus impactées sur l'ensemble des effets sanitaires étudiés.

Face à ce constat, l'Anses formule des recommandations structurées autour de quatre axes :

• une régulation stricte des plateformes pour protéger les mineurs,
• le renforcement de l'éducation aux médias,
• des campagnes de prévention en santé publique, et
• un soutien accru à la recherche pour combler les lacunes de connaissances.

L'Agence conclut que si l'accompagnement parental et l'éducation sont nécessaires, ils ne peuvent se substituer à un cadre de gouvernance contraignant pour les plateformes, dont la responsabilité dans les impacts sanitaires observés est centrale.

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1. Contexte et Organisation de l'Expertise

1.1. Origine et Objectifs

Face à l'expansion massive des technologies numériques et aux interrogations sur leurs effets sanitaires, l'Anses s'est autosaisie le 12 septembre 2019 pour évaluer les risques liés à leurs usages.

L'expertise a été spécifiquement focalisée sur les risques pour la santé des adolescents (11-17 ans) liés à l'utilisation des réseaux sociaux numériques, en raison de la vulnérabilité particulière de cette période de la vie.

Les objectifs de l'expertise étaient de :

• Caractériser le fonctionnement et les usages des réseaux sociaux.

• Analyser les spécificités de la population adolescente.

• Décrire les effets sur la santé de certaines pratiques.

• Analyser les risques sanitaires globaux.

• Formuler des recommandations pour protéger la santé des adolescents.

1.2. Méthodologie

L'expertise a été menée par le groupe de travail "Effets de l’usage des outils numériques sur la santé des adolescents", créé en septembre 2020, et adoptée par le Comité d’experts spécialisé (CES) "Agents physiques et nouvelles technologies". La démarche s'est appuyée sur :

• Une revue exhaustive de la littérature scientifique académique (plus d'un millier d'articles analysés via les bases de données Scopus et Pubmed entre 2011 et 2021, complétée par des études antérieures et postérieures).

• L'analyse de la littérature grise (rapports institutionnels et associatifs).

• Une analyse du cadre législatif menée par l'Institut de recherche juridique de la Sorbonne.

1.3. Limites de la Littérature Scientifique

Le groupe de travail a identifié plusieurs limites aux études disponibles :

• Décalage temporel : De nombreuses études portent sur des réseaux sociaux moins populaires aujourd'hui (ex: Facebook) et peu sur des plateformes plus récentes comme TikTok.

• Mesure de l'utilisation : La plupart des études reposent sur le temps d'utilisation déclaré, une mesure sujette aux biais de mémoire et de désirabilité sociale. Un temps élevé n'est pas suffisant pour qualifier un usage de "préoccupant".

• Hétérogénéité des contextes : Les études proviennent de divers pays, mais les mécanismes d'action des plateformes étant similaires, les résultats ont été jugés transposables.

• Causalité : La majorité des études sont transversales, montrant des liens statistiques mais ne permettant pas d'établir de lien de cause à effet. Les études longitudinales, bien que moins nombreuses, apportent des éléments sur la temporalité des effets.

2. Le Fonctionnement des Réseaux Sociaux Numériques

2.1. Définition et Modèle Économique

En l'absence de définition consensuelle, l'expertise s'est adossée à une conception large, similaire à celle de la loi du 7 juillet 2023 : une plateforme permettant aux utilisateurs de se connecter, communiquer et partager des contenus.

Le modèle économique des plateformes majeures s'apparente à celui d'une régie publicitaire. La gratuité apparente du service est compensée par la monétisation des données personnelles et de l'attention des utilisateurs.

Ce modèle incite les plateformes à maximiser le temps passé et l'engagement des utilisateurs.

2.2. Stratégies de Captation de l'Attention

Pour maintenir l'engagement, les plateformes déploient des stratégies de conception spécifiques :

• Algorithmes de personnalisation : Ils proposent des contenus visant à retenir l'utilisateur, créant parfois un "effet silo" qui renforce l'exposition à des contenus potentiellement délétères.

• Interfaces trompeuses (ou dark patterns) : Ce sont des mécanismes persuasifs qui exploitent des biais psychologiques pour inciter les utilisateurs à des actions qu'ils ne feraient pas autrement.

• Fonctionnalités incitatives : Le défilement infini, les notifications et les contenus éphémères sont conçus pour inciter à un usage prolongé et induire une perte de contrôle.

Ces stratégies exploitent les vulnérabilités de l'adolescence : besoin d'interactions sociales, recherche de sensations et capacités de régulation émotionnelle encore limitées.

3. Usages des Réseaux Sociaux par les Adolescents

L'expertise distingue l'utilisation (interaction technique), l'usage (intégration sociale et culturelle) et la pratique (routines et savoir-faire). L'analyse se concentre sur les usages, qui sont des phénomènes complexes.

3.1. État des Lieux

| Donnée Clé | Valeur | Source / Année | | --- | --- | --- | | Adolescents (12-17 ans) utilisant un smartphone quotidiennement pour aller sur Internet | Près de 90 % | \- | | Adolescents (12-17 ans) passant entre 2 et 5h/jour sur leur smartphone | 42 % | Credoc, 2025 | | Adolescents (12-17 ans) passant plus de 5h/jour sur leur smartphone | 9 % | Credoc, 2025 | | Utilisation quotidienne des réseaux sociaux chez les 12-17 ans (2023) | 53 % | CREDOC, Baromètre du numérique | | Utilisation quotidienne des réseaux sociaux chez les 12-17 ans (2024) | 58 % | CREDOC, Baromètre du numérique |

Les usages varient selon l'âge, le genre et le milieu social. Les filles consacrent plus de temps aux réseaux sociaux que les garçons, qui privilégient les jeux vidéo.

3.2. Rôle dans la Socialisation

Les réseaux sociaux répondent aux aspirations des adolescents (interactions, recherche d'informations auprès des pairs, prise de risques) et participent à l'exploration de leur identité. Ils prolongent et transforment les processus de socialisation, s'inscrivant dans la continuité des dynamiques familiales, scolaires et amicales.

La sphère familiale peut jouer un rôle de régulation et de ressource, mais les usages configurent aussi un territoire informationnel propre à l'adolescent.

4. Principaux Effets sur la Santé des Adolescents

L'expertise révèle des conséquences négatives significatives, avec une prévalence plus marquée chez les filles pour la majorité des effets sanitaires étudiés.

4.1. Usage Problématique et Addiction

Le terme "addiction aux réseaux sociaux" n'est pas reconnu dans les classifications internationales (DSM-5R, ICD-11) et fait l'objet de débats. Le rapport opte pour la notion d'"usage problématique", la plus fréquente dans la littérature.

Les outils de mesure sont hétérogènes mais s'accordent sur deux dimensions caractéristiques d'une addiction :

• Les répercussions négatives sur la santé et les activités quotidiennes.

• L'impossibilité de maîtriser le temps passé sur les plateformes (perte de contrôle).

4.2. Perturbation du Sommeil

Un impact négatif clair est démontré. Les mécanismes sont :

• Réduction de la durée du sommeil par un retard de l'heure du coucher.

• Stimulation de l'éveil (physiologique, cognitif, émotionnel) qui entrave l'endormissement.

• Exposition à la lumière bleue des écrans le soir, qui inhibe la sécrétion de mélatonine.

Une perturbation chronique du sommeil est un facteur de risque pour des maladies physiques et mentales, et un médiateur clé entre l'usage des réseaux sociaux et les symptômes anxiodépressifs.

4.3. Image du Corps et Troubles des Conduites Alimentaires (TCA)

Certaines pratiques, notamment sur les réseaux "hautement visuels", sont corrélées à :

• L'intériorisation d'idéaux corporels irréalistes.

• La comparaison sociale ascendante (se comparer à des personnes perçues comme plus désirables).

• L'auto-objectification (se percevoir comme un objet à regarder).

Ces facteurs renforcent l'insatisfaction corporelle et la surveillance de son apparence, particulièrement chez les filles, et constituent des facteurs intermédiaires des TCA.

Les algorithmes peuvent amplifier l'exposition à des contenus valorisant la maigreur ou la musculature, exacerbant les comportements délétères.

4.4. Troubles Anxiodépressifs et Idées Suicidaires

L'usage des réseaux sociaux est identifié comme un facteur contributif aux troubles anxiodépressifs, sans être une cause unique. La relation est complexe et médiée par :

• L'altération du sommeil.

• Le cyberharcèlement.

• La comparaison sociale.

• Le FoMO (Fear Of Missing Out), qui peut entraîner une perte de contrôle.

Une spirale délétère est souvent observée : un mal-être initial peut conduire à un usage compulsif des réseaux ("escapisme"), qui à son tour détériore la santé mentale.

Les algorithmes peuvent enfermer les jeunes en détresse dans des "silos" de contenus négatifs (automutilation, suicide), banalisant ces comportements (effet Werther).

4.5. Conduites à Risques et Cyberviolences

• Consommation de substances : Les réseaux sociaux participent à la normalisation de la consommation d'alcool, de tabac et de cannabis en exposant les jeunes à des contenus valorisants et en renforçant les normes sociales perçues.

• Défis (challenges) : La recherche de reconnaissance par les pairs peut inciter à la participation à des défis dangereux.

• Cyberharcèlement : Il s'agit souvent d'une extension du harcèlement scolaire, amplifiée par l'anonymat, la persistance des contenus et l'ampleur de leur diffusion.

La cybervictimation est associée à une augmentation des symptômes dépressifs, des idées suicidaires et de l'automutilation.

• Sexting non consenti : La diffusion d'images intimes sans consentement est une forme de cyberviolence sexuelle aux conséquences graves, en particulier pour les filles.

4.6. Résultats Scolaires

L'expertise met en évidence une association négative faible entre le temps passé sur les réseaux sociaux et les résultats scolaires.

Cependant, les limites méthodologiques des études empêchent de conclure à un lien causal direct. Le multitâche numérique et la perturbation du sommeil sont des facteurs explicatifs probables.

5. Autres Impacts Soulignés par le Comité d'Experts

Le CES a rappelé la pertinence d'autres enjeux sanitaires et sociétaux :

• Sédentarité et inactivité physique : Bien que l'usage nomade des smartphones ne soit pas directement synonyme de sédentarité, les longues durées d'utilisation y contribuent probablement.

• Lumière bleue : Les adolescents sont plus sensibles à la lumière bleue des écrans, ce qui augmente le risque de perturbation des rythmes circadiens et, à long terme, de troubles métaboliques ou de santé mentale.

• Impacts environnementaux : Le numérique représente près de 4 % des émissions mondiales de gaz à effet de serre, un chiffre en hausse, notamment à cause du streaming vidéo encouragé par les réseaux sociaux.

• Enjeux démocratiques : Les algorithmes peuvent polariser les opinions, diffuser de la désinformation et manipuler l'information, soulevant des questions majeures pour la construction citoyenne des adolescents.

6. Recommandations de l'Anses

Face à ces constats, l'Agence formule des recommandations structurées selon quatre axes d'action complémentaires.

6.1. Réguler et Sécuriser l'Environnement Numérique

• Imposer un cahier des charges aux plateformes pour qu'elles soient accessibles aux mineurs, incluant des mécanismes fiables de vérification de l'âge.

• Encadrer légalement les interfaces persuasives et les algorithmes de personnalisation pour interdire les techniques d'influence trompeuse et limiter l'amplification de contenus préjudiciables.

• Instaurer un paramétrage par défaut protecteur pour les comptes des mineurs (limitation des notifications, etc.).

• Mettre en place des procédures simples et efficaces de signalement et de modération des contenus délétères.

• Étendre aux réseaux sociaux l'encadrement des publicités prévu pour la télévision.

6.2. Éduquer aux Médias Numériques

• Fournir des repères de bonnes pratiques aux parents et adolescents, coconstruits avec eux.

• Renforcer l'éducation au numérique à l'école, en formant du personnel dédié et en développant l'esprit critique et les compétences socio-émotionnelles des élèves.

• Promouvoir des espaces de parole entre pairs pour réfléchir collectivement aux pratiques numériques.

6.3. Prévenir les Effets sur la Santé

• Mener des campagnes de santé publique sur l'hygiène de vie (sommeil, activité physique) et l'hygiène numérique (risques liés à l'image de soi, au consentement).

• Renforcer la prévention en santé mentale en formant les professionnels au contact des adolescents et en dotant les systèmes scolaire et de santé de moyens suffisants.

• Intensifier la lutte contre les cyberviolences et toutes les formes de discrimination.

• Développer des alternatives de socialisation hors ligne (infrastructures sportives, culturelles) adaptées aux adolescents.

6.4. Soutenir la Recherche

• Garantir l'accès aux données des plateformes pour les chercheurs, comme le prévoit le Digital Services Act (DSA) européen.

• Améliorer la méthodologie des études scientifiques en diversifiant les approches et en développant des outils de mesure plus fiables.

• Financer la recherche sur des thèmes clés comme le cyberharcèlement, les interfaces trompeuses, les populations vulnérables et l'efficacité des actions de prévention.

• Étudier la pertinence de qualifier l'usage problématique des réseaux sociaux comme une addiction comportementale.

What is FERPA exactly?

Why do some students feel imposter syndrome more than others ?

Annotation 2: Reading about imposter syndrome really stood out to me because it’s something a lot of students probably experience but don’t talk about. I have experienced it myself, especially going back to school after many years. Knowing that feeling unsure or overwhelmed is normal makes college feel less intimidating. It reminded me to be patient with myself while adjusting and to not give up when things get hard.

Synthèse de l'Avis du Conseil d'État sur la Proposition de Loi "Protéger les Mineurs en Ligne"

1. Contexte et Objectifs de la Proposition de Loi

Cette proposition de loi a été élaborée en réponse à des constats alarmants concernant les risques auxquels les réseaux sociaux exposent les mineurs.

Faisant directement suite aux recommandations du rapport de la commission d’enquête sur TikTok, le texte met en lumière les dangers d'addiction et les effets psychologiques néfastes de certaines plateformes sur la santé mentale des jeunes.

L'objectif principal du législateur est donc de renforcer de manière significative le cadre de protection des mineurs dans l'environnement numérique, en instaurant des mesures contraignantes et préventives.

Les deux mesures phares de la proposition initiale sont les suivantes :

• Interdiction d'accès pour les moins de 15 ans : Le texte visait à imposer une obligation directe aux fournisseurs de services de réseaux sociaux de refuser l'inscription des mineurs de moins de 15 ans.

Pour ce faire, les plateformes auraient dû mettre en œuvre des dispositifs de contrôle d'âge robustes, sous peine de sanctions financières et d'injonctions judiciaires.

• Couvre-feu numérique pour les 15-18 ans : Pour cette tranche d'âge, la proposition prévoyait une obligation de désactivation automatique de l'accès aux comptes entre 22 heures et 8 heures du matin, en s'appuyant sur les mêmes solutions techniques de vérification de l'âge.

En complément de ce dispositif central, le texte comprend plusieurs autres mesures structurantes :

| Mesure | Objectif Stratégique | | --- | --- | | Lutte contre la publicité pro-suicide | Compléter la liste des contenus illicites pour inclure la propagande en faveur de moyens de se donner la mort. | | Renforcement des peines | Augmenter la durée de suspension des comptes d'accès aux plateformes en cas d'infraction. | | Messages sanitaires | Imposer des informations préventives sur les publicités pour les réseaux sociaux et sur les emballages de smartphones. | | Formation scolaire | Étendre la formation sur l'usage du numérique à la sensibilisation aux enjeux de santé mentale. | | Interdiction des téléphones dans les lycées | Généraliser l'interdiction déjà en vigueur dans les collèges pour favoriser la concentration et prévenir le harcèlement. | | Création d'un délit de négligence parentale | Sanctionner les parents en cas d'usage excessif, inadapté ou non surveillé des outils numériques par leur enfant. |

L'analyse juridique approfondie du Conseil d'État révèle cependant que, si l'intention est louable, les mécanismes proposés soulèvent des difficultés majeures de compatibilité avec le droit européen et les libertés fondamentales.

2. Analyse Critique du Conseil d'État : Compatibilité avec le Droit Européen

La conformité au droit de l'Union européenne est une condition essentielle de la validité de toute loi nationale.

Le Conseil d'État souligne que le Règlement sur les Services Numériques (DSA) harmonise pleinement les règles pour les plateformes opérant dans l'UE, limitant drastiquement la capacité des États membres à leur imposer des obligations supplémentaires.

L'avis du Conseil se révèle être une véritable leçon d'ingénierie juridique, démontrant comment atteindre un objectif de politique nationale dans le cadre contraignant d'un droit européen harmonisé.

Le Conseil d'État met en évidence une incompatibilité juridique frontale : en imposant une obligation directe aux plateformes de refuser l'inscription des mineurs, la proposition de loi initiale violerait le principe d'harmonisation maximale du DSA, rendant la mesure juridiquement fragile et susceptible d'être invalidée.

Pour surmonter cet obstacle majeur, le Conseil d'État propose une reformulation décisive, qui constitue le pivot de sa stratégie. Au lieu d'obliger les plateformes, la loi doit directement interdire l'accès au mineur : `

« Il est interdit au mineur de quinze ans d’accéder à un service de réseau social en ligne »`.

Cet acte de prohibition qualifie automatiquement un tel accès de "contenu illicite" au sens de la définition large du DSA.

Cette reclassification est la clé de voûte de la stratégie du Conseil : elle permet de mobiliser les puissants mécanismes de régulation du DSA (injonctions de l'Arcom, signalements, sanctions) contre les plateformes sans créer une nouvelle obligation nationale, interdite par le droit européen.

Le cadre de l'UE devient ainsi le principal outil d'application d'une politique nationale française.

