Make "Administration"

Mike to update this section

Images of the students need to be updated.

Adita's CTA needs to say "Learn More About Adita"

"See All Events" button (I couldn't highlight it, sorry) is going to a broken link. Please update to go to https://calendar.stonybrook.edu/

Can these each link to the event pages?

Link these three buttons (Majors and Minors, Schedule a Tour, Apply Now)

Change and link to "Facts and Rankings"

Change to "Areas of Study" and link to Majors & Minors

Make Major plural (Majors)

Change to Deadline for Early Action is Nov. 11, link to https://www.stonybrook.edu/undergraduate-admissions/apply/first-year.php

Link to Admissions page.

Broken link.

As faculty who are considering what it means to bring OEP into our classrooms in this particular moment (fall of 2025) this note about perpetrating harm unintentionally resonates with our conversations. Specifically, how do we balance the risks that are changing and evolving for putting information into the public sphere (eg. depending on your context individuals can be targeted)? What does the evolution of how AI uses our information that's publicly available mean for the risks of doing OEPs in our classes?

How the Trump administration is dramatically reshaping education in America<br /> by [[John Yang]] of PBS at PBS News Weekend accessed on 2025-10-22T12:01:15

Interview with Jennifer Smith Richards of ProPublica

Anti-DEI, Anti-CRT bills

This part shows that the crisis wasn’t just social but emotional. Uncertainty caused peoples strife after the war. Even the universe seemed unstable. This new worldview influenced all types of art, suggesting that truth is known and knowledge is limited.

The magical source behind your favorite pastrami sandwiches and chili dogs<br /> by [[By Josh Heller]] for LAist<br /> accessed on 2025-10-22T11:56:41

uitproberen

I can see how this is true. Meta using selling information to ad agencies can easily lead to leaks and scams from other sources.

I find it shocking that the personal information that you willingly give to social media platforms can be so easily stolen and posted publicly on the internet. That is a huge breach of trust as people put their trust into these companies to save and protect their personal information.

I think the sentence "Although we are concerned about information privacy, we often share information with social media platforms and believe they will safely keep these details" is particularly true. We always say we want to protect privacy, but in reality, we still readily click "agree" and hand over our information without hesitation. Seeing this sentence made me reflect a bit - perhaps we have become too accustomed to convenience, and thus have overlooked the aspect of security.

This post is very interesting because it does not technically have any means for a lawsuit. If celebrities who could have gotten their information leaked, that could ruin their phones with the number of people who try to contact them after their information is leaked.

This article is documenting that Facebook stored passwords of users in a way that made them accessible to around 200,000 employees. I don't fear my information being "leaked" due to lack of assets that are desirable but that could just be a lack of understanding but this did concern me.

This source discusses the right to privacy and each person's right to privacy. It discusses the law and the United States rights that make sure that each citizen has the right to have their own personal and private information and lives.

I read this report from NPR titled "After Data Breach Exposes 530 Million, Facebook Says It Will Not Notify Users". I was really shocked. The data of 53 million people was leaked, and yet the company chose not to notify the users? This made me realize that big companies claim to "value privacy", but when problems occur, their first reaction is usually to protect themselves rather than protect the users. After reading it, I will be more cautious about the personal information on social media. After all, sometimes "the sense of security" is just an illusion.

In cryptography, salt is random information fed into a type of function called a one way function to get an effect called hashing. This apparently helps with security and solves some logistical problems of having a lot of passwords all in one database.

This article talks about the right to privacy, and everyone in this country has the right to privacy on the internet. The internet should protect users' privacy. This right encourage more users to participate in any social media. I personally agree with this right, and I feel safe with the right to privacy.

This connects to not having a fixed mindset when doing research. During the research process it is important to test your original hypothesis because you never know what alternative solution you could come up with after the previous prediction.

It is especially important for science to be shared freely. This is because it will help people form networks with each other to solve complicated solutions during these difficult times of our society.

It seems like there is an overlap where some people think social media is news media, and they believe everything they read on social media as the real news sometimes.

its in truth a deep analysis and the the way or articulation that is to be considred as a critical thinking stage

there has been a change over time in how the media has passed the informantion and the changes explain the differences between the articles

I think it’s interesting how the video points out that ADHD is found from kids before they are 12. I would think if symptoms start coming in later closer to puberty, it wouldn’t be different. I would even think it’s possible for younger kids to have mask traits such as procrastination due to the help of their parents so it might spark more obvious signs when they reach adulthood when there are less people helping them succeed.

I have a lot of fears around privacy on social media. I worry that social media companies are constantly listening and tracking what I say and do. I worry that they have a profile on me with everything about me. And I believe they are doing these things because I will be talking about something with my friends or parents, and then I will get an ad for it on instagram. I didn't plug anything into instagram to make them know that I was thinking about it yet they will somehow know. This is the kind of thing I don't like about social media.

At the moment there are still a good amount of legal protections that restrict companies from being too flagrant with your privacy but over the lat few decades that has become more and more of a market is companies actually selling people's information. I also think that people don't like the idea of a company having all their information so maybe another thing stopping them is the public sentiment toward private companies doing these massive privacy violations. But there is a lot of money on the other side of that fence and more and more and more companies are deciding that they want to take that leap and violate privacy.

http://johnsargentbarnard.com/Ribbon_Shutter/

Interesting UI for IndieWeb Challenges or events like 100 Days (https://indieweb.org/100_days).

This made me curious, are there any programs trying to fund science just for science's sake, or are all the funders profit based?

I have never thought about science as being inherently social. I think this idea is contradictory to the personality type and abilities most people associate with scientists.

This is very real. Society's image of a scientist is still very much limited to a white man. I think part of this problem is also that women and people in marginalized communities are more likely to be convinced younger that they are not good at science and math by those around them, limiting their abilities and motivation young.

I find this interesting. This system encourages advertising, and I see how that could easily lead to disingenuous work.

https://www.reddit.com/r/RetroFuturism/comments/1nreyfx/letters_by_radio_science_and_invention_artist/

“Letters by radio”, Science and Invention, (Artist Unknown) 1922<br />

https://www.reddit.com/r/typewriters/comments/1o8lzdm/trying_to_id_a_typewriter_from_a_video_game/

Folklore (2007, PlayStation 3)

https://www.instagram.com/nprfreshair/reel/DNVlf2tMstg/?hl=en

Terry Gross reading a book has rendered it useless for others to read.

Dog earing of top corner for interesting sections or questions she may have for the author.

Dog earing bottom corner as an indicator of remembering facts for the intro or for sentences she wants to quote.

Uses front of book for connecting themes and focus, so she won't forget it.

Introductions/prologues for quick overviews of what the book is about and why they wrote it.

https://www.instagram.com/reel/DNYh6b_uBwd/?hl=en

Terry Gross reads slowly to start and speeds up as she continues. She annotates and dog-ears as she reads and then makes notes and questions after she's done.

<br /> via https://www.facebook.com/misfitsmarket/posts/1196992682465751

I think it’s strange that disabled people aren’t able to design for themselves considering they have first hand experience with most of the issues. One of the first parts of designing is to empathize with a certain demographic so most disabled people would be able to understand the problems. I don’t get how you would need higher qualifications for someone designing a ramp as a disabled person rather than a non-disabled designer.

Colonial soldiers often joined for short times. The army made them stay longer, which made some unhappy with British control.

Clough is talking about the French and Indian War, when the British and colonists fought the French and Native Americans for land in North America.

A military court where soldiers are tried for breaking rules or laws.

Ye” just means “the.” People used it in old English writing.

Sounds like pedagogy. I think i'm still not getting the difference between inclusive design and universal design for learning.

one, extendto manyby focusing on what’suniversally important toall humans.

Downright philosophical.

1. Increased access2. Reduced friction3. More emotional contextThe impact of inclusive design is morethan just the products that people use. It’salso a shift in our mindset, methods, andbehaviors.

Realm of psychology, spirituality, libera; arts/

studying the family life cycle will help with creating family interventions (Purpose of article???)

If we want to research extended family's impact on ecological systems, we need to start here by developing our understanding of families better.

Still not much research on the impact of extended family on adolescent mood :(

Purpose for the article (???)

This info can improve the help black people get in therapy

Broadening our research and definition of families could reduce some of the negative effects extended family may have regarding socioeconomic status

This is going in the conclusion, all 4 sources back this up.

the negative effects aren't tied to race, but socioeconomic status (which can be tied back to race in some ways but getting into that gets us off track of the research question)

This is the only negative effect I've seen for children/adolescents

The negative effects seem to be geared more towards the adults than the adolescents

Black and White families can't be compared the same way

Affirm the importance of extended family in single parent homes

The benefits of extended family are shown clearly through black families based off origin, values, beliefs, and varying familial structures that are distinct from the white "standard"

researchers turned their focus to tradition and culture, and less of the white "norm"

Important quote to include

The main problems of research on black families based on the nuclear definition was caused by assumptions that a) all black people will possess cultural attributes to the same degree, b) research on lower socioeconomic black families represented the entire black population (they generalized all of their research) and c) That white and black people had a cultural equivalence and that their behaviors could be compared the same way based off of a common standard

not related to my question, just guessing that this resulted in harmful stereotypes for black people (now that I think about it maybe it is related because the results would've been different if they didn't skew the data)

skewed results because they forced black families into their standards

Bias and beliefs hinder research of black families

These familial bonds have importance, but because they aren't included in the "standard" definition, research on the effects are limited

is he Lenoro-Otero suggesting that there's a connection between diverse family structure and diverse cultures and ethnicities?

This will cause many families to deviate from the "norm", the "standard" definition will exclude many families in the US that may not be white or middle to upperclass

aligns with how other sources explained the changes in our definition of family overtime

Black Americans have different family life (close with extended family) than White Americans. Their norms are seen as atypical and divergent from the "norm", causing limited research on the effects of extended family on Black Americans

New Yorker Staff. 2007. “The Typing Life: How Writers Used to Write.” The New Yorker. https://www.newyorker.com/magazine/2007/04/09/the-typing-life (October 22, 2025).

typewriter plays a central role in the story

source? original quote?

via Darren Herschler-Henry's book

看了直想按怒

羽兔盒 Frabbitbox 唉... 2h Reply 陳咚咚 專業是一回事，班級經營閣是另外一回事 3h Reply Tsiā Uán Tsin 垃圾老師 4h Reply Karen Cheng 古早無學校 去學漢學？ 3h Reply Harvey Chen 有時候，學校真的是不得已的…… 3h Reply

Good example of basic Teams bot interaction

Interesting option for showcasing capabilities / FAQ, etc. without needing to manage a separate website.

https://www.facebook.com/groups/705152958470148/posts/1098284832490290/

https://www.reddit.com/r/typewriters/comments/1o8o98j/comment/njx2pgo/?context=1

Haul Out the Halloween (2025), Hallmark Movie

via Aaron Mitchell at https://www.facebook.com/groups/705152958470148/posts/1102482705403836/

Typewriter Typebar Holding Rack by grifter7 - Thingiverse<br /> by [[grifter7]] on [[Thingiverse.com]]<br /> accessed on 2025-10-22T10:26:22

Tool: Typebar Sorting Rack

This not only affects their health but also their education. If a student cannot afford to buy glasses they will likely continue to struggle seeing the board in school. Similarly if a student has a hard time hearing, they will face disadvantages which can affect their academic performance. It is unfair for intelligent students to fall behind because they can't afford the luxury of getting medical attention.