Pour renforcer l'effectivité de cette interdiction, le Conseil suggère d'ouvrir un second flanc de mise en conformité. Il préconise de prévoir la nullité de plein droit des contrats passés par un mineur en violation de cette interdiction.

Une telle nullité priverait de base légale tout traitement de ses données personnelles, exposant les plateformes à des contrôles et sanctions de la part de la CNIL au titre du RGPD, ce qui augmente considérablement la pression en faveur du respect de la loi.

Enfin, le Conseil recommande que la Commission européenne élabore des lignes directrices pour s'assurer que les plateformes gèrent correctement la restitution des contenus et des données aux mineurs dont les comptes sont résiliés, afin de ne pas porter atteinte à leurs droits de propriété intellectuelle.

Cette refonte juridique est présentée comme une condition sine qua non à la viabilité du texte.

3. Analyse Critique du Conseil d'État : Équilibre avec les Droits et Libertés Fondamentaux

Au-delà de la conformité européenne, le Conseil d'État analyse la conciliation entre l'objectif de protection de l'enfance — une exigence constitutionnelle — et le respect des libertés fondamentales du mineur (liberté d'expression, d'information) et des droits des parents.

Sur ce plan, le Conseil juge le dispositif initial déséquilibré et disproportionné pour trois raisons principales :

1. Caractère général et absolu : L'interdiction s'appliquerait à tous les "réseaux sociaux" sans distinction, y compris ceux ne présentant aucun risque avéré (plateformes collaboratives, éducatives), ce qui est jugé excessif.

2. Absence de discernement et de rôle parental : Le mécanisme initial ignore le degré de maturité de l'enfant et écarte totalement les parents de leur rôle d'accompagnement, en contradiction avec le Code civil et la Convention relative aux droits de l’enfant.

3. Manque de justification du couvre-feu : Les bornes horaires du couvre-feu pour les 15-18 ans (22h-8h) sont jugées insuffisamment documentées et donc disproportionnées.

Pour rééquilibrer le texte, le Conseil d'État propose une refonte qui incarne un changement de philosophie réglementaire : passer d'une interdiction étatique, brute et centrée sur la plateforme, à un système nuancé, responsabilisant les parents et centré sur le terminal. Ce mécanisme alternatif repose sur deux volets :

• Volet 1 - Interdiction Ciblée Le Gouvernement pourrait, par décret en Conseil d’État pris après avis de l’Arcom, interdire l'accès aux mineurs de moins de 15 ans à des réseaux sociaux spécifiquement identifiés comme dangereux en raison de leurs systèmes de recommandation.

L'État utilise ici son pouvoir de prohibition de manière ciblée, là où le danger est avéré.

• Volet 2 - Autorisation Parentale Généralisée Pour tous les autres réseaux sociaux, l'accès serait interdit sauf autorisation expresse d'un parent.

Réalisée via des dispositifs installés sur les systèmes d’exploitation des équipements terminaux distribués par les fournisseurs d’accès à l’internet (à l'instar des mécanismes de contrôle parental existants), cette autorisation serait révocable et pourrait préciser une durée d'usage.

L'État délègue ici à une autorité parentale guidée le soin d'évaluer le risque.

Cette approche duale résout le problème de proportionnalité, transformant une interdiction fragile en un système de régulation juridiquement beaucoup plus solide.

4. Recommandations et Points de Vigilance sur les Autres Articles

Le Conseil d'État a également examiné les autres articles de la proposition de loi, formulant des recommandations d'ajustement ou des réserves importantes.

• Interdiction des téléphones dans les lycées (Art. 6) : La mesure est jugée nécessaire et proportionnée.

Le Conseil recommande d'exclure explicitement de son champ les formations de l'enseignement supérieur et de différer son entrée en vigueur à la rentrée scolaire 2026.

• Formation scolaire (Art. 4) : Jugée conforme, la mesure est cependant qualifiée de potentiellement redondante avec des dispositions déjà existantes.

Une entrée en vigueur différée à la rentrée 2026 est également suggérée pour permettre l'adaptation des enseignants.

• Délit de négligence numérique (Art. 7) : Le Conseil exprime de fortes réserves.

À titre principal, il estime que le droit pénal existant est suffisant.

À titre subsidiaire, si le délit était maintenu, ses termes ("usage excessif", "outils numériques") sont jugés trop vagues et contraires au principe constitutionnel de légalité des délits et des peines.

• Publicité et emballages (Art. 3) : Ces dispositions devront être notifiées à la Commission européenne au titre de la directive "TRIS", une étape procédurale cruciale destinée à prévenir la création de barrières techniques inopinées au sein du marché unique.

• Rapport au Parlement (Art. 5) : Il est suggéré de restreindre le champ du rapport pour le concentrer sur le respect par les plateformes de leurs obligations spécifiques envers les mineurs dans le cadre du DSA.

Ces ajustements visent à garantir la sécurité juridique et l'applicabilité concrète de l'ensemble du texte.

5. Conclusion : Synthèse Stratégique pour la Décision

L'avis du Conseil d'État valide sans équivoque la nécessité d'agir face aux dangers documentés que les réseaux sociaux font peser sur les mineurs et reconnaît la pertinence de l'objectif poursuivi par le législateur.

Cependant, cette validation de l'objectif s'accompagne d'une censure quasi totale du dispositif initialement proposé. Celui-ci est jugé doublement fragile :

1. Incompatible avec le droit de l'Union européenne, en raison de la violation du principe d'harmonisation maximale du DSA.

2. Déséquilibré au regard des droits fondamentaux, car l'interdiction générale et le couvre-feu sont jugés disproportionnés et écartent indûment l'autorité parentale.

En définitive, les amendements du Conseil d'État ne sont pas de simples ajustements.

Ils constituent une refondation juridique et une véritable feuille de route stratégique et législative offerte au Parlement. Ils transforment un projet juridiquement précaire en une loi conforme, proportionnée et, par conséquent, viable et réellement efficace pour protéger les mineurs dans l'espace numérique.

key point for review

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save work via Peergos

make it work with peersuite.space

I really like how the whole class is centered around community and I wonder how different it will be to write about community in 3 different ways for each paper.

Carefully align model selection with data availability, acknowledging that both significantly influence analytical outcomes and policy relevance.

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Referee #3

Evidence, reproducibility and clarity

In this work Neupane et al used large-scale robust CRISPR-based gene activation and ablation screens to identify novel regulators of α-synuclein pathology in synucleinopathies using as read-out p-αSyn129 signals by high-throughput fluorescence microscopy. The authors reveal that mitochondrial protein OXR1 promotes Ser129-phosphorylated αSyn aggregation, while ER-associated EMC4 suppresses it via enhanced autophagic clearance, highlighting new possible mechanistic pathways in disease progression of alpha-synucleinopathies.

Major comments:

1. As correctly pointed out by the authors in Introduction p-Syn is associated with aggregates, but its functional role is far to be clear and both neuroprotective or pro-aggregations effects have been proposed. Further it has been shown that, physiological neuronal activity augments Ser129-phospho αSyn, which is a trigger for protein-protein interactions, which in turn is necessary for mediating αSyn function at the synapse (https://doi.org/10.1016/j.neuron.2023.11.020). As a consequence modulation of p- p-αSyn as possible therapeutical target for PD and synucleinopathies is quite a complicate matter. The assumption, on which the whole paper is based, that increase in p-αSyn equates to αSyn aggregation and disease progression is rather weak to this reviewer, unless further validation of it is provided. Indeed while the authors performed experiments on human iPSC-derived cortical and dopaminergic neurons on p-αSyn analysis, any measurement of αSyn aggregates/oligomers, and neuronal degeneration is provided. It is recommended to provid this experiments ideally using different tecnique like αSyn-Proximity Ligation Assay for measurements of oligomers, as it has been largely validated in autoptic brains of PD, MSA and DLB patients (doi: 10.1007/s00401-025-02871-w.), as well as cell viability/apoptosis and neurites degeneration measurements upon OXR1 and EMC4 modulation in iPSC derived cortical and dopaminergic neurons.
2. The authors claims in Results page 5: "The absence of cytoplasmic pSyn129 signal in HEK293 cells lacking α-Syn overexpression demonstrates that elevated α-Syn levels are essential to drive robust and rapid aggregation. Moreover, it indicates that the 81A antibody selectively recognizes de novo aggregates rather than the recombinant seeds". The fact that ab81A recognize deNovo aggregates and not rec seeds is quite speculative, not supported by data, and might rather indicate that ab 81A does not recognize aggregates. Thus this further implays that other technology like for example Seeding amplification assays are being employed by the authors in addition to p-αSyn129 signals in validation experiments for example in genetic PD (ideally GBA1 or LRRK2) IPSC-derived dopaminergic neurons.

Minor comments:

1. The strain-specific effects especially from patients-derived fibrils of OXR1 activation and EMC4 depletion on pSyn levels is rather weak in comparison with RAB3 and PIKFYVE (fig 3F-G) and therefore the expected relevance of these results especially in vivo in patients should be better clarified and modulated in discussion
2. In discussion authors write "We observed that OXR1 activation preferentially increases α-Syn aggregates phosphorylation (EP1536Y) in neuronal somata, suggesting that mitochondrial dysfunction exacerbates α-Syn phosphorylation in later-stage aggregates." This is quite a surprising result since distal axonal endings are particularly susceptible to mitochondrial impairments for anatomical and physiologically reasons and if p-αSyn129 accumulation is driven by mithocondrial disfunction as suggested by this paper, this should be detected in neurites as well. Please clarify.
3. Authors say that they targeted mitochondrial, trafficking, and motility (MTM) genes in human cellular models. While mitochondrial and trafficking is clear in the context of Parkinson and neurodegnerative disease, less clear is the motility genes. Please expand on this.

Significance

This is a well written, comprehensive study with a well characterized, robust CRISPR-based gene activation and ablation screening pipeline to identify novel regulators of α-synuclein pathology. Methodology is rigorous and clearly described and results are well presented. The major limitation relays in the validation experiments where only one main read-out that is p-αSyn129 fluorescence signal is employed, limiting the significance and impact of the presented results. I believe that the basic science community might benefit principally of the proposed methodology of a large high-throughput screening to modulate a large set of genes, a platform that in principle might be used also for other scientific questions.

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Referee #2

Evidence, reproducibility and clarity

In the present study, Neupane et al. performed arrayed CRISPR activation and ablation screens, targeting genes related to mitochondria, trafficking and motility, to identify genes that modulate the presence of Ser 129 phosphorylated alpha-synuclein aggregates (pSyn129) upon administration of exogenous preformed alpha-synuclein fibrils. The screens have been performed in HEK cells stably overexpressing alpha-synuclein in two independent replicates, and hits have been further validated in induced pluripotent stem cell derived forebrain and dopaminergic neurons. Following functional validations, the authors conclude that enhancing the expression of OXR1 results in a modest increase in the number of pSyn129 puncta within cells, and their size, while partial loss of EMC4 expression reduces these puncta. To date some pre-print studies have used genome-wide CRISPR screening to identify modifiers of the accumulation of alpha-synuclein preformed fibrils in cells, suggesting the importance of uptake and endolysosomal trafficking for the propagation of alpha-synuclein aggregates in recipient cells. Although the topic is of interest in the field of Parkinson's disease and synucleinopathies in general, the readout of the present screen (presence of pSyn129) is not very sensitive and without investigating endogenous alpha-synuclein or cell homeostasis in neuronal models limits the stated conclusions.

Major comments:

• Please clarify whether the positive control genes RAB13 and PIKFYVE were nominated hits within the CRISPR screens. Specifically, the authors state that the positive control of the CRISPRa screen was RAB13, expected to reduce pSyn129 upon overexpression, nevertheless this gene does not appear as a hit in the CRISPRa volcano plot (although present in table S1 but not making the cutoff). In figure 2D, activation of RAB13 does not seem to impact the main readout phenotype. Moreover, in the CRISPRo screen, PIKFYVE was used, but this gene is also not presented as a hit linked to reducing pSyn129 in the CRISPRo plot. If these control genes do not come up as hits, it is difficult to support the conclusions of the screen.
• The effect size for screen hits presented in figure 2A/B is rather small. It is difficult to interpret the power of these findings in the absence of uptake efficiency controls, such as dextrans of appropriate molecular weights.
• The readout of the screen is not very sensitive, and it is unclear what it represents. Specifically, in Figure 2F, G the authors validate the hits OXR1 and EMC4, showing a small effect, albeit statistically significant. The authors should strengthen this data by adding more experiments addressing, for instance, what the pSyn spot area and spot intensity signify for the cell. Some experiments in a neuronal context are important, including SNCA KO as a negative control.
• It is unclear why the authors chose to follow up on the OXR1 and EMC4 hits. Please explain the rationale for follow-up studies.
• Generally, the notable difference in the number of pSyn129+ cells in the non-targeting across various experiments (including Fig.1G/I compared to Fig.2G/I or Fig. 3F/G or flow cytometry experiment) suggests the readout is not very sensitive.

For instance, in figure S3 it would be important to add an experiment controlling for cell number as opposed to LDH release, as the micrographs show some differences in cells number, e.g. in the ntg vs. EMC4 condition. - The data is not sufficient to suggest that OXR1 and EMC4 are strong modulators of alpha-synuclein aggregation, as the authors suggest based on figures 2 and 3 that show statistically significant difference and a rather small effect size. It is important to provide more insight into how these genes may affect endogenous alpha-synuclein and cellular homeostasis in more detail, especially in neuronal models. Further investigating the hits in this direction in additional genetic backgrounds would also increase the relevance of the findings, e.g. in SNCA triplication or GBA-PD neurons.<br /> In Fig. S8B the immunoblot analysis shows there may be an effect of EMC4 and OXR1 CRISPRa on α-synuclein levels; please quantify for both iPSC-derived cortical neurons and dopaminergic neurons. - The pattern of tyrosine hydroxylase staining in Figure 5F does not seem specific or as expected for iPSC-derived dopaminergic neurons. Furthermore, since endogenous SNCA expression is expected to be analogous to the expression of TH (with TH+ cells expressing higher SNCA), it would be important to compare pSyn129 between TH+ cells and/or relative to the TH+ area.

Minor comments:

• The authors report that RAB13 overexpression reduces pSyn129⁺ prevalence, whereas RAB13 ablation (CRISPRo screen) enhances pSyn129⁺ levels (Figures 2D-2E). Please revise as these specific figures show no effects for this gene.
• Please specify how many individual cells (approximately) were quantified in each figure legend.
• Figure 3F/G may be better as a supplemental figure since it does not add to the conclusions of the study.
• It would be good to clarify for the reader some of the genes that serve as positive controls for the screen's readout (as shown in Fig. S2D/G).
• It would be helpful to further clarify which cell type was used in each figure legend.

Significance

Important topic but their experimental design limits the significance of their findings. Hard to improve the work in a reasonable amount of time. Also many technical issues.

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Referee #1

Evidence, reproducibility and clarity

Summary:

This study by Neupane et al. investigates modulators of α-synuclein aggregation, focusing on Ser129-phosphorylated α-synuclein (pSyn129), a pathological hallmark of Parkinson's disease (PD). The authors performed high-content image-based, arrayed CRISPR activation (CRISPRa) and knockout (CRISPRo) screens targeting > 2300 genes related to mitochondrial function, intracellular trafficking, and cytoskeletal reorganization. Using α-Syn overexpressing HEK293 cells, they identified OXR1 and EMC4 as novel modulators of pSyn129 abundance. Key findings were that activation of the mitochondrial protein OXR1 increased pSyn129 by decreasing ATP levels, while ablation of the ER-associated protein EMC4 reduced pSyn129 by enhancing autophagic flux and lysosomal clearance. These findings were validated in human iPSC-derived cortical and dopaminergic neurons.

My major comments have to do with statistical methods and with significance of their findings.

Major comments:

Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?

The claims and conclusions are generally well-supported by the presented data. The dual CRISPRa/CRISPRo screen provides a robust initial discovery platform, and the validation in iPSC-derived neurons strengthens the findings and their translational relevance. The mechanistic insights into OXR1 (ATP levels) and EMC4 (autophagic flux, lysosomal clearance) are supported by the described experiments. The use of two antibodies (81A and EP1536Y) for pSyn129 also enhances confidence in the measurements. I had a few questions about the statistical methods. The main concern I have about methodology for the screen is whether the authors have corrected for multiple hypotheses in their discovery screen. This is not clear from the text, methods, or legends (for Figures 2A/2B/2C).

• Figure 1B suggests a very large range of activation (multiple orders of magnitude) in the initial screen. What is the relationship between level of expression change and functional effect across the screen? How upregulated/downregulated are OXR1 and EMC4 at the mRNA and protein levels?
• Supplemental Figure S2D: Why do the non-targeting controls differ from the majority of the CRISPRa genes? If I am reading the figure correctly, it seems strange that the vast majority of the CRISPRa gene targets reduces pSyn pathology relative to the non-targeting controls (which is why I am wondering whether the level of increased expression correlates with the level of functional effect).
• In Figure 2A/B/C, is the p-value adjusted in any way for multiple comparisons? If so, this should be indicated in the legend. If not, why not? (The potential for false positives in a screen is very large and requires correction for multiple comparisons.)
• Figure 3: It's interesting that different seeding materials have different effects. However, it's quite surprising that the authors find less seeding with MSA-derived material in both the CRISPRa and CRISPRo context. This contradicts the work of Peng and coauthors (PMID 29743672) who find that MSA-derived material is much more potent in seeding aggregates in a number of different cell types. Do the authors have any thoughts about why this is the case?
• Figure 7A: pSyn129 image in the non-targeting control is poor - the very bright dots look like artifact. Not clear why the authors don't corroborate with EP1536Y antibody as they do in Figure 5.
• Overall methodology: Are the pSyn inclusions soluble? This could be easily determined by performing 1% TritonX extraction, for example, and it helps us understand how "pathological" the inclusions are.
• OPTIONAL: The authors perform some interesting experiments looking at genes affected downstream by, for example, OXR1 over-expression. It would be useful to understand whether the upstream effect is dependent on downstream effect. This could be tested by performing double perturbations (e.g. OXR1 overexpression and CCL8 knockout or ALDOC upregulation).
• OPTIONAL: The link between EMC4 ablation and enhanced ER-driven autophagic flux/lysosomal clearance could be corroborated with additional experiments. E.g.: Does EMC4 normally inhibit this pathway? Or only in the context of aSyn fibril seeding?