Many, like my family have come in the pursuit of the American Dream. While I do think it's possible, it is much easier said than done, especially for those who come from a low-income. Not everyone has access to resources that can help guide those new to this country. Because of that, many left to fend for themselves, not knowing what is necessary to set themselves for success. Yes, many can find employment, but employment does not equate to the American dream.

pbtpocket<br /> by [[Kaveh Shahbazian]]<br /> accessed on 2025-10-22T10:25:25

Make Pocketmods (little Booklets) With MS Word : 3 Steps<br /> by [[Instructables]]<br /> accessed on 2025-10-22T10:24:10

UPDATED Watch Woody Allen's Interview with Bill Maher: "Sunset Boulevard" (the Movie) is "Fun Junk," "Streetcar" is "Perfect" - Showbiz411<br /> by [[Roger Friedman]]<br /> accessed on 2025-10-22T10:20:51

AKA, LLM prompt: make it sound academic.

Does this mean cognitive disability or lack of needed vocab?

Look at that one memo again. It's unusefully fusty

I need examples

It's interesting how people so early on in our lives play a huge role in our future achievement. I see this being true in many aspects. Hearing my teachers talk about their higher education experience inspired me to do the same. Not only do educators play a huge role in influencing students for higher education, but often times help create a welcoming and safe environment for students who don't feel safe anywhere else. Because of this I strongly believe elementary school and its educators are what shape student success.

I have always wondered what the implications are from parents who work all the time and have little time to see their kids. I have seen this on both sides, low-income students whose parents work a lot to survive, and well-off students whose parents are always on business trips they rarely see their kids. While both are done for different reasons, I think the experience of the kids is similar emotionally.The lack of a parent figure in the home leads many to become independent. While the experiences can be different, I find many of the students in similar situations are able to find a common ground In how they felt, but plan to go about their lives in different ways. Low-inccome students often want a well paying job that allows them to see their kids, while wealthier students follow a similar path made by their parents.m

https://www.reddit.com/r/typewriters/comments/1o9bpgu/isnt_it_woddys_long_lost_ribbon_cover/

Woody Allen reported in a documentary that he'd lost his cover for his Olympia SM3, but it appears to be on his desk in this undated photo.

facts are boring but overall good

bad seem bland

this is important because it gives you a guide on what to do

good shows a compare and contrast

to personal bad

good something that is relatable

shows what to use and how to use it

This is the introduction to the paper, and discusses the main problem that the research aims to solve.

This sentence clearly states the author's main argument: single stories create stereotypes, and they can be challenged through real interaction.

This section introduces the source (Adichie’s TED Talk), explains her experience, and sets the stage for the main idea by showing how a “single story” can limit perception.

This engages the reader by asking a reflective question and raising curiosity about assumptions and stereotypes.

thesis

hook

background

Although I was always fond of art projects in preparation for mother and father's day, I always felt bad for the students who did not have one or the other. While it is important to teach young students the importance of appreciation and giving gifts during an important day, it can make others feel uncomfortable and secluded from the rest. During my time working on such crafts, I never realized there could be students next to me who were participating just to fit in, but had no one to give the gift to.

I dont think it is necessarily their fault they are unaware of others situation. There is a lack of awareness and lack of emphasis on educating others about financial hardships many face. Many are oblivious to the fact that someone could be living in poverty because they have never encountered someone in that situation. It is unfair to judge those who are unaware, rather it's important to focus on the education system which is meant to inform students but often fails to do its job.

This is unfortunately true for many, and became clear to me when I came to university. Many first-generation students and students who come from poverty have been too worried figuring out how to pay for college or simply how to survive, which forces them to take time away from hanging out with friends and exploring their own curiosity. This is why many of these students either go crazy their first year or feel as if they don't belong.

This is a reality for many people, especially across California, on of the most expensive states to live in. However, I don't believe this accounts for changes in income. Yes, if someone living in California were to move to a state like Kentucky they'd likely have the financial stability to live well. But, as time continues and people settle in, they'll be forced to work the wages within that state, not California. This can potentially cause financial hardships because of the sudden change in lifestyle. Although it could be beneficial to move out of state, it is important to be cautions.

Q3. Kaitlin Breuchel Ball and Loewe’s ideas connect well with “Only Geniuses Can Be Writers.” Both challenge the belief that writing is something only a few talented people can do. They remind us that everyone can be a writer and that creativity shows up in many different kinds of writing, not just in what’s seen as “artistic.”

Q4. Kaitlin Breuchel In concluding their argument, Ball and Loewe assert that there is creativity in all writing, and not just what we term "creative writing." They argue that setting a definition for creativity to be used in some forms of writing makes people imagine and thought that enter ordinary writing. From what I have read, I view "creative writing" differently i think it cannot just be referring to poetry or fiction but any form of writing where a person is deciding, communicating ideas, and speaking with others.

Q1. Kaitlin Breuchel I have authored styles like academic reports and technical reports, which Cheryl E. Ball and Drew M. Loewe have termed weaker in terms of creativity. I used creativity in organizing arguments, picking examples, and crafting my papers yet persuasive explanations in an effort to make complex ideas comprehensible and persuasive.

Q2. Kaitlin Breuchel I'd identify more with being a writer because I like to think about how to communicate my ideas and engage other individuals with them. Most individuals are okay with labeling themselves as readers because reading is natural and simple it's something that we all do every single day without even thinking about it. Writing, is intimidating since people seem to think that it needs to be perfect or "creative." I think that expectation makes a lot of people wonder if they are really writers, even though everyone writes in some capacity.

HI y'all what up

Version 2 of this preprint has been peer-reviewed and recommended by Peer Community in Ecology.<br /> See the peer reviews and the recommendation.

the first priniciple that the author's argue that contribute to a successful multinational federation is Staatsvolk. it's not a pancea but the evidence demonstrates that the more heterogenous a federation is the more likely it will be unstable, face secessionism, or break up becuase the minorities are more likely to think they can prevail. the authors suggest that multinational federations without staatsvolk to survive as democratic entites they must develop consociational practices to protect interests of all comm. adding Gannon into this paragraph because they share similar views, the majority nation must appropriately behave to maintain stability.

mentions india's refusal to recognize religion, not ethnicity, as the basis of state formation. india is a successful multinational federation but due to their refusal to recognize religion they have had issues with Kashmir and Punjab. violoence would be a result of centralising decisions.

canada is a great example of a successful multinational federation that has had its up and downs regarding quebec. the problems arose as the authors argue, due to centralising movements away from multinational federalist principles. in 1982 the canadian prime minister Trudeau introduced two major changes to the constituion to weaken Quebec's powers. tensions rose and a there was a close referendum result in 1995 that lessened the tensions.

introduces the idea that these failed multinational federations had a lack of Staatsvolk and were the most heterogenous. this combine created major tensions. in O'Leary's past research they have found that the world's stable and durably dem majoritatian federatiosn are so because that have straatsvolk (dominant people/identity)

authors argue that collapses were caused by insufficient implementation of pluri-national federal principles. for example war started in croatia because the serb population wanted to stay in Yugoslavia (this sentiment also spread to bosnia).

the authors even argue that it is because of these centralising movements and majoritarian policies of the dominant groups that contribute to the wars and conflict of the existing nations that came out of the federations.

wrong historical causation leads to prominent arguments against multinational federation. the authors in this article argues its attempts to unitarise and centralise multinational federations that lead to secession and violence. For Yugoslavia, the successive Serbian-dominated moves against autonomy of the others lead to Kosovo to de facto breakaway. it could be said that federal constitutions with procedural and negotiable secession rules might have avoided violence and even succession better.

the authors of this paper counters the argument that mono-nation-building strategies can be used as an alternative for deeply diverse states is that these strategies have not been successful. UK's civic and unitary state did not prevent the nationalism of its different nations. Therefore the UK had to use a devolution strategy but it still does not quell nationalism.

counterfactuals = relating to or expressing what has not happened or is not the case. authors against multinational federation argue that it was unnecessary to accommodate diversity through federation and that there were democratic civic or unitarists (one nation) alternatives.

the econoic systems within these failed federations eventually proved incapable of providing a reasonable or growing standard of living for citizens. different economic systems of the different regions of the state caused resentment.

colonial federations still were imprinted by the departing metropolitan. s decision to federate instead of the indigenous elites. this case includes Nigeria and Cameroon.

Yugoslavia isn't actually a multinational federation, it was decentralised but that doesn't mean its was democratic, it was held together by the League of Communists. Other "multinational federations" which are more like pseudo-federations include USSR, Czechoslovakia, and Nigeria. they all had weak or no overarching identities and no democratic mechanism for developing those identities

this harks back to an article i read a little bit of (i think it was the Yugoslavia one) where america's motivation relied on ideological differences to further break up that federation. but that's an interpretation. regarding the paragraph, american academic argue that the break-up of former communist federations are due to their implemenation of "ethno-federal" strcutures. Jack Snyder argues that ethnofederalism tends to heighten and politicise ethnic consciousness, creating self-conscious intelligentsia and org strucutres of an ethnic state in waiting. implying that federalism leaves ethnic groups waiting for something they will not receiving leading to nationalism and tensions. additionally snyder notes that nationalist violence happened only where ethnofederal institutions channelled pol activity along ethnic lines (ex: USSR and Yugoslavia).

to manage divisions in thnically heterogenous soc many different authors have suggests that federations can be partly designed to prevent ethnic minorities from becoming local provincial majorities. a balance of power principles, proliferating points of power away from one focal centre, encouraging intra-ethnic divisions, nad creating alignments based on non-ehtnic interests. america suggested this to iraq.

brings in the disagreement that american academy and policy making have with multinational federations that is unlike Jacobins because americans reject the strong state idea. The disagreement is that America's position on federalism is that it shoud be more national than pluri-national. Instead american praise federalism for its ability to diffuse power to multiple points and it protects liberalism and enhances markets. americans even encouraged federalism for post-war germany. additionally, american federalism's goal is the same as Jacobinism which is to contruct a single poeple out of many.

breaksdown where the negativity toward multinational federations are coming from. Specifically from France's Jacobins where he thinks that pol recognition of multiple nations/ethnic comm institutionalizes and reinforces division, endangers national/state unity, and leads to state breka up. Then refers to practical examples which include several eastern euro states that have moved to replacing pluri-naitonal federations with "nationalising" states (tightly centralised, controlled by dom national comm, and intent on honogenisation of deviant identities). these nationalist seek indep as unitary, sovereign and indivisible nation-staets with some able to consider confederation.

New idea to me. Useful

UDL

final rule

The two laws. And isn't there one in Washington?

cool phrase

Main areas of accessibility?

policy frame; limits

A big storm brings things into focus

What engineers were doing

social history of the above

Geography, infrastructure, history policy cf Bangkok floodplain - People shoudl be left with an overview of the situation Bkk faces vis-a-vis climate flooding hydrology "broader stakes" of innovation

3 sentences, tight

1. Snacks (I do not really snack a whole lot, but they are nice to have just in case).
2. Books (I love buying new books, but I have so many that I already have and should read).
3. Make up (I do not wear makeup often, so why do I have so much?)