Are the suggested experiments realistic in terms of time and resources?

The OPTIONAL experiments are generally feasible as they employ methods that the lab is already using in this paper.

Are the experiments adequately replicated and statistical analysis adequate?

See comment about multiple hypothesis testing above.

Significance

This is a well-designed, difficult-to-accomplish study that expands the landscape of pS129Syn modulators. The validation of the primary hits identified in HEK293 cells in iPSC-derived neurons gives the findings greater relevance.

Strengths:

• Novelty: Using an unbiased and high-throughput approach, the study identifies two novel regulators of α-Syn aggregation, namely OXR1 and EMC4.
• Methodological Rigor: The use of arrayed CRISPRa/CRISPRo screens with high-content imaging is powerful and difficult to accomplish. Methodologically, this is a tour de force.
• Orthogonal Validation: The use of multiple α-Syn fibril polymorphs/strains and different antibodies (81A, EP1536Y) strengthens the robustness of the findings.

Limitations:

• It's not clear to me that pSyn129 is the ultimate readout. At a minimum, we should know something about the solubility of the inclusions. Some panels (e.g. Figure 7A) are not very informative in terms of what the authors are calling pSyn129+.
• The study relies on in vitro cellular models. While iPSC-derived neurons are relevant, the complexity of the brain environment, including glial cell interactions is not fully captured. This is fine for an initial report, but it does limit the significance.
• OXR1 and EMC4 seem to be very generic modulators. It's not clear to me that their effects are specific to aSyn or to PD in any way - they might just be effects on very basic cellular functions that would be applicable to a number of stressors or proteinopathies. Maybe that is fine (we probably need to get rid of tau aggregates, too!), but I don't think the authors can claim that they have identified "organelle-specific genetic nodes of aSyn pathology" since they biased their screen towards mitochondria and they don't test any other pathological aggregates. Moreover, from a translational perspective, it's not clear to me that implicating the antioxidant pathway or lysosomal/autophagosomal pathways in the pathogenesis of PD is new, and it's not clear that the specific genes identified would make good therapeutic targets.

OKAY -> takeaways - What Voting Influence tool reveals 1. ID must NOT be internally fractured in order to be pivotal (is not often the case) 2. For radical right to wield influence, THE PLENARY MUST BE VERY POLARISED 3. Overall voting influence remains limited in absolute terms -> usually joins coalitions that win by a LARGE majority, where it can therefore NOT wield a major influence. 4. In some cases where grand coalition is not formed, rad right is "crucial partner" for EPP (one of the mainstream preogressive parties)

"For the radical right group to hold voting influence, there must be a minimum level of polarisation in plenary, and the group itself must be internally cohesive."

So parties don't want to work w/ ID as opposed to CR (other less radical conservative party)

BUT voting share has increased much in past five years.

THIS article is going to MEASURE / EXAMINE THE VOTING INFLUENCE OF ID IN THE EP -> doing so by lookin at the "final stage of decision making in the EP" which is PLENARY VOTING

Voting influence (introduce term here): - "ability of a party group to sway the outcome of a vote" - Basically applies to PLENARY voting WITHIN the EP -. doesn't refer to ballot box.

Questions 1. What steps can be implemented when a social worker is attempting to understand the intersectionality of the client while maintaining mutual respect? 2. As an African American social worker that serves majority Caucasian clients, explain how cultural humility can help shape cultural competency.

How can educators create a classroom environment that helps children feel a sense of belonging and agency?

This explains that children may temporarily struggle or regress before mastering new skills, which is a normal part of development.

This sentence emphasizes that play is a powerful way children learn important skills, not just something for fun.

How can educators identify which children may be more sensitive to environmental influences without labeling them?

This sentence explains why early childhood is such an important time for learning and brain development.

This shows that children’s development is shaped by both their biology and their surroundings, not just one or the other.

This explains how inconsistent or missing care can interrupt healthy brain development during early childhood.

This sentence stood out because it shows that a lack of responsive caregiving can have serious long term effects on a child’s growth and health.

This sentence explains how everyday interactions between a child and a caregiver directly support the brain development and showing that learning starts through simple responsive communication.

Canon because they wanted to politically oppose to keep power

indyweb.link/hyperpost/~/indyweb/📓/20/25/11/3/setup/-/indy0wiki.pad/

Reviewer #2 (Public review):

Summary:

In this work, Gupta & Murphy present several parallel efforts. On one side, they present the hardware and software they use to build a head-fixed mouse experimental setup that they use to track in "real-time" the calcium activity in one or two spots at the surface of the cortex. On the other side, they present another setup that they use to take advantage of the "real-time" version of DeepLabCut with their mice. The hardware and software that they used/develop is described at length, both in the article and in a companion GitHub repository. Next, they present experimental work that they have done with these two setups, training mice to max out a virtual cursor to obtain a reward, by taking advantage of auditory tone feedback that is provided to the mice as they modulate either (1) their local cortical calcium activity, or (2) their limb position.

Strengths:

This work illustrates the fact that thanks to readily available experimental building blocks, body movement and calcium imaging can be carried out using readily available components, including imaging the brain using an incredibly cheap consumer electronics RGB camera (RGB Raspberry Pi Camera). It is a useful source of information for researchers that may be interested in building a similar setup, given the highly detailed overview of the system. Finally, it further confirms previous findings regarding the operant conditioning of the calcium dynamics at the surface of the cortex (Clancy et al. 2020) and suggests an alternative based on deeplabcut to the motor tasks that aim to image the brain at the mesoscale during forelimb movements (Quarta et al. 2022).

Weaknesses:

This work covers 3 separate research endeavors: (1) The development of two separate setups, their corresponding software. (2) A study that is highly inspired from the Clancy et al. 2021 paper on the modulation of the local cortical activity measured through a mesoscale calcium imaging setup. (3) A study of the mesoscale dynamics of the cortex during forelimb movements learning. Sadly, the analyses of the physiological data appears incomplete, and more generally, the paper shows weaknesses regarding several points:

The behavioral setups that are presented are representative of the state of the art in the field of mesoscale imaging/head fixed behavior community, rather than a highly innovative design. Still, they definitely have value as a starting point for laboratories interested in implementing such approaches.

Throughout the paper, there are several statements that point out how important it is to carry out this work in a closed-loop setting with an auditory feedback, but sadly there is no "no feedback" control in cortical conditioning experiments, while there is a no-feedback condition in the forelimb movement study, which shows that learning of the task can be achieved in the absence of feedback.

The analysis of the closed-loop neuronal data behavior lacks controls. Increased performance can be achieved by modulating actively only one of the two ROIs, this is not really analyzed, while this finding which does not match previous reports (Clancy et al. 2020) would be important to further examine.

Reviewer #3 (Public review):

Summary:

The study demonstrates the effectiveness of a cost-effective closed-loop feedback system for modulating brain activity and behavior in head-fixed mice. Authors have tested real-time closed-loop feedback system in head-fixed mice two types of graded feedback: 1) Closed-loop neurofeedback (CLNF), where feedback is derived from neuronal activity (calcium imaging), and 2) Closed-loop movement feedback (CLMF), where feedback is based on observed body movement. It is a python based opensource system, and the authors call it CLoPy. Authors also claim to provide all software, hardware schematics, and protocols to adapt it to various experimental scenarios. This system is capable and can be adapted for a wide use case scenarios.

Authors have shown that their system can control both positive (water drop) and negative reinforcement (buzzer-vibrator). This study also shows that using the closed-loop system, mice have shown to better performance, learnt arbitrary tasks and can adapt to changes in the rules as well. By integrating real-time feedback based on cortical GCaMP imaging and behavior tracking authors have provided strong evidence that such closed-loop systems can be instrumental in exploring the dynamic interplay between brain activity and behavior.

Strengths:

Simplicity of feedback systems design. Simplicity of implementation and potential adoption.

Weaknesses:

Long latencies, due to slow Ca2+ dynamics and slow imaging (15 FPS), may limit the application of the system.

Author response:

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

Public reviews:

Reviewer #1 (Public review):

Summary: 

The authors provide a resource to the systems neuroscience community, by offering their Python-based CLoPy platform for closed-loop feedback training. In addition to using neural feedback, as is common in these experiments, they include a capability to use real-time movement extracted from DeepLabCut as the control signal. The methods and repository are detailed for those who wish to use this resource. Furthermore, they demonstrate the efficacy of their system through a series of mesoscale calcium imaging experiments. These experiments use a large number of cortical regions for the control signal in the neural feedback setup, while the movement feedback experiments are analyzed more extensively.

Strengths:

The primary strength of the paper is the availability of their CLoPy platform. Currently, most closed-loop operant conditioning experiments are custom built by each lab and carry a relatively large startup cost to get running. This platform lowers the barrier to entry for closed-loop operant conditioning experiments, in addition to making the experiments more accessible to those with less technical expertise.

Another strength of the paper is the use of many different cortical regions as control signals for the neurofeedback experiments. Rodent operant conditioning experiments typically record from the motor cortex and maybe one other region. Here, the authors demonstrate that mice can volitionally control many different cortical regions not limited to those previously studied, recording across many regions in the same experiment. This demonstrates the relative flexibility of modulating neural dynamics, including in non-motor regions.

Finally, adapting the closed-loop platform to use real-time movement as a control signal is a nice addition. Incorporating movement kinematics into operant conditioning experiments has been a challenge due to the increased technical difficulties of extracting real-time kinematic data from video data at a latency where it can be used as a control signal for operant conditioning. In this paper they demonstrate that the mice can learn the task using their forelimb position, at a rate that is quicker than the neurofeedback experiments.

Weaknesses:

There are several weaknesses in the paper that diminish the impact of its strengths. First, the value of the CLoPy platform is not clearly articulated to the systems neuroscience community. Similarly, the resource could be better positioned within the context of the broader open-source neuroscience community. For an example of how to better frame this resource in these contexts, I recommend consulting the pyControl paper. Improving this framing will likely increase the accessibility and interest of this paper to a less technical neuroscience audience, for instance by highlighting the types of experimental questions CLoPy can enable.

We appreciate the editor’s feedback regarding the clarity of the CLoPy platform's value and its positioning within the broader neuroscience community. We agree and understand the importance of effectively communicating the utility of CLoPy to both the systems neuroscience field and the wider open-source neuroscience community.

To address this, we have revised the introduction and discussion sections of the manuscript to more clearly articulate the unique contributions of the CLoPy platform. Specifically:

(1) We have emphasized how CLoPy can address experimental questions in systems neuroscience by highlighting its ability to enable real-time closed-loop experiments, such as investigating neural dynamics during behavior or studying adaptive cortical reorganization after injury. These examples are aimed at demonstrating its practical utility to the neuroscience audience.

(2) We have positioned CLoPy within the broader open-source neuroscience ecosystem, drawing comparisons to similar resources like pyControl. We describe how CLoPy complements existing tools by focusing on real-time optical feedback and integration with genetically encoded indicators, which are becoming increasingly popular in systems neuroscience. We also emphasize its modularity and ease of adoption in experimental settings with limited resources.

(3) To make the manuscript more accessible to a less technically inclined audience, we have restructured certain sections to focus on the types of experiments CLoPy enables, rather than the technical details of the implementation.

We have consulted the pyControl paper, as suggested, and have used it as a reference point to improve the framing of our resource. We believe these changes will increase the accessibility and appeal of the paper to a broader neuroscience audience.

While the dataset contains an impressive amount of animals and cortical regions for the neurofeedback experiment, and an analysis of the movement-feedback experiments, my excitement for these experiments is tempered by the relative incompleteness of the dataset, as well as its description and analysis in the text. For instance, in the neurofeedback experiment, many of these regions only have data from a single mouse, limiting the conclusions that can be drawn. Additionally, there is a lack of reporting of the quantitative results in the text of the document, which is needed to better understand the degree of the results. Finally, the writing of the results section could use some work, as it currently reads more like a methods section.

Thank you for your thoughtful and constructive feedback on our manuscript. We appreciate the time and effort you took to review our work and provide detailed suggestions for improvement. Below, we address the key points raised in your review:

(1) Dataset Completeness: We acknowledge that some of the neurofeedback experiments include data from only a single mouse for some cortical regions while for some cortical regions, there are several animals. This was due to practical constraints during the study, and we understand the limitations this poses for drawing broad conclusions. We felt it was still important to include these data sets with smaller sample sizes as they might be useful for others pursuing this direction in the future. To address this, we have revised the text to explicitly acknowledge these limitations and clarify that the results for some regions are exploratory in nature. We believe our flexible tool will provide a means for our lab and others include more animals representing additional cortical regions in future studies. Importantly, we have included all raw and processed data as well as code for future analysis.

(2) Quantitative Results: We recognize the importance of reporting quantitative results in the text for better clarity and interpretation. In response, we have added more detailed description of the quantitative findings from both the neurofeedback and movement-feedback experiments. This will include effect sizes, statistical measures, and key numerical results to provide a clearer understanding of the degree and significance of the observed effects.

(3) Results Section Writing: We appreciate your observation that parts of the results section read more like a methods section. To improve clarity and focus, we have restructured the results section to present the findings in a more concise and interpretative manner, while moving overly detailed descriptions of experimental procedures to the methods section.

Suggestions for improved or additional experiments, data or analyses:

Not necessary for this paper, but it would be interesting to see if the CLNF group could learn without auditory feedback.

This is a great suggestion and certainly something that could be done in the future.

There are no quantitative results in the results section. I would add important results to help the reader better interpret the data. For example, in: "Our results indicated that both training paradigms were able to lead mice to obtain a significantly larger number of rewards over time," You could show a number, with an appropriate comparison or statistical test, to demonstrate that learning was observed.

Thank you for pointing this out. We have mentioned quantification values in the results now, along with being mentioned in the figure legends, and we are quoting it in following sentences. “A ΔF/F0 threshold value was calculated from a baseline session on day 0 that would have allowed 25% performance. Starting from this basal performance of around 25% on day 1, mice (CLNF No-rule-change, N=23, n=60 and CLNF Rule-change, N=17, n=60) were able to discover the task rule and perform above 80% over ten days of training (Figure 4A, RM ANOVA p=2.83e-5), and Rule-change mice even learned a change in ROIs or rule reversal (Figure 4A, RM ANOVA p=8.3e-10, Table 5 for different rule changes). There were no significant differences between male and female mice (Supplementary Figure 3A).”

For: "Performing this analysis indicated that the Raspberry Pi system could provide reliable graded feedback within ~63 {plus minus} 15 ms for CLNF experiments." The LED test shows the sending of the signal, but the actual delay for the audio generation might be longer. This is also longer than the 50 ms mentioned in the abstract.

We appreciate the reviewer’s insightful comment. The latency reported (~63ms) was measured using the LED test, which captures the time from signal detection to output triggering on the Raspberry Pi GPIO. We agree that the total delay for auditory feedback generation could include an additional latency component related to the digital-to-analog conversion and speaker response. In our setup, we employ a fast Audiostream library written in C to generate the audio signal and expect the delay contribution to be negligible compared to the GPIO latency. Though we did not do this, it can be confirmed by an oscilloscope-based pilot measurement (for additional delay calculation). We have updated the manuscript to clarify that the 63 ± 15 ms value reflects the GPIO-triggered output latency, and we have revised the abstract to accurately state the delay as “~63 ms” rather than 50 ms. This ensures consistency and avoids underestimation of the latency. We have corrected the LED latency for CLNF and CLMF experiments in the abstract as well.

It could be helpful to visualize an individual trial for each experiment type, for instance how the audio frequency changes as movement speed / calcium activity changes.

We have added Supplementary Figure 8 that contains this data where you can see the target cortical activity trace, target paw speed, rewards, along with the audio frequency generated.

The sample sizes are small (n=1) for a few groups. I am excited by the variety of regions recorded, so it could be beneficial for the authors to collect a few more animals to beef up the sample sizes.

We've acknowledged that some of the sample sizes are small. Importantly, we have included raw and processed data as well as code for future analysis. We felt it was still important to still include these data sets with smaller sample sizes as they might be useful for others pursuing this direction in the future.

I am curious as to why 60 trials sessions were used. Was it mostly for the convenience of a 30 min session, or were the animals getting satiated? If the former, would learning have occurred more rapidly with longer sessions?

This is a great observation and the answer is it was mostly due to logistical reasons. We tried to not keep animals headfixed for more than 45 minutes in each session as they become less engaged with long duration headfixed sessions. After headfixing them, it takes about 15 minutes to get the experiment going and therefore 30 - 40 minutes long recorded sessions seemed appropriate before they stop being engaged or before they get satiated in the task. We provided supplemental water after the sessions and we observed that they consumed water after the sessions so they were not fully satiated during the sessions even when they performed well in the task and got maximum rewards. We also had inter-trial rest periods of 10s that elongated the session duration. We think it would be interesting to explore the relationship between session duration(number of trials) and task learning progression over the days in a separate study.

Figure 4E is interesting, it seems like the changes in the distribution of deltaF was in both positive and negative directions, instead of just positive. I'd be curious as to the author's thoughts as to why this is the case. Relatedly, I don't see Figure 4E, and a few other subplots, mentioned in the text. As a general comment, I would address each subplot in the text.

We have split Figure 4 into two to keep the figures more readable. Previous Figure 4E-H are now Figure 5A-D in the revised manuscript. The online real-time CLNF sessions were using a moving window average to calculate ΔF/F₀  and the figures were generated by averaging the whole recorded sessions. We have added text in Methods under “Online ΔF/F₀calculation” and “Offline ΔF/F₀ calculation” sections making it clear about how we do our ΔF/F₀ normalization based on average fluorescence over the entire session. Using this method of normalization does increase the baseline so that some peaks appear to be below zero. Additionally, it is unclear what strategy animals are employing to achieve the rule specific target activity. The task did not constrain them to have a specific strategy for cortical activation - they were rewarded as long as they crossed the threshold in target ROI(s). For example, in 2-ROI experiments, to increase ROI1-ROI2 target activity, they could increase activity of ROI1 relative to ROI2 or decreased activity of ROI1 relative to ROI1 - both would have led to a reward as long as the result crossed the threshold.

We have now addressed and added reference to the figures in the text in Results under “Mice can explore and learn an arbitrary task, rule, and target conditions” and “Mice can rapidly adapt to changes in the task rule” sections - thanks for pointing this out.

For: "In general, all ROIs assessed that encompassed sensory, pre-motor, and motor areas were capable of supporting increased reward rates over time," I would provide a visual summary showing the learning curves for the different types of regions.

We have rewritten this section to emphasize that these conclusions were based on pooled data from multiple regions of interest. The sample sizes for each type of region are different and some are missing. We believe it would be incomplete and not comparable to present this as a regular analysis since the sample sizes were not balanced. We would be happy to dive deeper into this and point to the raw and processed dataset if anyone would like to explore this further by GitHub or other queries.

Relatedly, I would further explain the fast vs slow learners, and if they mapped onto certain regions.

Mice were categorized into fast or slow learners based on the slope of learning over days (reward progression over the days) as shown in Supplementary Figure 3C,D. Our initial aim was not to probe cortical regions that led to fast vs slow learning but this was a grouping we did afterwards. Based on the analysis we did, the fast learners included the sensory (V1), somatosensory (BC, HL), and motor (M1, M2) areas, while the slow learners included the motor (M1, M2), and higher order (TR, RL) cortical areas. Testing all dorsal cortical areas would be prudent to establish their role in fast or slow learning and it is an interesting future direction.

Also I would make the labels for these plots (e.g. Supp Fig3) more intuitive, versus the acronyms currently used.

We have made more expressive labels and explained the acronyms below the Supplementary Figure 3.

The CLMF animals showed a decrease in latency across learning, what about the CLNF animals? There is currently no mention in the text or figures.

We have now incorporated the CLNF task latency data into both the Results text and Figure 4C. Briefly, task latency decreased as performance improved, increased following a rule change, and then decreased again as the animals relearned the task. The previous Figure 4C has been updated to Figure 4D, and the former Figure 4D has been moved to Supplementary Figure 4E.

Reviewer #2 (Public review):

Summary:

In this work, Gupta & Murphy present several parallel efforts. On one side, they present the hardware and software they use to build a head-fixed mouse experimental setup that they use to track in "real-time" the calcium activity in one or two spots at the surface of the cortex. On the other side, the present another setup that they use to take advantage of the "real-time" version of DeepLabCut with their mice. The hardware and software that they used/develop is described at length, both in the article and in a companion GitHub repository. Next, they present experimental work that they have done with these two setups, training mice to max out a virtual cursor to obtain a reward, by taking advantage of auditory tone feedback that is provided to the mice as they modulate either (1) their local cortical calcium activity, or (2) their limb position.

Strengths:

This work illustrates the fact that thanks to readily available experimental building blocks, body movement and calcium imaging can be carried using readily available components, including imaging the brain using an incredibly cheap consumer electronics RGB camera (RGB Raspberry Pi Camera). It is a useful source of information for researchers that may be interested in building a similar setup, given the highly detailed overview of the system. Finally, it further confirms previous findings regarding the operant conditioning of the calcium dynamics at the surface of the cortex (Clancy et al. 2020) and suggests an alternative based on deeplabcut to the motor tasks that aim to image the brain at the mesoscale during forelimb movements (Quarta et al. 2022).

Weaknesses:

This work covers 3 separate research endeavors: (1) The development of two separate setups, their corresponding software. (2) A study that is highly inspired from the Clancy et al. 2020 paper on the modulation of the local cortical activity measured through a mesoscale calcium imaging setup. (3) A study of the mesoscale dynamics of the cortex during forelimb movements learning. Sadly, the analyses of the physiological data appears uncomplete, and more generally the paper tends to offer overstatements regarding several points:

In contrast to the introductory statements of the article, closed-loop physiology in rodents is a well-established research topic. Beyond auditory feedback, this includes optogenetic feedback (O'Connor et al. 2013, Abbasi et al. 2018, 2023), electrical feedback in hippocampus (Girardeau et al. 2009), and much more.

We have included and referenced these papers in our introduction section (quoted below) and rephrased the part where our previous text indicated there are fewer studies involving closed-loop physiology.

“Some related studies have demonstrated the feasibility of closed-loop feedback in rodents, including hippocampal electrical feedback to disrupt memory consolidation (Girardeau et al.2009), optogenetic perturbations of somatosensory circuits during behavior (O'Connor et al.2013), and more recent advances employing targeted optogenetic interventions to guide behavior (Abbasi et al. 2023).”

The behavioral setups that are presented are representative of the state of the art in the field of mesoscale imaging/head fixed behavior community, rather than a highly innovative design. In particular, the closed-loop latency that they achieve (>60 ms) may be perceived by the mice. This is in contrast with other available closed-loop setups.

We thank the reviewer for this thoughtful comment and fully agree that our closed-loop latency is larger than that achieved in some other contemporary setups. Our primary aim in presenting this work, however, is not to compete with the lowest possible latencies, but to provide an open-source, accessible, and flexible platform that can be readily adopted by a broad range of laboratories. By building on widely available and lower-cost components, our design lowers the barrier of entry for groups that wish to implement closed-loop imaging and behavioral experiments, while still achieving latencies well within the range that can support many biologically meaningful applications.

For example, our latency (~60 ms) remains compatible with experimental paradigms such as:

Motor learning and skill acquisition, where sensorimotor feedback on the scale of tens to hundreds of milliseconds is sufficient to modulate performance.

Operant conditioning and reward-based learning, in which reinforcement timing windows are typically broader and not critically dependent on sub-20 ms latencies.

Cortical state dependent modulation, where feedback linked to slower fluctuations in brain activity (hundreds of milliseconds to seconds) can provide valuable insight.

Studies of perception and decision-making, in which stimulus response associations often unfold on behavioral timescales longer than tens of milliseconds.

We believe that emphasizing openness, affordability, and flexibility will encourage widespread adoption and adaptation of our setup across laboratories with different research foci. In this way, our contribution complements rather than competes with ultra-low-latency closed-loop systems, providing a practical option for diverse experimental needs.

Through the paper, there are several statements that point out how important it is to carry out this work in a closed-loop setting with an auditory feedback, but sadly there is no "no feedback" control in cortical conditioning experiments, while there is a no-feedback condition in the forelimb movement study, which shows that learning of the task can be achieved in the absence of feedback.

We fully agree that such a control would provide valuable insight into the contribution of feedback to learning in the CLNF paradigm. In designing our initial experiments, we envisioned multiple potential control conditions, including No-feedback and Random-feedback. However, our first and primary objective was to establish whether mice could indeed learn to modulate cortical ROI activation through auditory feedback, and to further investigate this across multiple cortical regions. For this reason, we focused on implementing the CLNF paradigm directly, without the inclusion of these additional control groups. To broaden the applicability of the system, we subsequently adapted the platform to the CLMF experiments, where we did incorporate a No-feedback group. These results, as the reviewer notes, strengthen the evidence for the role of feedback in shaping task performance. We agree that the inclusion of a No-feedback control group in the CLNF paradigm will be crucial in future studies to further dissect the specific contribution of feedback to cortical conditioning.

The analysis of the closed-loop neuronal data behavior lacks controls. Increased performance can be achieved by modulating actively only one of the two ROIs, this is not clearly analyzed (for instance looking at the timing of the calcium signal modulation across the two ROIs. It seems that overall ROIs1 and 2 covariate, in contrast to Clancy et al. 2020. How can this be explained?

We agree that the possibility of increased performance being driven by modulation of a single ROI is an important consideration. Our study indeed began with 1-ROI closed-loop experiments. In those early experiments, while we did observe animals improving performance across days, we realized that daily variability in ongoing cortical GCaMP activity could lead to fluctuations in threshold-crossing events. The 2-ROI design was subsequently introduced to reduce this variability, as the target activity was defined as the relative activity between the two ROIs (e.g., ROI1 – ROI2). This approach offered a more stable signal by normalizing ongoing fluctuations. In our analysis of the early 2-ROI experiments, we observed that animals adopted diverging strategies to achieve threshold crossings. Specifically, some animals increased activity in ROI1 relative to ROI2, while others decreased activity in ROI2 to accomplish the same effect. Once discovered, each animal consistently adhered to its chosen strategy throughout subsequent training sessions. This was an early and intriguing observation, but as the experiments were not originally designed to systematically test this effect, we limited our presentation to the analysis of a small number of animals (shown in Figure 11). We have added details about this observation in our Results section as well, quoted below-

“In the 2-ROI experiment where the task rule required “ROI1 - ROI2” activity to cross a threshold for reward delivery, mice displayed divergent strategies. Some animals predominantly increased ROI1 activity, whereas others reduced ROI2 activity, both approaches leading to successful threshold crossing (Figure 11)”.

We hope this clarifies how the use of two ROIs helps explain the apparent covariation of the signals, and why some divergence from the observations of Clancy et al. (2020) may be expected.

Reviewer #3 (Public review):

Summary:

The study demonstrates the effectiveness of a cost-effective closed-loop feedback system for modulating brain activity and behavior in head-fixed mice. Authors have tested real-time closed-loop feedback system in head-fixed mice two types of graded feedback: 1) Closed-loop neurofeedback (CLNF), where feedback is derived from neuronal activity (calcium imaging), and 2) Closed-loop movement feedback (CLMF), where feedback is based on observed body movement. It is a python based opensource system, and authors call it CLoPy. The authors also claim to provide all software, hardware schematics, and protocols to adapt it to various experimental scenarios. This system is capable and can be adapted for a wide use case scenario.

Authors have shown that their system can control both positive (water drop) and negative reinforcement (buzzer-vibrator). This study also shows that using the close loop system mice have shown better performance, learnt arbitrary task and can adapt to change in the rule as well. By integrating real-time feedback based on cortical GCaMP imaging and behavior tracking authors have provided strong evidence that such closed-loop systems can be instrumental in exploring the dynamic interplay between brain activity and behavior.

Strengths:

Simplicity of feedback systems designed. Simplicity of implementation and potential adoption.

Weaknesses:

Long latencies, due to slow Ca2+ dynamics and slow imaging (15 FPS), may limit the application of the system.

We appreciate the reviewer’s comment and agree that latency is an important factor in our setup. The latency arises partly from the inherent slow kinetics of calcium signaling and GCaMP6s, and partly from the imaging rate of 15 FPS (every 66 ms). These limitations can be addressed in several ways: for example, using faster calcium indicators such as GCaMP8f, or adapting the system to electrophysiological signals, which would require additional processing capacity. In our implementation, image acquisition was fixed at 15 FPS to enable real-time frame processing (256 × 256 resolution) on Raspberry Pi 4B devices. With newer hardware, such as the Raspberry Pi 5, substantially higher acquisition and processing rates are feasible (although we have not yet benchmarked this extensively). More powerful platforms such as Nvidia Jetson or conventional PCs would further support much faster data acquisition and processing.

Major comments:

(1) Page 5 paragraph 1: "We tested our CLNF system on Raspberry Pi for its compactness, general-purpose input/output (GPIO) programmability, and wide community support, while the CLMF system was tested on an Nvidia Jetson GPU device." Can these programs and hardware be integrated with windows-based system and a microcontroller (Arduino/ Tency). As for the broad adaptability that's what a lot of labs would already have (please comment/discuss)?

While we tested our CLNF system on a Raspberry Pi (chosen for its compactness, GPIO programmability, and large user community) and our CLMF system on an Nvidia Jetson GPU device (to leverage real-time GPU-based inference), the underlying software is fully written in Python. This design choice makes the system broadly adaptable: it can be run on any device capable of executing Python scripts, including Windows-based PCs, Linux machines, and macOS systems. For hardware integration, we have confirmed that the framework works seamlessly with microcontrollers such as Arduino or Teensy, requiring only minor modifications to the main script to enable sending and receiving of GPIO signals through those boards. In fact, we are already using the same system in an in-house project on a Linux-based PC where an Arduino is connected to the computer to provide GPIO functionality. Furthermore, the system is not limited to Raspberry Pi or Arduino boards; it can be interfaced with any GPIO-capable devices, including those from Adafruit and other microcontroller platforms, depending on what is readily available in individual labs. Since many neuroscience and engineering laboratories already possess such hardware, we believe this design ensures broad accessibility and ease of integration across diverse experimental setups.

(2) Hardware Constraints: The reliance on Raspberry Pi and Nvidia Jetson (is expensive) for real-time processing could introduce latency issues (~63 ms for CLNF and ~67 ms for CLMF). This latency might limit precision for faster or more complex behaviors, which authors should discuss in the discussion section.

In our system, we measured latencies of approximately ~63 ms for CLNF and ~67 ms for CLMF. While such latencies indeed limit applications requiring millisecond precision, such as fast whisker movements, saccades, or fine-reaching kinematics, we emphasize that many relevant behaviors, including postural adjustments, limb movements, locomotion, and sustained cortical state changes, occur on timescales that are well within the capture range of our system. Thus, our platform is appropriate for a range of mesoscale behavioral studies that probably needs to be discussed more. It is also important to note that these latencies are not solely dictated by hardware constraints. A significant component arises from the inherent biological dynamics of the calcium indicator (GCaMP6s) and calcium signaling itself, which introduce slower temporal kinetics independent of processing delays. Newer variants, such as GCaMP8f, offer faster response times and could further reduce effective biological latency in future implementations.

With respect to hardware, we acknowledge that Raspberry Pi provides a low-cost solution but contributes to modest computational delays, while Nvidia Jetson offers faster inference at higher cost. Our choice reflects a balance between accessibility, cost-effectiveness, and performance, making the system deployable in many laboratories. Importantly, the modular and open-source design means the pipeline can readily be adapted to higher-performance GPUs or integrated with electrophysiological recordings, which provide higher temporal resolution. Finally, we agree with the reviewer that the issue of latency highlights deeper and interesting questions regarding the temporal requirements of behavior classification. Specifically, how much data (in time) is required to reliably identify a behavior, and what is the minimum feedback delay necessary to alter neural or behavioral trajectories? These are critical questions for the design of future closed-loop systems and ones that our work helps frame.

We have added a slightly modified version of our response above in the discussion section under “Experimental applications and implications”.

(3) Neurofeedback Specificity: The task focuses on mesoscale imaging and ignores finer spatiotemporal details. Sub-second events might be significant in more nuanced behaviors. Can this be discussed in the discussion section?

This is a great point  and we have added the following to the discussion section. “In the case of CLNF we have focused on regional cortical GCAMP signals that are relatively slow in kinetics. While such changes are well suited for transcranial mesoscale imaging assessment, it is possible that cellular 2-photon imaging (Yu et al. 2021) or preparations that employ cleared crystal skulls (Kim et al. 2016) could resolve more localized and higher frequency kinetic signatures.”

(4) The activity over 6s is being averaged to determine if the threshold is being crossed before the reward is delivered. This is a rather long duration of time during which the mice may be exhibiting stereotyped behaviors that may result in the changes in DFF that are being observed. It would be interesting for the authors to compare (if data is available) the behavior of the mice in trials where they successfully crossed the threshold for reward delivery and in those trials where the threshold was not breached. How is this different from spontaneous behavior and behaviors exhibited when they are performing the test with CLNF? 

We would like to emphasize that we are not directly averaging activity over 6 s to compare against the reward threshold. Instead, the preceding 6 s of activity is used solely to compute a dynamic baseline for ΔF/F₀ ( ΔF/F₀ = (F –F₀ )/F₀). Here, F₀is calculated as the mean fluorescence intensity over the prior 6 s window and is updated continuously throughout the session. This baseline is then subtracted from the instantaneous fluorescence signal to detect relative changes in activity. The reward threshold is therefore evaluated against these baseline-corrected ΔF/F₀ values at the current time point, not against an average over 6 s. This moving-window baseline correction is a standard approach in calcium imaging analyses, as it helps control for slow drifts in signal intensity, bleaching effects, or ongoing fluctuations unrelated to the behavior of interest. Thus, the 6-s window is not introducing a temporal lag in reward assignment but is instead providing a reference to detect rapid increases in cortical activity.  We have added the term dynamic baseline to the Methods to clarify.

Recommendations for the authors

Reviewer #1 (Recommendations for the authors):

Additional suggestions for improved or additional experiments, data or analyses.