Money Problems and debt.

AI Tools for each phase of analysis

von Wissen, Normen, Gesetzen, Gerichtsurteilen …

Prozesswandel?

Stronger alternatives: The central claim that, "AI-powered systems enable teams collaborate, explore new choices, and develop ideas in real time," is a good argument, however I would end the sentence with a stronger explanation. The ending of their sentence is vague and indistinct.

Consistency: This article is consistent, drawing all conclusion back to the thesis that AI improves firms. It also recognizes that AI needs to be implemented with care. By doing this the article is recognizing the counter claim that AI is "taking over," which improves the logic of the argument.

Ambiguity: The term "operations efficiency" is used quite a bit in this article. An explanation of what operations efficiency really means should be provided so that the reader understands the full context of the argument. Operations efficiency could refer to refer to product development, marketing, or sales.

Fallacies: I couldn't find a fallacy in this article. Evidence is used to support the claim, there aren't emotional/personal attacks, and evidence is broken down and explained. This article is thoroughly written with the intent to inform, not attack.

Link your ethics to your plans to grow the business sustainably--this allows you to both articulate your values and end with a hopeful note about the future. It's a good move that helps both describe and promote your vision.

Second-order headings (reflected in the Table of Contents) and bulleted lists help break up the clutter of the document.

You may wish to consider some bulleted lists to make the examples stand out more visually.

"Another thing" is generic sounding. Use strong beginnings to each sentence, e.g. "Bean There also provides."

For each value, list at least one practice that demonstrates that value. It need not be long, but it does make your commitment clearer to the reader of the document.

The story about COVID can demonstrate qualities like resilience and determination, which you can then describe as qualities that drive your business model.

So, tldr, you are always linking your company's actions and your company's values.

Can you briefly tell us what was wrong with the existing ones that created this opportunity for you to make a better (and more ethical) product? What did the opportunity look like?

Nah, you wanna exceed expectations rather than just meet them.

Use the "insert table of contents" function to create a symmetrical presentation with consistent columns.

eLife Assessment

This fundamental study presents a new method for longitudinally tracking cells in two-photon imaging data that addresses the specific challenges of imaging neurons in the developing cortex. It provides compelling evidence demonstrating reliable longitudinal identification of neurons across the second postnatal week in mice. The study should be of interest to development neuroscientists engaged in population-level recordings using two-photon imaging.

Reviewer #1 (Public review):

Summary:

This manuscript presents a compelling and innovative approach that combines Track2p neuronal tracking with advanced analytical methods to investigate early postnatal brain development. The work provides a powerful framework for exploring complex developmental processes such as the emergence of sensory representations, cognitive functions, and activity-dependent circuit formation. By enabling the tracking of the same neurons over extended developmental periods, this methodology sets the stage for mechanistic insights that were previously inaccessible.

Strengths:

(1) Innovative Methodology:

The integration of Track2p with longitudinal calcium imaging offers a unique capability to follow individual neurons across critical developmental windows.

(2) High Conceptual Impact:

The manuscript outlines a clear path for using this approach to study foundational developmental questions, such as how early neuronal activity shapes later functional properties and network assembly.

(3) Future Experimental Potential:

The authors convincingly argue for the feasibility of extending this tracking into adulthood and combining it with targeted manipulations, which could significantly advance our understanding of causality in developmental processes.

(4) Broad Applicability:

The proposed framework can be adapted to a wide range of experimental designs and questions, making it a valuable resource for the field.

Weaknesses:

None major. The manuscript is conceptually strong and methodologically sound. Future studies will need to address potential technical limitations of long-term tracking, but this does not detract from the current work's significance and clarity of vision

Comments on revisions:

I have no further requests. I think this is an excellent manuscript

Reviewer #2 (Public review):

Summary:

The manuscript by Majnik and colleagues introduces "Track2p", a new tool designed to track neurons across imaging sessions of two-photon calcium imaging in developing mice. The method addresses the challenge of tracking cells in the growing brain of developing mice. The authors showed that "Track2p" successfully tracks hundreds of neurons in the barrel cortex across multiple days during the second postnatal week. This enabled identification of the emergence of behavioral state modulation and desynchronization of spontaneous network activity around postnatal day 11.

Strengths

The authors have satisfactorily addressed the majority of our questions and comments, and the revisions substantially improve the manuscript. The expansion of Track2p to accept general NumPy array inputs makes the tool more accessible to researchers using different analysis pipelines. While the absence of benchmarking standards remains a limitation across the field, the release of the ground-truth dataset is an important step forward that will allow other researchers to evaluate and compare algorithms.

Minor point

(1) The authors tested the robustness of the algorithm across non-consecutive days. As expected, performance drops significantly under these conditions. We agree that this limitation reflects biological constraints due to brain growth rather than shortcomings of the algorithm itself. This is relevant for researchers planning to use Track2p for longitudinal imaging or benchmarking new algorithms, and we recommend including some of this information in the Supplementary Information along with a brief discussion.

Comments on revisions:

We acknowledge the extended documentation for using Track2p and converting between Suite2p outputs and NumPy arrays. This addition is of great utility. We would also suggest further expanding the documentation for the NumPy array implementation, as we ran into some errors when testing this feature using NumPy arrays generated from deltaF traces, TIFF FOVs, and Cellpose masks.