For: "Looking closely at their reward rate on day 5 (day of rule change), they had a higher reward rate in the second half of the session as compared to the first half, indicating they were adapting to the rule change within one session." It would be helpful to see this data, and would be good to see within-session learning on the rule change day

Thank you for pointing this out. We had missed referencing the figure in the text, and have now added a citation to Supplementary Figure 4A, which shows the cumulative rewards for each day of training. As seen in the plot for day 5, the cumulative rewards are comparable to those on day 1, with most rewards occurring during the second half of the session.

For: "These results suggest that motor learning led to less cortical activation across multiple regions, which may reflect more efficient processing of movement-related activity," it could also be the case that the behaviour became more stereotyped over learning, which would lead to more concentrated, correlated activity. To test this, it would be good to look at the limb variability across sessions. Similarly, if it is movement-related, there should be good decoding of limb kinematics.

Indeed, we observed that behavior became more stereotyped over the course of learning, as shown in Supplementary Figure 4C, 4D. One plausible explanation for the reduction in cortical activation across multiple regions is that behavior itself became more stereotyped, a possibility we have explored in the manuscript. Specifically, forelimb movements during the trial became increasingly correlated as mice improved on the task, particularly in the groups that received auditory feedback (Rule-change and No-rule-change groups; Figure 8). As movements became more correlated, overall body movements during trials decreased and aligned more closely with the task rule (Figure 9D). This suggests that reduced cortical activity may in part reflect changes in behavior. Importantly, however, in the Rule-change group, we observed that on the day of the rule switch (day 5), when the target shifted from the left to the right forelimb, cortical activity increased bilaterally (Figure 9A–C). This finding highlights our central point: groups that received feedback (Rule-change and No-rule-change) were able to identify the task rule more effectively, and both their behavior and cortical activity became more specifically aligned with the rule compared to the No-feedback group. We agree with the reviewers that additional analyses along these lines would be valuable future directions. To facilitate this, we have included the movement data for readers who may wish to pursue further analyses, details can be found under “Data and code availability” in Methods section. However, given the limited sample sizes in our dataset and the need to keep the manuscript focused on the central message, we felt that including these additional analyses here would risk obscuring the main findings.

For: "We believe the decrease in ΔF/F0peak is unlikely to be driven by changes in movement, as movement amplitudes did not decrease significantly during these periods (Figure 7D CLMF Rule-change)." I would formally compare the two conditions. This is an important control. Also, another way to see if the change in deltaF is related to movement would be to see if you can predict movement from the deltaF.

Figure 7D in the previous version is Figure 9D in the current revision of the manuscript. We've assessed this for the examples shown based on graphing the movement data, unfortunately there is not enough of that data to do a group analysis of movement magnitude. We would suggest that this would be an excellent future direction that would take advantage of the flexible open source nature of our tool.

Recommendations for improving the writing and presentation.

In the abstract there is no mention of the rationale for the project, or the resulting significance. I would modify this to increase readership by the behavioral neuroscience community. Similarly, the introduction also doesn't highlight the value of this resource for the field. Again, I think the pyControl paper does a good job of this. For readability, I would add more subheadings earlier in the results, to separate the different technical aspects of the system.

We have revised the introduction to include the rationale for the project, its potential implications, and its relevance for translational research. We have also framed the work within the broader context of the behavioral and systems neuroscience community. We greatly appreciate this suggestion, as we believe it enhances the clarity and accessibility of the manuscript for the community.

For: "While brain activity can be controlled through feedback, other variables such as movements have been less studied, in part because their analysis in real time is more challenging." I would highlight research that has studied the control of behavior through feedback, such as the Mathis paper where mice learn to pull a joystick to a virtual box, and adapt this motion to a force perturbation.

We have added a citation to the Mathis paper and describe this as an additional form of feedback. The text is quoted below:

“Opportunities also exist in extending real time pose classification (Forys et al. 2020; Kane et al. 2020) and movement perturbation (Mathis et al. 2017) to shape aspects of an animal’s motor repertoire.”

Some of the results content would be better suited for the methods, one example: "A previous version of the CLNF system was found to have non-linear audio generation above 10 kHz, partly due to problems in the audio generation library and partly due to the consumer-grade speaker hardware we were employing. This was fixed by switching to the Audiostream (https://github.com/kivy/audiostream) library for audio generation and testing the speakers to make sure they could output the commanded frequencies"

This is now moved to the Methods section.

For: "There are reports of cortical plasticity during motor learning tasks, both at cellular and mesoscopic scales (17-19), supporting the idea that neural efficiency could improve with learning," not sure I agree with this, the studies on cortical plasticity are usually to show a neural basis for the learning observed, efficiency is separate from this.

We have modified this statement to remove the concept of efficiency "There are reports of cortical plasticity during motor learning tasks, both at cellular and mesoscopic scales (17-19).”

The paragraph that opens "Distinct task- and reward-related cortical dynamics" that describes the experiment should appear in the previous section, as the data is introduced there.

We have moved the mentioned paragraphs in the previous section where we presented the data and other experiment details. This makes the text more readable and contextual.

I would present the different ROI rules with better descriptors and visualization to improve the readability.

We have added Supplementary Figure 7, which provides visualizations of the ROIs across all task rules used in the CLNF experiments.

Minor corrections to the text and figures.

Figure 1 is a little crowded, combining the CLNF and CLMF experiments, I would turn this into a 2 panel figure, one for each, similar to how you did figure 2.

We have revised Figure 1 to include two panels, one for CLNF and one for CLMF. The colored components indicate elements specific to each setup, while the uncolored components represent elements shared between CLNF and CLMF. Relevant text in the manuscript is updated to refer to these figures.

For Figure 2, the organization of the CLMF section is not intuitive for the reader. I would reorder it so it has a similar flow as the CLNF experiment.

We have revised the figure by updating the layout of panel B (CLMF) to align with panel A (CLNF), thereby creating a more intuitive and consistent flow between the panels. We appreciate this helpful suggestion, which we believe has substantially improved the clarity of the figure. The corresponding text in the manuscript has also been updated to reflect these changes.

For Figure 3, highlight that C and E are examples. They also seem a little out of place, so they could even be removed.

We have now explicitly labeled Figures 3C and 3E as representative examples (figure legend and on figure itself). We believe including these panels provides helpful context for readers: Figure 3C illustrates how the ROIs align on the dorsal cortical brain map with segmented cortical regions, while Figure 3E shows example paw trajectories in three dimensions, allowing visualization of the movement patterns observed during the trials.

In the plots, I would add sample sizes, for instance, in CLNF learning curve in Figure 4A, how many animals are in each group? 

We have labeled Figure 4 with number of animals used in CLNF (No-rule-change, N=23; Rule-change, N=17), and CLMF (Rule-change, N=8; No-rule-change, N=4; No-feedback, N=4).

Also, Figure 7 for example, which figures are single-sessions, versus across animals? For Figure 7c, what time bin is the data taken from?

We have clarified this now and mentioned it in all the figures. Figure 7 in the previous version is Figure 9 in the current updated manuscript. Figure 9A is from individual sessions on different days from the same mouse. Figure 9B is the group average reward centered ΔF/F₀ activity in different cortical regions (Rule-change, N=8; No-rule-change, N=4; No-feedback, N=4). Figure 9C shows average ΔF/F₀ peak values obtained within -1sec to +1sec centered around the reward point (N=8).

It says "punish" in Figure 3, but there is no punishment?

Yes, the task did not involve punishment. Each trial resulted in either a success, which is followed by a reward, or a failure, which is followed by a buzzer sound. To better reflect these outcomes, we have updated Figure 3 and replaced the labels “Reward” with “Success” and “Punish” with “Failure.”

The regression on 5c doesn't look quite right, also this panel is not mentioned in the text.

The figure referred to by the reviewer as Figure 5 is now presented as Figure 6 in the revised manuscript. Regarding the reviewer’s observation about the regression line in the left panel of Figure 5C, the apparent misalignment arises because the majority of the data points are densely clustered at the center of the scatter plot, where they overlap substantially. The regression line accurately reflects this concentration of overlapping data. To improve clarity, we have updated the figure and ensured that it is now appropriately referenced in the Results section.

Reviewer #2 (Recommendations for the authors):

(1) There would be many interesting observations and links between the peripheral and cortical studies if there was a body video available during the cortical study. Is there any such data available?

We agree that a detailed analysis of behavior during the CLNF task would be necessary to explore any behavior correlates with success in the task. Unfortunately, we do not have a sufficient video of the whole body to perform such an analysis.

(2) The text (p. 24) states: [intracortical GCAMP transients measured over days became more stereotyped in kinetics and were more correlated (to each other) as the task performance increased over the sessions (Figure 7E).] But I cannot find this quantification in the figures or text?

Figure 7 in the previous version of the manuscript now appears as Figure 9. In this figure, we present cortical activity across selected regions during trials, and in Figure 9E we highlight that this activity becomes more correlated. Since we did not formally quantify variability, we have removed the previous claim that the activity became stereotyped and revised the text in the updated manuscript accordingly.

Typos:

10-serest c (page 13)

Inverted color codes in figure 4E vs F

Reviewer #3 (Recommendations for the authors):

We have mostly attempted to limit the feedback to suggestions and posed a few questions that might be interesting to explore given the dataset the authors have collected.

Comments:

In close loop systems the latency is primary concern, and authors have successfully tested the latency of the system (Delay): from detection of an event to the reaction time was less than 67ms.

We have commented on the issues and limitations caused by latency, and potential future directions to overcome these challenges in responses to some of the previous comments.

Additional major comments:

"In general, all ROIs assessed that encompassed sensory, pre-motor, and motor areas were capable of supporting increased reward rates over time (Figure 4A, Animation 1)." Fig 4A is merely showing change in task performance over time and does not have information regarding the changes observed specific to CLNF for each ROI.

We acknowledge that the sample size for individual ROI rules was not sufficient for meaningful comparisons. To address this limitation, we pooled the data across all the rules tested. The manuscript includes a detailed list of the rules along with their corresponding sample sizes for transparency.

A ΔF/F₀ threshold value was calculated from a baseline session on day 0 that would have allowed 25% performance. Starting from this basal performance of around 25% on day 1, mice (CLNF No-rule-change, n=28 and CLNF Rule-change, n=13). It is unclear what the replicates here are. Trials or mice? The corresponding Figure legend has a much smaller n value.

Thank you for pointing this out. We realized that we had not indicated the sample replicates in the figure, and the use of n instead of N for the number of animals may have been misleading. We have now corrected the notation and clarified this information in the figure to resolve the discrepancy.

What were the replicates for each ROI pairs evaluated?

Each ROI rule and number of mice and trials are listed in Table 5 and Table 6.

Our analysis revealed that certain ROI rules (see description in methods) lead to a greater increase in success rate over time than others (Supplementary Figure 3D). The Supplementary figures 3C and 3D are blurry and could use higher resolution images. 

We have increased the font size of the text that was previously difficult to read and re-exported the figure at a higher resolution (300 DPI). We believe these changes will resolve the issue.

Also, It will help the reader is a visual representation of the ROI pairs are provided, instead of the text view. One interesting question is whether there are anatomical biases to fast vs slow learning pairs (Directionality - anterior/posterior, distance between the selected ROIs etc). This could be interesting to tease apart.

We have added Supplementary Figure 7, which provides visualizations of the ROIs across all task rules used in the CLNF experiments. While a detailed investigation of the anatomical basis of fast versus slow learning cortical ROIs is beyond the scope of the present study, we agree that this represents an exciting future direction for further research.

How distant should the ROIs be to achieve increased task performance?

We appreciate this insightful question. We did not specifically test this scenario. In our study, we selected 0.3 × 0.3 mm ROIs centered on the standard AIBS mouse brain atlas (CCF). At this resolution, ROIs do not overlap, regardless of their placement in a two-ROI experiment. Furthermore, because our threshold calculations are based on baseline recordings, we expect the system would function for any combination of ROI placements. Nonetheless, exploring this systematically would be an interesting avenue for future experiments.

Figures:

I would leave out some of the methodological details such as the protocol for water restriction (Fig. 3) out of the legend. This will help with readability.

We have removed some of the methodological details, including those mentioned above, from the legend of Figure 3 in the updated manuscript.

Fig 1 and Fig 2: In my opinion, It would be easier for the reader if the current Fig. 2, which provides a high level description of CLNF and CLBF is presented as Fig. 1. The current Fig. 1, goes into a lot of methodological implementation details, and also includes a lot of programming jargon that is being introduced early in the paper that is hard to digest early on in the paper's narrative.

Thank you for the suggestion. In the new manuscript, Figure 1 and Figure 2 have been swapped.

Higher-resolution images/ plots are needed in many instances. Unsure if this is the pdf compression done by the manuscript portal that is causing this.

All figures were prepared in vector graphics format using the open-source software Inkscape. For this manuscript, we exported the images at 300 DPI, which is generally sufficient for publication-quality documents. The submission portal may apply additional processing, which could have resulted in a reduction in image quality. We will carefully review the final submission files and ensure that all figures are clear and of high quality.

The authors repeatedly show ROI specific analysis M1_L, F1_R etc. It will be helpful to provide a key, even if redundant in all figures to help the reader.

We have now included keys to all such abbreviations in all the figures.

There are also instances of editorialization and interpretation e.g., "Surprisingly, the "Rule-change" mice were able to discover the change in rule and started performing above 70% within a day of the rule change, on day 6" that would be more appropriate in the main body of the paper.

Thank you for pointing this out in the figure legend, and we have removed it now since we already discussed this in the Results.

Minor comments

(1) The description of Figure 1 is hard to follow and can be described better based on how the information is processed and executed in the system from source to processing and back. Using separated colors (instead of shaded of grey) for the neuro feedback and movement feedback would help as well. Common components could have a different color. The specification like the description of the config file should come later.

Figure 1 in the previous version is Figure 2 in the updated version. We have taken suggestions from other reviewers and made the figure easier to understand and split it into two panels with color coding Green for CLNF, Pink for CLMF specific parts while common shared parts are left without any color.

(2) Page 20 last paragraph:

Authors are neglecting that the rule change is done one day prior and the results that you see in the second half on the 6th day are not just because of the first half of the 6th day instead combined training on the 5th day (rule change) and then the first half of the 6th day. Rephrasing this observation is essential.

We have revised the text for clarity to indicate that the performance increase observed on day 6 is not necessarily attributable to training on that day. In fact, we noted and mentioned that mice began to perform the task better during the second half of the session on day 5 itself.

(3)  The method section description of the CLMF setup (Page no 39 first paragraph) is more detailed, a diagram of this setup would make it easy to follow and a better read.

We have made changes to the CLMF setup (Figure 1B) and CLMF schematic (Figure 2B) to make it easier to understand parts of the setup and flow of control.

Conclusion p.28-29 - attitudes of landowners could delay development or could initiate development andprovide another terminous on the railway networks. when lanowners acquisesced in hthe developemtn without participating in planning, the resulting town tended to provide accommodation for the mass market - question of the availability of captial to deveop and extend a resort. the role of the merchants, millowners and commercial entreprenurs are evident. when capital failed to materialize, there was a hiatus in the development of the town. This dependence on capital from outside wales may be compared with the experience of industrialisation in nineteenth-century wales

in conclusion, he argues that agricultural wales was unabl or unwilling to fund the process of urbanisation, it wasn't unique to wales and needed capital injection from landowning elites or commercial capital from relatively new ndustries.

development had 3 major interlinking components - necessity to have incolvement of elites, capital to be invested for long periods before good rate of return, and railways to transport holiday makers quickly and cheaply two and from the resorts

page 3 i guess for this one, the main argument would be that industrialisation and tourism had a relationship through industrialisations facilitation of tourism (it goes with the post it note i wrote tbh)

Reviewer #3 (Public review):

Summary:

This manuscript investigates the regulation of host-seeking behavior in Anopheles stephensi females across different life stages and mating states. Through transcriptomic profiling, the authors identify differential gene expression between "blood-hungry" and "blood-sated" states. Two neuropeptides, sNPF and RYamide, are highlighted as potential mediators of host-seeking behavior. RNAi knockdown of these peptides alters host-seeking activity, and their expression is anatomically mapped in the mosquito brain (sNPF and RYamide) and midgut (sNPF only).

Strengths:

(1) The study addresses an important question in mosquito biology, with relevance to vector control and disease transmission.

(2) Transcriptomic profiling is used to uncover gene expression changes linked to behavioral states.

(3) The identification of sNPF and RYamide as candidate regulators provides a clear focus for downstream mechanistic work.

(3) RNAi experiments demonstrate that these neuropeptides are necessary for normal host-seeking behavior.

(4) Anatomical localization of neuropeptide expression adds depth to the functional findings.

Weaknesses:

(1) The title implies that the neuropeptides promote host-seeking, but sufficiency is not demonstrated and some conclusions appear premature based on the current data. The support for this conclusion would be strengthened with functional validation using peptide injection or genetic manipulation.

(2) The identification of candidate receptors is promising, but the manuscript would be significantly strengthened by testing whether receptor knockdowns phenocopy peptide knockdowns. Without this, it is difficult to conclude that the identified receptors mediate the behavioral effects.

(3) Some important caveats, such as variation in knockdown efficiency and the possibility of off-target effects, are not adequately discussed.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

Bansal et al. present a study on the fundamental blood and nectar feeding behaviors of the critical disease vector, Anopheles stephensi. The study encompasses not just the fundamental changes in blood feeding behaviors of the crucially understudied vector, but then uses a transcriptomic approach to identify candidate neuromodulation pathways which influence blood feeding behavior in this mosquito species. The authors then provide evidence through RNAi knockdown of candidate pathways that the neuromodulators sNPF and Rya modulate feeding either via their physiological activity in the brain alone or through joint physiological activity along the brain-gut axis (but critically not the gut alone). Overall, I found this study to be built on tractable, well-designed behavioral experiments.