Reviewer #3 (Public review):

Summary:

In this manuscript Majnik et al. developed a computational algorithm to track individual developing interneurons in the rodent cortex at postnatal stages. Considerable development in cortical networks takes place during the first postnatal weeks, however, tools to study them longitudinally at a single cell level are scarce. This paper provides a valuable approach to study both single cell dynamics across days and state-drive network changes. The authors used Gad67Cre mice together with virally introduced TdTom to track interneurons based on their anatomical location in the FOV and AAVSynGCaMP8m to follow their activity across the second postnatal week, a period during which the cortex is known to undergo marked decorrelation in spontaneous activity. Using Track2P, the authors show feasibility to track populations of neurons in the same mice capturing with their analysis previously described developmental decorrelation and uncovering stable representations of neuronal activity, coincident with the onset of spontaneous active movement. The quality of the imaging data is compelling, and the computational analysis is thorough, providing a widely applicable tool for the analysis of emerging neuronal activity in the cortex. Below are some points for the authors to consider.

Major points

The authors use a viral approach to label cortical interneurons. It is unclear how Track2P will perform in dense networks of excitatory cells using GCaMP transgenic mice.

The authors used 20 neurons to generate a ground truth data set. The rational for this sample size is unclear. Figure 1 indicates capability to track ~728 neurons. A larger ground truth data set will increase the robustness of the conclusions.

It is unclear how movement was scored in the analysis shown in Fig 5A. Was the time that the mouse spent moving scored after visual inspection of the videos? Were whisker and muscle twitches scored as movement or was movement quantified as amount of time in which the treadmill was displaced?

The rational for binning the data analysis in early P11 is unclear. As the authors acknowledged, it is likely that the decoder captured active states from P11 onwards. Because active whisking begins around P14, it is unlikely to drive this change in network dynamics at P11. Does pupil dilation in the pups change during locomotor and resting states? Does the arousal state of the pups abruptly change at P11?

Comments on revisions:

The authors have addressed carefully all my comments. This is an interesting paper.

Author response:

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

Reviewer #1 (Public review):

We thank the reviewer for very enthusiastic and supportive comments on our manuscript. 

Summary:

This manuscript presents a compelling and innovative approach that combines Track2p neuronal tracking with advanced analytical methods to investigate early postnatal brain development. The work provides a powerful framework for exploring complex developmental processes such as the emergence of sensory representations, cognitive functions, and activity-dependent circuit formation. By enabling the tracking of the same neurons over extended developmental periods, this methodology sets the stage for mechanistic insights that were previously inaccessible.

Strengths:

(1) Innovative Methodology:

The integration of Track2p with longitudinal calcium imaging offers a unique capability to follow individual neurons across critical developmental windows.

(2) High Conceptual Impact:

The manuscript outlines a clear path for using this approach to study foundational developmental questions, such as how early neuronal activity shapes later functional properties and network assembly.

(3) Future Experimental Potential:

The authors convincingly argue for the feasibility of extending this tracking into adulthood and combining it with targeted manipulations, which could significantly advance our understanding of causality in developmental processes.

(4) Broad Applicability:

The proposed framework can be adapted to a wide range of experimental designs and questions, making it a valuable resource for the field.

Weaknesses:

No major weaknesses were identified by this reviewer. The manuscript is conceptually strong and methodologically sound. Future studies will need to address potential technical limitations of long-term tracking, but this does not detract from the current work's significance and clarity of vision.

Reviewer #2 (Public review):

Summary:

The manuscript by Majnik and colleagues introduces "Track2p", a new tool designed to track neurons across imaging sessions of two-photon calcium imaging in developing mice. The method addresses the challenge of tracking cells in the growing brain of developing mice. The authors showed that "Track2p" successfully tracks hundreds of neurons in the barrel cortex across multiple days during the second postnatal week. This enabled the identification of the emergence of behavioral state modulation and desynchronization of spontaneous network activity around postnatal day 11.

Strengths:

The manuscript is well written, and the analysis pipeline is clearly described. Moreover, the dataset used for validation is of high quality, considering the technical challenges associated with longitudinal two-photon recordings in mouse pups. The authors provide a convincing comparison of both manual annotation and "CellReg" to demonstrate the tracking performance of "Track2p". Applying this tracking algorithm, Majnik and colleagues characterized hallmark developmental changes in spontaneous network activity, highlighting the impact of longitudinal imaging approaches in developmental neuroscience. Additionally, the code is available on GitHub, along with helpful documentation, which will facilitate accessibility and usability by other researchers.

Weaknesses:

(1) The main critique of the "Track2p" package is that, in its current implementation, it is dependent on the outputs of "Suite2p". This limits adoption by researchers who use alternative pipelines or custom code. One potential solution would be to generalize the accepted inputs beyond the fixed format of "Suite2p", for instance, by accepting NumPy arrays (e.g., ROIs, deltaF/F traces, images, etc.) from files generated by other software. Otherwise, the tool may remain more of a useful add-on to "Suite2p" (see https://github.com/MouseLand/suite2p/issues/933) rather than a fully standalone tool.

We thank the reviewer for this excellent suggestion. 

We have now implemented this feature, where Track2p is now compatible with ‘raw’ NumPy arrays for the three types of inputs. For more information, please check the updated documentation: https://track2p.github.io/run_inputs_and_parameters.html#raw-npy-arrays. We have also tested this feature using a custom segmentation and trace extraction pipeline using Cellpose for segmentation.

(2) Further benchmarking would strengthen the validation of "Track2p", particularly against "CaIMaN" (Giovannucci et al., eLife, 2019), which is widely used in the field and implements a distinct registration approach.

This reviewer suggested  further benchmarking of Track2P.  Ideally, we would want to benchmark Track2p against the current state-of-the-art method. However, the field currently lacks consensus on which algorithm performs best, with multiple methods available including CaIMaN, SCOUT (Johnston et al. 2022), ROICaT (Nguyen et al. 2023), ROIMatchPub (recommended by Suite2p documentation and recently used by Hasegawa et al. 2024), and custom pipelines such as those described by Sun et al. 2025. The absence of systematic benchmarking studies—particularly for custom tracking pipelines—makes it impossible to identify the current state-of-the-art for comparison with Track2p. While comparing Track2p against all available methods would provide comprehensive evaluation, such an analysis falls beyond the scope of this paper.

We selected CellReg for our primary comparison because it has been validated under similar experimental conditions—specifically, 2-photon calcium imaging in developing hippocampus between P17-P25 (Wang et al. 2024)—making it the most relevant benchmark for our developmental neocortex dataset.

That said, to support further benchmarking in mouse neocortex (P8-P14), we will publicly release our ground truth tracking dataset.

(3) The authors might also consider evaluating performance using non-consecutive recordings (e.g., alternate days or only three time points across the week) to demonstrate utility in other experimental designs.

Thank you for your suggestion. We have performed a similar analysis prior to submission, but we decided against including it in the final manuscript, to keep the evaluation brief and to not confuse the reader with too many different evaluation methods. We have included the results inAuthor response images 1 and 2 below.

To evaluate performance in experimental designs with larger time spans between recordings (>1 day) we performed additional evaluation of tracking from P8 to each of the consecutive days while omitting the intermediate days (e. g. P8 to P9, P8 to P10 … P8 to P14). The performance for the three mice from the manuscript is shown below:

Author response image 1.

As expected with increasing time difference between the two recordings the performance drops significantly (dropping to effectively zero for 2 out of 3 mice). This could also explain why CellReg struggles to track cells across all days, since it takes P8 as a reference and attempts to register all consecutive days to that time point before matching, instead of performing registration and matching in consecutive pairs of recordings (P8-P9, P9-P10 … P13-P14) as we do.

Finally for one of the three mice we also performed an additional test where we asked how adding an additional recording day might rescue the P8-P14 tracking performance. This corresponds to the comment from the reviewer, answering the question if we can only perform three days of recording which additional day would give the best tracking performance. 

Author response image 2.

As can be seen from the plot, adding the P10 or P11 recording shows the most significant improvement to the tracking performance, however the performance is still significantly lower than when including all days (see Fig. 4). This test suggests that including a day that is slightly skewed to earlier ages might improve the performance more than simply choosing the middle day between the two extremes. This would also be consistent with the qualitative observation that the FOV seems to show more drastic day-to-day changes at earlier ages in our recording conditions.

Reviewer #3 (Public review):

Summary:

In this manuscript, Majnik et al. developed a computational algorithm to track individual developing interneurons in the rodent cortex at postnatal stages. Considerable development in cortical networks takes place during the first postnatal weeks; however, tools to study them longitudinally at a single-cell level are scarce. This paper provides a valuable approach to study both single-cell dynamics across days and state-driven network changes. The authors used Gad67Cre mice together with virally introduced TdTom to track interneurons based on their anatomical location in the FOV and AAVSynGCaMP8m to follow their activity across the second postnatal week, a period during which the cortex is known to undergo marked decorrelation in spontaneous activity. Using Track2P, the authors show the feasibility of tracking populations of neurons in the same mice, capturing with their analysis previously described developmental decorrelation and uncovering stable representations of neuronal activity, coincident with the onset of spontaneous active movement. The quality of the imaging data is compelling, and the computational analysis is thorough, providing a widely applicable tool for the analysis of emerging neuronal activity in the cortex. Below are some points for the authors to consider.

We thank the reviewer for a constructive and positive evaluation of our MS. 

Major points:

(1) The authors used 20 neurons to generate a ground truth dataset. The rationale for this sample size is unclear. Figure 1 indicates the capability to track ~728 neurons. A larger ground truth data set will increase the robustness of the conclusions.

We think this was a misunderstanding of our ground truth dataset analysis which included 192 and not 20 neurons. Indeed, as explained in the methods section, since manually tracking all cells would require prohibitive amounts of time, we decided to generate sparse manual annotations, only tracking a subset of all cells from the first recording day onwards. To do this, we took the first recording (s0), and we defined a grid 64 equidistant points over the FOV and, for each point, identified the closest ROI in terms of euclidean distance from the median pixel of the ROI (see Fig. S3A). We then manually tracked these 64 ROIs across subsequent days. Only neurons that were detected and tracked across all sessions were taken into account and referred to as our ground truth dataset (‘GT’ in Fig. 4). This was done for 3 mice, hence 3X64 neurons and not 20 were used to generate our GT dataset. 

(2) It is unclear how movement was scored in the analysis shown in Figure 5A. Was the time that the mouse spent moving scored after visual inspection of the videos? Were whisker and muscle twitches scored as movement, or was movement quantified as the amount of time during which the treadmill was displaced?

Movement was scored using a ‘motion energy’ metric as in Stringer et al. 2019 (V1) or Inácio et al. 2025 (S1). This metric takes each two consecutive frames of the videography recordings and computes the difference between them by summing up the square of pixelwise differences between the two images. We made the appropriate changes in the manuscript to further clarify this in the main text and methods in order to avoid confusion.

Since this metric quantifies global movements, it is inherently biased to whole-body movements causing more significant changes in pixel values around the whole FOV of the camera. Slight twitches of a single limb, or the whisker pad would thus contribute much less to this metric, since these are usually slight displacements in a small region of the camera FOV. Additionally, comparing neural activity across all time points (using correlation or R²) also favours movements that last longer (such as wake movements / prolonged periods of high arousal) since each time point is treated equally.