Their study begins with a well-structured experiment to assess how the feeding behaviors of A. stephensi change over the course of its life history and in response to its age, mating, and oviposition status. The authors are careful and validate their experimental paradigm in the more well-studied Ae. aegypti, and are able to recapitulate the results of prior studies, which show that mating is a prerequisite for blood feeding behaviors in Ae. aegypt. Here they find A. Stephensi, like other Anopheline mosquitoes, has a more nuanced regulation of its blood and nectar feeding behaviors.

The authors then go on to show in a Y-maze olfactometer that ,to some degree, changes in blood feeding status depend on behavioral modulation to host cues, and this is not likely to be a simple change to the biting behaviors alone. I was especially struck by the swap in valence of the host cues for the blood-fed and mated individuals, which had not yet oviposited. This indicates that there is a change in behavior that is not simply desensitization to host cues while navigating in flight, but something much more exciting is happening.

The authors then use a transcriptomic approach to identify candidate genes in the blood-feeding stages of the mosquito's life cycle to identify a list of 9 candidates that have a role in regulating the host-seeking status of A. stephensi. Then, through investigations of gene knockdown of candidates, they identify the dual action of RYa and sNPF and candidate neuromodulators of host-seeking in this species. Overall, I found the experiments to be well-designed. I found the molecular approach to be sound. While I do not think the molecular approach is necessarily an all-encompassing mechanism identification (owing mostly to the fact that genetic resources are not yet available in A. stephensi as they are in other dipteran models), I think it sets up a rich line of research questions for the neurobiology of mosquito behavioral plasticity and comparative evolution of neuromodulator action.

We appreciate the reviewer’s detailed summary of our work. We thank them for their positive comments and agree with them on the shortcomings of our approach.

Strengths:

I am especially impressed by the authors' attention to small details in the course of this article. As I read and evaluated this article, I continued to think about how many crucial details could potentially have been missed if this had not been the approach. The attention to detail paid off in spades and allowed the authors to carefully tease apart molecular candidates of blood-seeking stages. The authors' top-down approach to identifying RYamide and sNPF starting from first principles behavioral experiments is especially comprehensive. The results from both the behavioral and molecular target studies will have broad implications for the vectorial capacity of this species and comparative evolution of neural circuit modulation.

We really appreciate that the reviewer has recognised the attention to detail we have tried to put, thank you!

Weaknesses:

There are a few elements of data visualizations and methodological reporting that I found confusing on a first few read-throughs. Figure 1F, for example, was initially confusing as it made it seem as though there were multiple 2-choice assays for each of the conditions. I would recommend removing the "X" marker from the x-axis to indicate the mosquitoes did not feed from either nectar, blood, or neither in order to make it clear that there was one assay in which mosquitoes had access to both food sources, and the data quantify if they took both meals, one meal, or no meals.

We thank the reviewer for flagging the schematic in figure 1F. As suggested, we have removed the “X” markers from the x-axis and revised the axis label from “choice of food” to “choice made” to better reflect what food the mosquitoes chose in the assay. For clarity, we have now also plotted the same data as stacked graphs at the bottom of Fig. 1F, which clearly shows the proportion of mosquitoes fed on each particular choice. We avoid the stacked graph as the sole representation of this data, as it does not capture the variability in the data.

I would also like to know more about how the authors achieved tissue-specific knockdown for RNAi experiments. I think this is an intriguing methodology, but I could not figure out from the methods why injections either had whole-body or abdomen-specific knockdown.

The tissue-specific knockdown (abdomen only or abdomen+head) emerged from initial standardisations where we were unable to achieve knockdown in the head unless we used higher concentrations of dsRNA and did the injections in older females. We realised that this gave us the opportunity to isolate the neuronal contribution of these neuropeptides in the phenotype produced. Further optimisations revealed that injecting dsRNA into 0-10h old females produced abdomen-specific knockdowns without affecting head expression, whereas injections into 4 days old females resulted in knockdowns in both tissues. Moreover, head knockdowns in older females required higher dsRNA concentrations, with knockdown efficiency correlating with the amount injected. In contrast, abdominal knockdowns in younger females could be achieved even with lower dsRNA amounts.

We have mentioned the knockdown conditions- time of injection and the amount dsRNA injected- for tissue-specific knockdowns in methods but realise now that it does not explain this well enough. We have now edited it to state our methodology more clearly (see lines 932-948).

I also found some interpretations of the transcriptomic to be overly broad for what transcriptomes can actually tell us about the organism's state. For example, the authors mention, "Interestingly, we found that after a blood meal, glucose is neither spent nor stored, and that the female brain goes into a state of metabolic 'sugar rest', while actively processing proteins (Figure S2B, S3)".

This would require a physiological measurement to actually know. It certainly suggests that there are changes in carbohydrate metabolism, but there are too many alternative interpretations to make this broad claim from transcriptomic data alone.

We thank the reviewer for pointing this out and agree with them. We have now edited our statement to read:

“Instead, our data suggests altered carbohydrate metabolism after a blood meal, with the female brain potentially entering a state of metabolic 'sugar rest' while actively processing proteins (Figure S2B, S3). However, physiological measurements of carbohydrate and protein metabolism will be required to confirm whether glucose is indeed neither spent nor stored during this period.” See lines 271-277.

Reviewer #2 (Public review):

Summary:

In this manuscript, Bansal et al examine and characterize feeding behaviour in Anopheles stephensi mosquitoes. While sharing some similarities to the well-studied Aedes aegypti mosquito, the authors demonstrate that mated females, but not unmated (virgin) females, exhibit suppression in their bloodfeeding behaviour. Using brain transcriptomic analysis comparing sugar-fed, blood-fed, and starved mosquitoes, several candidate genes potentially responsible for influencing blood-feeding behaviour were identified, including two neuropeptides (short NPF and RYamide) that are known to modulate feeding behaviour in other mosquito species. Using molecular tools, including in situ hybridization, the authors map the distribution of cells producing these neuropeptides in the nervous system and in the gut. Further, by implementing systemic RNA interference (RNAi), the study suggests that both neuropeptides appear to promote blood-feeding (but do not impact sugar feeding), although the impact was observed only after both neuropeptide genes underwent knockdown.

Strengths and/or weaknesses:

Overall, the manuscript was well-written; however, the authors should review carefully, as some sections would benefit from restructuring to improve clarity. Some statements need to be rectified as they are factually inaccurate.

Below are specific concerns and clarifications needed in the opinion of this reviewer:

(1) What does "central brains" refer to in abstract and in other sections of the manuscript (including methods and results)? This term is ambiguous, and the authors should more clearly define what specific components of the central nervous system was/were used in their study.

Central brain, or mid brain, is a commonly used term to refer to brain structures/neuropils without the optic lobes (For example: https://www.nature.com/articles/s41586-024-07686-5). In this study we have focused our analysis on the central brain circuits involved in modulating blood-feeding behaviour and have therefore excluded the optic lobes. As optic lobes account for nearly half of all the neurons in the mosquito brain (https://pmc.ncbi.nlm.nih.gov/articles/PMC8121336/), including them would have disproportionately skewed our transcriptomic data toward visual processing pathways. 

We have indicated this in figure 3A and in the methods (see lines 800-801, 812). We have now also clarified it in the results section for neurotranscriptomics to avoid confusion (see lines 236-237).

(2) The abstract states that two neuropeptides, sNPF and RYamide are working together, but no evidence is summarized for the latter in this section.

We thank the reviewer for pointing this out. We have now added a statement “This occurs in the context of the action of RYa in the brain” to end of the abstract, for a complete summary of our proposed model. 

(3) Figure 1

Panel A: This should include mating events in the reproductive cycle to demonstrate differences in the feeding behavior of Ae. aegypti.

Our data suggest that mating can occur at any time between eclosion and oviposition in An. stephensi and between eclosion and blood feeding in Ae. aegypti. Adding these into (already busy) 1A, would cloud the purpose of the schematic, which is to indicate the time points used in the behavioural assays and transcriptomics.

Panel F: In treatments where insects were not provided either blood or sugar, how is it that some females and males had fed? Also, it is unclear why the y-axis label is % fed when the caption indicates this is a choice assay. Also, it is interesting that sugar-starved females did not increase sugar intake. Is there any explanation for this (was it expected)?

We apologise for the confusion. The experiment is indeed a choice assay in which sugar-starved or sugar-sated females, co-housed with males, were provided simultaneous access to both blood and sugar, and were assessed for the choice made (indicated on the x-axis): both blood and sugar, blood only, sugar only, or neither. The x-axis indicates the choice made by the mosquitoes, not the choice provided in the assay, and the y-axis indicates the percentage of males or females that made each particular choice. We have now removed the “X” markers from the x-axis and revised the axis label from “choice of food” to “choice made” to better reflect what food the mosquitoes chose to take.

In this assay, we scored females only for the presence or absence of each meal type (blood or sugar) and are therefore unable to comment on whether sugar-starved females consumed more sugar than sugarsated females. However, when sugar-starved, a higher proportion of females consumed both blood and sugar, while fewer fed on blood alone.

For clarity, we have now also plotted the same data as stacked graphs at the bottom of Fig. 1F, which clearly shows the proportion of mosquitoes fed on each particular choice. We avoid the stacked graph as the sole representation of this data as it does not capture the variability in the data.

(4) Figure 3

In the neurotranscriptome analysis of the (central) brain involving the two types of comparisons, can the authors clarify what "excluded in males" refers to? Does this imply that only genes not expressed in males were considered in the analysis? If so, what about co-expressed genes that have a specific function in female feeding behaviour?

This is indeed correct. We reasoned that since blood feeding is exclusive to females, we should focus our analysis on genes that were specifically upregulated in them. As the reviewer points out, it is very likely that genes commonly upregulated in males and females may also promote blood feeding and we will miss out on any such candidates based on our selection criteria. 

(5) Figure 4

The authors state that there is more efficient knockdown in the head of unfed females; however, this is not accurate since they only get knockdown in unfed animals, and no evidence of any knockdown in fed animals (panel D). This point should be revised in the results test as well.

Perhaps we do not understand the reviewer’s point or there has been a misunderstanding. In figure 4D, we show that while there is more robust gene knockdown in unfed females, blood-fed females also showed modest but measurable knockdowns ranging from 5-40% for RYamide and 2-21% for sNPF. 

Relatedly, blood-feeding is decreased when both neuropeptide transcripts are targeted compared to uninjected (panel C) but not compared to dsGFP injected (panel E). Why is this the case if authors showed earlier in this figure (panel B) that dsGFP does not impact blood feeding?

We realise this concern stems from our representation of the data. Since we had earlier determined that dsGFP-injected females fed similarly to uninjected females (fig 4B), we used these controls interchangeably in subsequent experiments. To avoid confusion, we have now only used the label ‘control’ in figure 4 (and supplementary figure S9) and specified which control was used for each experiment in the legend.

In addition to this, we wanted to clarify that fig 4C and 4E are independent experiments. 4C is the behaviour corresponding to when the neuropeptides were knocked down in both heads and abdomens. 4E is the behaviour corresponding to when the neuropeptides were knocked down in only the abdomens. We have now added a schematic in the plots to make this clearer.

In addition, do the uninjected and dsGFP-injected relative mRNA expression data reflect combined RYa and sNPF levels? Why is there no variation in these data,…

In these qPCRs, we calculated relative mRNA expression using the delta-delta Ct method (see line 975). For each neuropeptide its respective control was used. For simplicity, we combined the RYa and sNPF control data into a single representation. The value of this control is invariant because this method sets the control baseline to a value of 1.

…and how do transcript levels of RYa and sNPF compare in the brain versus the abdomen (the presentation of data doesn't make this relationship clear).

The reviewer is correct in pointing out that we have not clarified this relationship in our current presentation. While we have not performed absolute mRNA quantifications, we extracted relative mRNA levels from qPCR data of 96h old unmanipulated control females. We observed that both sNPF and RYa transcripts are expressed at much lower levels in the abdomens, as compared to those in the heads, as shown in Author response Image 1 below. 

Author response image 1.

(6) As an overall comment, the figure captions are far too long and include redundant text presented in the methods and results sections.

We thank the reviewer for flagging this and have now edited the legends to remove redundancy.  

(7) Criteria used for identifying neuropeptides promoting blood-feeding: statement that reads "all neuropeptides, since these are known to regulate feeding behaviours". This is not accurate since not all neuropeptides govern feeding behaviors, while certainly a subset do play a role.

We agree with the reviewer that not all neuropeptides regulate feeding behaviours. Our statement refers to the screening approach we used: in our shortlist of candidates, we chose to validate all neuropeptides.

(8) In the section beginning with "Two neuropeptides - sNPF and RYa - showed about 25% and 40% reduced mRNA levels...", the authors state that there was no change in blood-feeding and later state the opposite. The wording should be clarified as it is unclear.

Thank you for pointing this out. We were referring to an unchanged proportion of the blood fed females. We have now edited the text to the following: 

“Two neuropeptides - sNPF and RYa - showed about 25% and 40% reduced mRNA levels in the heads but the proportion of females that took blood meals remained unchanged”. See lines 338-340.

(9) Just before the conclusions section, the statement that "neuropeptide receptors are often ligandpromiscuous" is unjustified. Indeed, many studies have shown in heterologous systems that high concentrations of structurally related peptides, which are not physiologically relevant, might cross-react and activate a receptor belonging to a different peptide family; however, the natural ligand is often many times more potent (in most cases, orders of magnitude) than structurally related peptides. This is certainly the case for various RYamide and sNPF receptors characterized in various insect species.

We agree with the reviewer and apologise for the mistake. We have now removed the statement.

(10) Methods

In the dsRNA-mediated gene knockdown section, the authors could more clearly describe how much dsRNA was injected per target. At the moment, the reader must carry out calculations based on the concentrations provided and the injected volume range provided later in this section.

We have now edited the section to reflect the amount of dsRNA injected per target. Please see lines 921-931.

It is also unclear how tissue-specific knockdown was achieved by performing injection on different days/times. The authors need to explain/support, and justify how temporal differences in injection lead to changes in tissue-specific expression. Does the blood-brain barrier limit knockdown in the brain instead, while leaving expression in the peripheral organs susceptible?

To achieve tissue-specific knockdowns of sNPF and RYa, we optimised both the time of injection as well as the dsRNA concentration to be injected. Injecting dsRNA into 0-10h females produced abdomen-specific knockdowns without affecting head expression, whereas injections into 96h old females resulted in knockdowns in both tissues. Head knockdowns in older females required higher dsRNA concentrations, with knockdown efficiency correlating with the amount injected. In contrast, abdominal knockdowns in younger females could be achieved even with lower dsRNA amounts, reflecting the lower baseline expression of sNPF in abdomens compared to heads and the age-dependent increase in head expression (as confirmed by qPCR). It is possible that the blood-brain barrier also limits the dsRNA entering the brain, thereby requiring higher amounts to be injected for head knockdowns. 

We have now edited this section to state our methodology more clearly (see lines 932-948).

For example, in Figure 4, the data support that knockdown in the head/brain is only effective in unfed animals compared to uninjected animals, while there is no evidence of knockdown in the brain relative to dsGFP-injected animals. Comparatively, evidence appears to show stronger evidence of abdominal knockdown mostly for the RYa transcript (>90%) while still significantly for the sNPF transcript (>60%).

As we explained earlier, this concern likely stems from our representation of the data. Since we had earlier determined that dsGFP-injected females fed similarly to uninjected females (fig 4B), we used these controls interchangeably in subsequent experiments. To avoid confusion, we have now only used the label ‘control’ in figure 4 (and supplementary figure S9) and specified which control was used for each experiment in the legend.

In addition to this, we wanted to clarify that fig 4C and 4E are independent experiments. 4C is the behaviour corresponding to when the neuropeptides were knocked down in both heads and abdomens.  4E is the behaviour corresponding to when the neuropeptides were knocked down in only the abdomen. We have now added a schematic in the plots to make this clearer.

Reviewer #3 (Public review):

Summary:

This manuscript investigates the regulation of host-seeking behavior in Anopheles stephensi females across different life stages and mating states. Through transcriptomic profiling, the authors identify differential gene expression between "blood-hungry" and "blood-sated" states. Two neuropeptides, sNPF and RYamide, are highlighted as potential mediators of host-seeking behavior. RNAi knockdown of these peptides alters host-seeking activity, and their expression is anatomically mapped in the mosquito brain (sNPF and RYamide) and midgut (sNPF only).

Strengths:

(1) The study addresses an important question in mosquito biology, with relevance to vector control and disease transmission.

(2) Transcriptomic profiling is used to uncover gene expression changes linked to behavioral states.

(3) The identification of sNPF and RYamide as candidate regulators provides a clear focus for downstream mechanistic work.

(4) RNAi experiments demonstrate that these neuropeptides are necessary for normal host-seeking behavior.

(5) Anatomical localization of neuropeptide expression adds depth to the functional findings.

Weaknesses:

(1) The title implies that the neuropeptides promote host-seeking, but sufficiency is not demonstrated (for example, with peptide injection or overexpression experiments).

Demonstrating sufficiency would require injecting sNPF peptide or its agonist. To date, no small-molecule agonists (or antagonists) that selectively mimic sNPF or RYa neuropeptides have been identified in insects. An NPY analogue, TM30335, has been reported to activate the Aedes aegypti NPY-like receptor 7 (NPYLR7; Duvall et al., 2019), which is also activated by sNPF peptides at higher doses (Liesch et al., 2013). Unfortunately, the compound is no longer available because its manufacturer, 7TM Pharma, has ceased operations. Synthesising the peptides is a possibility that we will explore in the future.

(2) The proposed model regarding central versus peripheral (gut) peptide action is inconsistently presented and lacks strong experimental support.

The best way to address this would be to conduct tissue-specific manipulations, the tools for which are not available in this species. Our approach to achieve head+abdomen and abdomen only knockdown was the closest we could get to achieving tissue specificity and allowed us to confirm that knockdown in the head was necessary for the phenotype. However, as the reviewer points out, this did not allow us to rule out any involvement of the abdomen. This point has been addressed in lines 364-371.

(3) Some conclusions appear premature based on the current data and would benefit from additional functional validation.

The most definitive way of demonstrating necessity of sNPF and RYa in blood feeding would be to generate mutant lines. While we are pursuing this line of experiments, they lie beyond the scope of a revision. In its absence, we relied on the knockdown of the genes using dsRNA. We would like to posit that despite only partial knockdown, mosquitoes do display defects in blood-feeding behaviour, without affecting sugar-feeding. We think this reflects the importance of sNPF in promoting blood feeding.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Overall, I found this manuscript to be well-prepared, visually the figures are great and clearly were carefully thought out and curated, and the research is impactful. It was a wonderful read from start to finish. I have the following recommendations:

Thank you very much, we are very pleased to hear that you enjoyed reading our manuscript!

(1) For future manuscripts, it would make things significantly easier on the reviewer side to submit a format that uses line numbers.

We sincerely apologise for the oversight. We have now incorporated line numbers in the revised manuscript.

(2) There are a few statements in the text that I think may need clarification or might be outside the bounds of what was actually studied here. For example, in the introduction "However, mating is dispensable in Anophelines even under conditions of nutritional satiety". I am uncertain what is meant by this statement - please clarify.

We apologise for the lack of clarity in the statement and have now deleted it since we felt it was not necessary.

(3) Typo/Grammatical minutiae:

(a) A small idiosyncrasy of using hyphens in compound words should also be fixed throughout. Typically, you don't hyphenate if the words are being used as a noun, as in the case: e.g. "Age affects blood feeding.". However, you would hyphenate if the two words are used as a compound adjective "Age affects blood-feeding behavior". This may not be an all-inclusive list, but here are some examples where hyphens need to either be removed or added. Some examples:

"Nutritional state also influences other internal state outputs on blood-feeding": blood-feeding -> blood feeding

"... the modulation of blood-feeding": blood-feeding -> blood feeding

"For example, whether virgin females take blood-meals...": blood-meals -> blood meals

".... how internal and external cues shape meal-choice"-> meal choice

"blood-meal" is often used throughout the text, but is correctly "blood meal" in the figures.

There are many more examples throughout.

We apologise for these errors and appreciate the reviewer’s keen eye. We have now fixed them throughout the manuscript.  

(b) Figure 1 Caption has a typo: "co-housed males were accessed for sugar-feeding" should be "co-housed males were assessed for sugar feeding"

We apologise for the typo and thank the reviewer for spotting it. We have now corrected this.  

(c) It would be helpful in some other figure captions to more clearly label which statement is relevant to which part of the text. For example, in Figure 4's caption.

"C,D. Blood-feeding and sugar-feeding behaviour of females when both RYa and sNPF are knocked down in the head (C). Relative mRNA expressions of RYa and sNPF in the heads of dsRYa+dssNPF - injected blood-fed and unfed females, as compared to that in uninjected females, analysed via qPCR (D)."

I found re-referencing C and D at the end of their statements makes it look as thought C precedes the "Relative mRNA expression" and on a first read through, I thought the figure captions were backwards. I'd recommend reformatting here and throughout consistently to only have the figure letter precede its relevant caption information, e.g.:

"C. Blood-feeding and sugar-feeding behaviour of females when both RYa and sNPF are knocked down in the head. D. Relative mRNA expressions of RYa and sNPF in the heads of dsRYa+dssNPF - injected bloodfed and unfed females, as compared to that in uninjected females, analysed via qPCR."

We have now edited the legends as suggested.

Reviewer #2 (Recommendations for the authors):

Separately from the clarifications and limitations listed above, the authors could strengthen their study and the conclusions drawn if they could rescue the behavioural phenotype observed following knockdown of sNPF and RYamide. This could be achieved by injection of either sNPF or RYa peptide independently or combined following knockdown to validate the role of these peptides in promoting blood-feeding in An. stephensi. Additionally, the apparent (but unclear) regionalized (or tissue-specific) knockdown of sNPF and RYamide transcripts could be visualized and verified by implementing HCR in situ hyb in knockdown animals (or immunohistochemistry using antibodies specific for these two neuropeptides). 

In a follow up of this work, we are generating mutants and peptides for these candidates and are planning to conduct exactly the experiments the reviewer suggests.

Reviewer #3 (Recommendations for the authors):

The loss-of-function data suggest necessity but not sufficiency. Synthetic peptide injection in non-hostseeking (blood-fed mated or juvenile) mosquitoes would provide direct evidence for peptide-induced behavioral activation. The lack of these experiments weakens the central claim of the paper that these neuropeptides directly promote blood feeding.

As noted above, we plan to synthesise the peptide to test rescue in a mutant background and sufficiency.  

Some of the claims about knockdown efficiency and interpretation are conflicting; the authors dismiss Hairy and Prp as candidates due to 30-35% knockdown, yet base major conclusions on sNPF and RYamide knockdowns with comparable efficiencies (25-40%). This inconsistency should be addressed, or the justification for different thresholds should be clearly stated.

We have not defined any specific knockdown efficacy thresholds in the manuscript, as these can vary considerably between genes, and in some cases, even modest reductions can be sufficient to produce detectable phenotypes. For example, knockdown efficiencies of even as low as about 25% - 40% gave us observable phenotypes for sNPF and RYa RNAi (Figure S9B-G).

No such phenotypes were observed for Hairy (30%) or Prp (35%) knockdowns. Either these genes are not involved in blood feeding, or the knockdown was not sufficient for these specific genes to induce phenotypes. We cannot distinguish between these scenarios. 

The observation that knockdown animals take smaller blood meals is interesting and could reflect a downstream effect of altered host-seeking or an independent physiological change. The relationship between meal size and host-seeking behavior should be clarified.

We agree with the reviewer that the reduced meal size observed in sNPF and RYa knockdown animals could result from their inability to seek a host or due to an independent effect on blood meal intake. Unfortunately, we did not measure host-seeking in these animals. We plan to distinguish between these possibilities using mutants in future work.

Several figures are difficult to interpret due to cluttered labeling and poorly distinguishable color schemes. Simplifying these and improving contrast (especially for co-housed vs. virgin conditions) would enhance readability. 

We regret that the reviewer found the figures difficult to follow. We have now revised our annotations throughout the manuscript for enhanced readability. For example, “D1^B” is now “D1^PBM” (post-bloodmeal) and “D1^O” is now “D1^PO” (post-oviposition). Wherever mated females were used, we have now appended “(m)” to the annotations and consistently depicted these females with striped abdomens in all the schematics. We believe these changes will improve clarity and readability.

The manuscript does not clearly justify the use of whole-brain RNA sequencing to identify peptides involved in metabolic or peripheral processes. Given that anticipatory feeding signals are often peripheral, the logic for brain transcriptomics should be explained.

The reviewer is correct in pointing out that feeding signals could also emerge from peripheral tissues. Signals from these tissues – in response to both changing nutritional and reproductive states – are then integrated by the central brain to modulate feeding choices. For example, in Drosophila, increased protein intake is mediated by central brain circuitry including those in the SEZ and central complex (Munch et al., 2022; Liu et al., 2017; Goldschmidt et al., 202ti). In the context of mating, male-derived sex peptide further increases protein feeding by acting on a dedicated central brain circuitry (Walker et al., 2015). We, therefore focused on the central brain for our studies.

The proposed model suggests brain-derived peptides initiate feeding, while gut peptides provide feedback. However, gut-specific knockdowns had no effect, undermining this hypothesis. Conversely, the authors also suggest abdominal involvement based on RNAi results. These contradictions need to be resolved into a consistent model.

We thank the reviewer for raising this point and recognise their concern. Our reasons for invoking an involvement of the gut were two-fold:

(1) We find increased sNPF transcript expression in the entero-endocrine cells of the midgut in blood-hungry females, which returns to baseline after a blood-meal (Fig. 4L, M).

(2) While the abdomen-only knockdowns did not affect blood feeding, every effective head knockdown that affected blood feeding also abolished abdominal transcript levels (Fig. S9C, F). (Achieving a head-only reduction proved impossible because (i) systemic dsRNA delivery inevitably reaches the abdomen and (ii) abdominal expression of both peptides is low, leaving little dynamic range for selective manipulation.) Consequently, we can only conclude the following: 1) that brain expression is required for the behaviour, 2) that we cannot exclude a contributory role for gut-derived sNPF. We have discussed this in lines 364-371.

The identification of candidate receptors is promising, but the manuscript would be significantly strengthened by testing whether receptor knockdowns phenocopy peptide knockdowns. Without this, it is difficult to conclude that the identified receptors mediate the behavioral effects.

We agree that functional validation of the receptors would strengthen the evidence for sNPF and RYa-mediated control of blood feeding in An. stephensi. We selected these receptors based on sequence homology. A possibility remains that sNPF neuropeptides activate more than one receptor, each modulating a distinct circuit, as shown in the case of Drosophila Tachykinin (https://pmc.ncbi.nlm.nih.gov/articles/PMC10184743/). This will mean a systematic characterisation and knockdown of each of them to confirm their role. We are planning these experiments in the future.  

The authors compared the percentage changes in sugar-fed and blood-fed animals under sugar-sated or sugar-starved conditions. Figure 1F should reflect what was discussed in the results.

Perhaps this concern stems from our representation of the data in figure 1F? We have now edited the xaxis and revised its label from “choice of food” to “choice made” to better reflect what food the mosquitoes chose to take.

For clarity, we have now also plotted the same data as stacked graphs at the bottom of Fig. 1F, which clearly shows the proportion of mosquitoes fed on each particular choice. We avoid the stacked graph as the sole representation of this data because it does not capture the variability in the data.

Minor issues:

(1) The authors used mosquitoes with belly stripes to indicate mated females. To be consistent, the post-oviposition females should also have belly stripes.

We thank the reviewer for pointing this out. We have now edited all the figures as suggested.

(2) In the first paragraph on the right column of the second page, the authors state, "Since females took blood-meals regardless of their prior sugar-feeding status and only sugar-feeding was selectively suppressed by prior sugar access." Just because the well-fed animals ate less than the starved animals does not mean their feeding behavior was suppressed.

Perhaps there has been a misunderstanding in the experimental setup of figure 1F, probably stemming from our data representation. The experiment is a choice assay in which sugar-starved or sugar-sated females, co-housed with males, were provided simultaneous access to both blood and sugar, and were assessed for the choice made (indicated on the x-axis): both blood and sugar, blood only, sugar only, or neither. We scored females only for the presence or absence of each meal type (blood or sugar) and did not quantify the amount consumed.

(3) The figure legend for Figure 1A and the naming convention for different experimental groups are difficult to follow. A simplified or consistently abbreviated scheme would help readers navigate the figures and text.

We regret that the reviewer found the figure difficult to follow. We have now revised our annotations throughout the manuscript for enhanced readability. For example, “D1^B” is now “D1^PBM” (post-bloodmeal) and “D1^O” is now “D1^PO” (post-oviposition).

(4) In the last paragraph of the Y-maze olfactory assay for host-seeking behaviour in An. stephensi in Methods, the authors state, "When testing blood-fed females, aged-matched sugar-fed females (bloodhungry) were included as positive controls where ever possible, with satisfactory results." The authors should explicitly describe what the criteria are for "satisfactory results".

We apologise for the lack of clarity. We have now edited the statement to read:

“When testing blood-fed females, age-matched sugar-fed females (blood-hungry) were included wherever possible as positive controls. These females consistently showed attraction to host cues, as expected.” See lines 786-790.

(5) In the first paragraph of the dsRNA-mediated gene knockdown section in Methods, dsRNA against GFP is used as a negative control for the injection itself, but not for the potential off-target effect.

We agree with the reviewer that dsGFP injections act as controls only for injection-related behavioural changes, and not for off-target effects of RNAi. We have now corrected the statement. See lines 919-920.

To control for off-target effects, we could have designed multiple dsRNAs targeting different parts of a given gene. We regret not including these controls for potential off-target effects of dsRNAs injected. 

(6) References numbers 48, 89, and 90 are not complete citations.

We thank the reviewer for spotting these. We have now corrected these citations.

Author response:

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

First, we thank the reviewers for the valuable and constructive reviews. Thanks to these, we believe the article has been considerably improved. We have organized our response to address points that are relevant to both reviewers first, after which we address the unique concerns of each individual reviewer separately. We briefly paraphrase each concern and provide comments for clarification, outlining the precise changes that we have made to the text.

Common Concerns (R1 & R2):

Can you clarify how NREM and REM sleep relate to the oneirogen hypothesis?

Within the submission draft we tried to stay agnostic as to whether mechanistically similar replay events occur during NREM or REM sleep; however, upon a more thorough literature review, we think that there is moderately greater evidence in favor of Wake-Sleep-type replay occurring during REM sleep which is related to classical psychedelic drug mechanisms of action.

First, we should clarify that replay has been observed during both REM and NREM sleep, and dreams have been documented during both sleep stages, though the characteristics of dreams differ across stages, with NREM dreams being more closely tied to recent episodic experience and REM dreams being more bizarre/hallucinatory (see Stickgold et al., 2001 for a review). Replay during sleep has been studied most thoroughly during NREM sharp-wave ripple events, in which significant cortical-hippocampal coupling has been observed (Ji & Wilson, 2007). However, it is critical to note that the quantification methods used to identify replay events in the hippocampal literature usually focus on identifying what we term ‘episodic replay,’ which involves a near-identical recapitulation of neural trajectories that were recently experienced during waking experimental recordings (Tingley & Peyrach, 2020). In contrast, our model focuses on ‘generative replay,’ where one expects only a statistically similar reproduction of neural activity, without any particular bias towards recent or experimentally controlled experience. This latter form of replay may look closer to the ‘reactivation’ observed in cortex by many studies (e.g. Nguyen et al., 2024), where correlation structures of neural activity similar to those observed during stimulus-driven experience are recapitulated. Under experimental conditions in which an animal is experiencing highly stereotyped activity repeatedly, over extended periods of time, these two forms of replay may be difficult to dissociate.

Interestingly, though NREM replay has been shown to couple hippocampal and cortical activity, a similar study in waking animals administered psychedelics found hippocampal replay without any obvious coupling to cortical activity (Domenico et al., 2021). This could be because the coupling was not strong enough to produce full trajectories in the cortex (psychedelic administration did not increase ‘alpha’ enough), and that a causal manipulation of apical/basal influence in the cortex may be necessary to observe the increased coupling. Alternatively, as Reviewer 1 noted, it may be that psychedelics induce a form of hippocampus-decoupled replay, as one would expect from the REM stage of a recently proposed complementary learning systems model (Singh et al., 2022). 

Evidence in favor of a similarity between the mechanism of action of classical psychedelics and the mechanism of action of memory consolidation/learning during REM sleep is actually quite strong. In particular, studies have shown that REM sleep increases the activity of soma-targeting parvalbumin (PV) interneurons and decreases the activity of apical dendrite-targeting somatostatin (SOM) interneurons (Niethard et al., 2021), that this shift in balance is controlled by higher-order thalamic nuclei, and that this shift in balance is critical for synaptic consolidation of both monocular deprivation effects in early visual cortex (Zhou et al., 2020) and for the consolidation of auditory fear conditioning in the dorsal prefrontal cortex (Aime et al., 2022). These last studies were not discussed in our previous text–we have added them, in addition to a more nuanced description of the evidence connecting our model to NREM and REM replay. 

Relevant modifications: Page 4, 1st paragraph; Page 11, 1st paragraph.

Can you explain how synaptic plasticity induced by psychedelics within your model relates to learning at a behavioral level?

While the Wake-Sleep algorithm is a useful model for unsupervised statistical learning, it is not a model of reward or fear-based conditioning, which likely occur via different mechanisms in the brain (e.g. dopamine-dependent reinforcement learning or serotonin-dependent emotional learning). The Wake-Sleep algorithm is a ‘normative plasticity algorithm,’ that connects synaptic plasticity to the formation of structured neural representations, but it is not the case that all synaptic plasticity induced by psychedelic administration within our model should induce beneficial learning effects. According to the Wake-Sleep algorithm, plasticity at apical synapses is enhanced during the Wake phase, and plasticity at basal synapses is enhanced during the Sleep phase; under the oneirogen hypothesis, hallucinatory conditions (increased ‘alpha’) cause an increase in plasticity at both apical and basal sites. Because neural activity is in a fundamentally aberrant state when ‘alpha’ is increased, there are no theoretical guarantees that plasticity will improve performance on any objective: psychedelic-induced plasticity within our model could perhaps better be thought of as ‘noise’ that may have a positive or negative effect depending on the context.

In particular, such ‘noise’ may be beneficial for individuals or networks whose synapses have become locked in a suboptimal local minimum. The addition of large amounts of random plasticity could allow a system to extricate itself from such local minima over subsequent learning (or with careful selection of stimuli during psychedelic experience), similar to simulated annealing optimization approaches. If our model were fully validated, this view of psychedelic-induced plasticity as ‘noise’ could have relevance for efforts to alleviate the adverse effects of PTSD, early life trauma, or sensory deprivation; it may also provide a cautionary note against repeated use of psychedelic drugs within a short time frame, as the plasticity changes induced by psychedelic administration under our model are not guaranteed to be good or useful in-and-of themselves without subsequent re-learning and compensation.

We should also note that we have deliberately avoided connecting the oneirogen hypothesis model to fear extinction experimental results that have been observed through recordings of the hippocampus or the amygdala (Bombardi & Giovanni, 2013; Jiang et al., 2009; Kelly et al., 2024; Tiwari et al., 2024). Both regions receive extensive innervation directly from serotonergic synapses originating in the dorsal raphe nucleus, which have been shown to play an important role in emotional learning (Lesch & Waider, 2012); because classical psychedelics may play a more direct role in modulating this serotonergic innervation, it is possible that fear conditioning results (in addition to the anxiolytic effects of psychedelics) cannot be attributed to a shift in balance between apical and basal synapses induced by psychedelic administration. We have provided a more detailed review of these results in the text, as well as more clarity regarding their relation to our model.

Relevant modifications: Page 9, final paragraph; Page 12, final paragraph.

Reviewer 1 Concerns:

Is it reasonable to assign a scalar parameter ‘alpha’ to the effects of classical psychedelics? And is your proposed mechanism of action unique to classical psychedelics? E.g. Could this idea also apply to kappa opioid agonists, ketamine, or the neural mechanisms of hallucination disorders?

We have clarified that within our model ‘alpha’ is a parameter that reflects the balance between apical and basal synapses in determining the activity of neurons in the network. For the sake of simplicity we used a single ‘alpha’ parameter, but realistically, each neuron would have its own ‘alpha’ parameter, and different layers or individual neurons could be affected differentially by the administration of any particular drug; therefore, our scalar ‘alpha’ value can be thought of as a mean parameter for all neurons, disregarding heterogeneity across individual neurons.

There are many different mechanisms that could theoretically affect this ‘alpha’ parameter, including: 5-HT2a receptor agonism, kappa opioid receptor binding, ketamine administration, or possibly the effects of genetic mutations underlying the pathophysiology of complex developmental hallucination disorders. We focused exclusively on 5-HT2a receptor agonism for this study because the mechanism is comparatively simple and extensively characterized, but similar mechanisms may well be responsible for the hallucinatory symptoms of a variety of drugs and disorders.

Relevant modifications: Page 4, first paragraph; Page 13, first paragraph.

Can you clarify the role of 5-HT2a receptor expression on interneurons within your model?

While we mostly focused on the effects of 5-HT2a receptors on the apical dendrites of pyramidal neurons, these receptors are also expressed on soma-targeting parvalbumin (PV) interneurons. This expression on PV interneurons is consistent with our proposed psychedelic mechanism of action, because it could lead to a coordinated decrease in the influence of somatic and proximal dendritic inputs while increasing the influence of apical dendritic inputs. We have elaborated on this point, and moved the discussion earlier in the text.

Relevant modifications: Page 1, 1st paragraph; Page 4, 2nd paragraph.

Discussions of indigenous use of psychedelics over millenia may amount to over-romanticization.

We ultimately decided to remove these discussions from the main text, as they had little bearing on the content of our work. Within the Ethics Declarations section we softened our claims from “millenia” to “centuries,” as indigenous psychedelic use over this latter period of time is well-substantiated.

Relevant modifications: removed from introduction; modified Ethics Declarations

You isolate the 5-HT2a agonism as the mechanism of action underlying ‘alpha’ in your model, but there exist 5-HT2a agonists that do not have hallucinatory effects (e.g. lisuride). How do you explain this?

Lisuride has much-reduced hallucinatory effects compared to other psychedelic drugs at clinical doses (though it does indeed induce hallucinations at high doses; Marona-Lewicka et al., 2002), and we should note that serotonin (5-HT) itself is pervasive in the cortex without inducing hallucinatory effects during natural function. Similarly, MDMA is a partial agonist for 5-HT2a receptors, but it has much-reduced perceptual hallucination effects relative to classical psychedelics (Green et al., 2003) in addition to many other effects not induced by classical psychedelics.

Therefore, while we argue that 5-HT2a agonism induces an increase in influence of apical dendritic compartments and a decrease in influence of basal/somatic compartments, and that this change induces hallucinations, we also note that there are many other factors that control whether or not hallucinations are ultimately produced, so that not all 5-HT2a agonists are hallucinogenic. There are two possible additional factors that could contribute to this phenomenon: 5-HT receptor binding affinity and cellular membrane permeability.

Importantly, many 5-HT2a receptor agonists are also 5-HT1a receptor agonists (e.g. serotonin itself and lisuride), while MDMA has also been shown to increase serotonin, norepinephrine, and dopamine release (Green et al., 2003). While 5-HT2a receptor agonism has been shown to reduce sensory stimulus responses (Michaiel et al., 2019), 5-HT1a receptor agonism inhibits spontaneous cortical activity (Azimi et al., 2020); thus one might expect the net effect of administering serotonin or a nonselective 5-HT receptor agonist to be widespread inhibition of a circuit, as has been observed in visual cortex (Azimi et al., 2020). Therefore, selective 5-HT2a agonism is critical for the induction of hallucinations according to our model, though any intervention that jointly excites pyramidal neurons’ apical dendrites and inhibits their basal/somatic compartments across a broad enough area of cortex would be predicted to have a similar effect. Lisuride has a much higher binding affinity for 5-HT1a receptors than, for instance, LSD (Marona-Lewicka et al., 2002).

Secondly, it has recently been shown that both the head-twitch effect (a coarse behavioral readout of hallucinations in animals) and the plasticity effects of psychedelics are abolished when administering 5-HT2a agonists that are impermeable to the cellular membrane because of high polarity, and that these effects can be rescued by temporarily rendering the cellular membrane permeable (Vargas et al., 2023). This suggests that the critical hallucinatory effects of psychedelics (apical excitation according to our model) may be mediated by intracellular 5-HT2a receptors. Notably, serotonin itself is not membrane permeable in the cortex.

Therefore, either of these two properties could play a role in whether a given 5-HT2a agonist induces hallucinatory effects. We have provided an extended discussion of these nuances in our revision.

Relevant modifications: Page 1, paragraph 2.

Your model proposes that an increase in top-down influence on neural activity underlies the hallucinatory effects of psychedelics. How do you explain experimental results that show increases in bottom-up functional connectivity (either from early sensory areas or the thalamus)?

Firstly, we should note that our proposed increase in top-down influence is a causal, biophysical property, not necessarily a statistical/correlative one. As such, we will stress that the best way to test our model is via direct intervention in cortical microcircuitry, as opposed to correlative approaches taken by most fMRI studies, which have shown mixed results with regard to this particular question. Correlative approaches can be misleading due to dense recurrent coupling in the system, and due to the coarse temporal and spatial resolution provided by noninvasive recording technologies (changes in statistical/functional connectivity do not necessarily correspond to changes in causal/mechanistic connectivity, i.e. correlation does not imply causation).

There are two experimental results that appear to contradict our hypothesis that deserve special consideration. The first shows an increase in directional thalamic influence on the distributed cortical networks after psychedelic administration (Preller et al., 2018). To explain this, we note that this study does not distinguish between lower-order sensory thalamic nuclei (e.g. the lateral and medial geniculate nuclei receiving visual and auditory stimuli respectively) and the higher-order thalamic nuclei that participate in thalamocortical connectivity loops (Whyte et al., 2024). Subsequent more fine-grained studies have noted an increase in influence of higher order thalamic nuclei on the cortex (Pizzi et al., 2023; Gaddis et al., 2022), and in fact extensive causal intervention research has shown that classical psychedelics (and 5-HT2a agonism) decrease the influence of incoming sensory stimuli on the activity of early sensory cortical areas, indicating decoupling from the sensory thalamus (Evarts et al., 1955; Azimi et al., 2020; Michaiel et al. 2019). The increased influence of higher-order thalamic nuclei is consistent with both the cortico-striatal-thalamo-cortical (CTSC) model of psychedelic action as well as the oneirogen hypothesis, since higher-order thalamic inputs modulate the apical dendrites of pyramidal neurons in cortex (Whyte et al., 2024).

The second experimental result notes that DMT induces traveling waves during resting state activity that propagate from early visual cortex to deeper cortical layers (Alamia et al., 2020). There are several possibilities that could explain this phenomenon: 1) it could be due to the aforementioned difficulties associated with directed functional connectivity analyses, 2) it could be due to a possible high binding affinity for DMT in the visual cortex relative to other brain areas, or 3) it could be due to increases in apical influence on activity caused by local recurrent connectivity within the visual cortex which, in the absence of sensory input, could lead to propagation of neural activity from the visual cortex to the rest of the brain. This last possibility is closest to the model proposed by (Ermentrout & Cowan, 1979), and which we believe would be best explained within our framework by a topographically connected recurrent network architecture trained on video data; a potentially fruitful direction for future research.

Relevant modifications: Page 9, paragraph 1; Page 10, final paragraph; Page 11, final paragraph.

Shouldn’t the hallucinations generated by your model look more ‘psychedelic,’ like those produced by the DeepDream algorithm?

We believe that the differences in hallucination visualization quality between our Wake-Sleep-trained models and DeepDream are mostly due to differences in the scale and power of the models used across these two studies. We are confident that with more resources (and potentially theoretical innovations to improve the Wake-Sleep algorithm’s performance) the produced hallucination visualizations could become more realistic.

We note that more powerful generative models trained with backpropagation are able to produce surreal images of comparable quality (Rezende et al., 2014; Goodfellow et al., 2020; Vahdat & Kautz, 2020), though these have not yet been used as a model of psychedelic hallucinations. However, the DeepDream model operates on top of large pretrained image processing models, and does not provide an biologically mechanistic/testable interpretation of its hallucination effects. When training smaller models with a local synaptic plasticity rule (as opposed to backpropagation), the hallucination effects are less visually striking due to the reduced quality of our trained generative model, though they are still strongly tied to the statistics of sensory inputs, as quantified by our correlation similarity metric (Fig. 5b).

To demonstrate that our proposed hallucination mechanism is capable of producing more complex hallucinations in larger, more powerful models, we employed our same hallucination generation mechanism in a pretrained Very Deep Variational Autoencoder (VDVAE) (Child et al., 2021), which is a hierarchical variational autoencoder with a nearly identical structure compared to our Wake-Sleep-trained networks, with both a bottom-up inference pathway and a top-down generative pathway that maps cleanly onto our multicompartmental neuron model. VDVAEs are trained on the same objective function as our Wake-Sleep-trained networks, but using the backpropagation algorithm. The VDVAE models were able to generate much more complex hallucinations (emergence of complex geometric patterns, smooth deformations of objects and faces), whose complexity arguably exceeds those produced by the DeepDream algorithm. Therefore while the VDVAEs are less biologically realistic (they do not learn via local synaptic plasticity), they function as a valuable high-level model of hallucination generation that complements our Wake-Sleep-trained approach. As further validation, we were also able to replicate our key results and testable predictions with these models.

Relevant modifications: Results section “Modeling hallucinations in large-scale pretrained networks”; Figure 6, S7, S8; Page 12, paragraph 3; Methods section “Generating hallucinations in hierarchical variational autoencoders.”

Your model assumes domination by entirely bottom-up activity during the ‘wake’ phase, and domination entirely by top-down activity during ‘sleep,’ despite experimental evidence indicating that a mixture of top-down and bottom-up inputs influence neural activity during both stages in the brain. How do you explain this?

Our use of the Wake-Sleep algorithm, in which top-down inputs (Sleep) or bottom-up inputs (Wake) dominate network activity is an over-simplification made within our model for computational and theoretical reasons. Models that receive a mixture of top-down and bottom-up inputs during ‘Wake’ activity do exist (in particular the closely related Boltzmann machine (Ackley et al., 1985)), but these models are considerably more computationally costly to train due to a need to run extensive recurrent network relaxation dynamics for each input stimulus. Further, these models do not generalize as cleanly to processing temporal inputs. For this reason, we focused on the Wake-Sleep algorithm, at the cost of some biological realism, though we note that our model should certainly be extended to support mixed apical-basal waking regimes. We have added a discussion of this in our ‘Model Limitations’ section.

Relevant modifications: Page 12, paragraph 4.

Your model proposes that 5-HT2a agonism enhances glutamatergic transmission, but this is not true in the hippocampus, which shows decreases in glutamate after psychedelic administration.

We should note that our model suggests only compartment specific increases in glutamatergic transmission; as such, our model does not predict any particular directionality for measures of glutamatergic transmission that includes signaling at both apical and basal compartments in aggregate, as was measured in the provided study (Mason et al., 2020).

You claim that your model is consistent with the Entropic Brain theory, but you report increases in variance, not entropy. In fact, it has been shown that variance decreases while entropy increases under psychedelic administration. How do you explain this discrepancy?

Unfortunately, ‘entropy’ and ‘variance’ are heavily overloaded terms in the noninvasive imaging literature, and the particularities of the method employed can exert a strong influence on the reported effects. The reduction in variance reported by (Carhart-Harris et al., 2016) is a very particular measure: they are reporting the variance of resting state synchronous activity, averaged across a functional subnetwork that spans many voxels; as such, the reduction in variance in this case is a reduction in broad, synchronous activity. We do not have any resting state synchronous activity in our network due to the simplified nature of our model (particularly an absence of recurrent temporal dynamics), so we see no reduction in variance in our model due to these effects.

Other studies estimate ‘entropy’ or network state disorder via three different methods that we have been able to identify. 1) (Carhart-Harris et al., 2014) uses a different measure of variance: in this case, they subtract out synchronous activity within functional subnetworks, and calculate variability across units in the network. This measure reports increases in variance (Fig. 6), and is the closest measure to the one we employ in this study. 2) (Lebedev et al., 2016) uses sample entropy, which is a measure of temporal sequence predictability. It is specifically designed to disregard highly predictable signals, and so one might imagine that it is a measure that is robust to shared synchronous activity (e.g. resting state oscillations). 3) (Mediano et al., 2024) uses Lempel-Ziv complexity, which is, similar to sample entropy, a measure of sequence diversity; in this case the signal is binarized before calculation, which makes this method considerably different from ours. All three of the preceding methods report increases in sequence diversity, in agreement with our quantification method. Our strongest explanation for why the variance calculation in (Carhart-Harris et al., 2016) produces a variance reduction is therefore due to a reduction in low-rank synchronous activity in subnetworks during resting state.

As for whether the entropy increase is meaningful: we share Reviewer 1’s concern that increases in entropy could simply be due to a higher degree of cognitive engagement during resting state recordings, due to the presence of sensory hallucinations or due to an inability to fall asleep. This could explain why entropy increases are much more minimal relative to non-hallucinating conditions during audiovisual task performance (Siegel et al., 2024; Mediano et al., 2024). However, we can say that our model is consistent with the Entropic Brain Theory without including any form of ‘cognitive processing’: we observe increases in variability during resting state in our model, but we observe highly similar distributions of activity when averaging over a wide variety of sensory stimulus presentations (Fig. 5b-c). This is because variability in our model is not due to unstructured noise: it corresponds to an exploration of network states that would ordinarily be visited by some stimulus. Therefore, when averaging across a wide variety of stimuli, the distribution of network states under hallucinating or non-hallucinating conditions should be highly similar.

One final point of clarification: here we are distinguishing Entropic Brain Theory from the REBUS model–the oneirogen hypothesis is consistent with the increase in entropy observed experimentally, but in our model this entropy increase is not due to increased influence of bottom-up inputs (it is due instead to an increase in top-down influence). Therefore, one could view the oneirogen hypothesis as consistent with EBT, but inconsistent with REBUS.

Relevant modifications: Page 10, paragraph 1.

You relate your plasticity rule to behavioral-timescale plasticity (BTSP) in the hippocampus, but plasticity has been shown to be reduced in the hippocampus after psychedelic administration. Could you elaborate on this connection?

When we were establishing a connection between our ‘Wake-Sleep’ plasticity rule and BTSP learning, the intended connection was exclusively to the mathematical form of the plasticity rule, in which activity in the apical dendrites of pyramidal neurons functions as an instructive signal for plasticity in basal synapses (and vice versa): we will clarify this in the text. Similarly, we point out that such a plasticity rule tends to result in correlated tuning between apical and basal dendritic compartments, which has been observed in hippocampus and cortex: this is intended as a sanity check of our mapping of the Wake-Sleep algorithm to cortical microcircuitry, and has limited further bearing on the effects of psychedelics specifically.

Reduction in plasticity in the hippocampus after psychedelic administration could be due to a complementary learning systems-type model, in which the hippocampus becomes partly decoupled from the cortex during REM sleep (Singh et al., 2022); were this to be the case, it would not be incompatible with our model, which is mostly focused on the cortex. Notably, potentiating 5HT-2a receptors in the ventral hippocampus does not induce the head-twitch response, though it does produce anxiolytic effects (Tiwari et al., 2024), indicating that the hallucinatory and anxiolytic effects of classical psychedelics may be partly decoupled. 

Reviewer 2 Concerns:

Could you provide visualizations of the ‘ripple’ phenomenon that you’re referring to?

In our revised submission, ‘ripple’ phenomena are now visible in two places: Fig 2c-d, and Fig 6 (rows 2 and 3). Because the VDVAE models used to generate Figure 6 produce higher quality generated images, the ripples appearing in these plots are likely more prototypical, but it is not easy to evaluate the quality of these visualizations relative to subjective hallucination phenomena.

Could you provide a more nuanced description of alternative roles for top-down feedback, beyond being used exclusively for learning as depicted in your model?

For the sake of simplicity, we only treat top-down inputs in our model as a source of an instructive teaching signal, the originator of generative replay events during the Sleep phase, and as the mechanism of hallucination generation. However, as discussed in a response to a previous question, in the cortex pyramidal neurons receive and respond to a mixture of top-down and bottom-up processing.