As we suggested in the discussion, in further analysis it would be interesting to look at the link between twitches and neural activity, but this would likely require extensive manual scoring. We could then treat movements not as continuous across all time-points, but instead using event-based analysis for example peri-movement time histograms for different types of movements at different ages, which is however outside of the scope of this study.

(3) The rationale for binning the data analysis in early P11 is unclear. As the authors acknowledged, it is likely that the decoder captured active states from P11 onwards. Because active whisking begins around P14, it is unlikely to drive this change in network dynamics at P11. Does pupil dilation in the pups change during locomotor and resting states? Does the arousal state of the pups abruptly change at P11?

We agree that P11 does not match any change in mouse behavior that we have been able to capture. However, arousal state in mice does change around postnatal day 11. This period marks a transition from immature, fragmented states to more organized and regulated sleep-wake patterns, along with increasing influence from neuromodulatory and sensory systems. All of these changes have been recently reviewed in Wu et al. 2024 (see also Martini et al. 2021). In addition, in the developing somatosensory system, before postnatal day 11 (P11), wake-related movements (reafference) are actively gated and blocked by the external cuneate nucleus (ECN, Tiriac et al. 2016 and all excellent recent work from the Blumberg lab). This gating prevents sensory feedback from wake movements from reaching the cortex, ensuring that only sleep-related twitches drive neural responses. However, around P11, this gating mechanism abruptly lifts, enabling sensory signals from wake movements to influence cortical processing—signaling a dramatic developmental shift from Wu et al. 2024

Reviewer #1 (Recommendations for the authors):

This manuscript represents a significant advancement in the field of developmental neuroscience, offering a powerful and elegant framework for longitudinal cellular tracking using the Track2p method combined with robust analytical approaches. The authors convincingly demonstrate that this integrated methodology provides an invaluable template for investigating complex developmental processes, including the emergence of sensory representations and higher cognitive functions.

A major strength of this work is its emphasis on the power of longitudinal imaging to illuminate activity-dependent development. By tracking the same neurons over time, the authors open up new possibilities to uncover how early activity patterns shape later functional outcomes and the organization of neuronal assemblies-insights that would be inaccessible using conventional cross-sectional designs.

Importantly, the manuscript highlights the potential for this approach to be extended even further, enabling continuous tracking into adulthood and thus offering an unprecedented window into long-term developmental trajectories. The authors also underscore the exciting opportunity to incorporate targeted perturbation experiments, allowing researchers to causally link early circuit dynamics to later outcomes.

Given the increasing recognition that early postnatal alterations can underlie the etiology of various neurodevelopmental disorders, this work is especially timely. The methods and perspectives presented here are poised to catalyze a new generation of developmental studies that can reveal mechanistic underpinnings of both typical and atypical brain development.

In summary, this is a technically impressive and conceptually forward-looking study that sets the stage for transformative advances in developmental neuroscience.

Thank you for the thoughtful feedback—it's greatly appreciated!

Reviewer #2 (Recommendations for the authors):

Minor points:

(1) Figure 1. Consider merging or moving to Supplemental, as its rationale is well described in the text.

We would like to retain the current figure as we believe it provides an effective visual illustration of our rationale that will capture readers' attention and could serve as a valuable reference for others seeking to justify longitudinal tracking of the developing brain. We hope the reviewer will understand our decision.

(2) Some axis labels and panels are difficult to read due to small font sizes (e.g. smaller panels in Figures 5-7).

Modified, thanks 

(3) Supplementary Figures. The order of appearance in the main text is occasionally inconsistent.

This was modified, thanks

(4) Line 132. Add a reference to the registration toolbox used (elastix). A brief description of the affine transformation would also be helpful, either here or in the Methods section (p. 27).

We have added reference to Ntatsis et al. 2023 and described affine transformation in the main text (lines 133-135): 

Firstly, we estimate the spatial transformation between s0 and s1 using affine image registration (i.e. allowing shifting, rotation, scaling and shearing, see Fig. 2B, the transformation is denoted as T).

(5) Lines 147-151. If this method is adapted from another work, please cite the source.

Computing the intersection over union of two ROIs for tracking is a widely established and intuitive method used across numerous studies, representing standard practice rather than requiring specific citation. We have however included the reference to the paper describing the algorithm we use to solve the linear sum assignment problem used for matching neurons across a pair of consecutive days (Crouse 2016).

(6) Line 218. "classical" or automatic?

We meant “classical” in the sense of widely used. 

(7) Lines 220-231. Did the authors find significant variability of successfully tracked neurons across mice? While the data for successfully tracked cells is reported (Figure 5B), the proportions are not. Could differences in neuron dropout across days and mice affect the analysis of neuronal activity statistics?

We thank the reviewer for raising this important point. We computed the fraction of successfully tracked cells in our dataset and found substantial variability:

Cells detected on day 0: [607, 1849, 2190, 1988, 1316, 2138] 

Proportion successfully tracked: [0.47, 0.20, 0.36, 0.37, 0.41, 0.19]

Notably, the number of cells detected on the first day varies considerably (607–2138 cells). There appears to be a trend whereby datasets with fewer initially detected cells show higher tracking success rates, potentially because only highly active cells are identified in these cases.

To draw more definitive conclusions about the proportion of active cells and tracking dropout rates, we would require activity-independent cell detection methods (such as Cellpose applied to isosbestic 830 nm fluorescence, or ideally a pan-neuronal marker in a separate channel, e.g., tdTomato). We have incorporated the tracking success proportions into the revised manuscript.

(8) Line 260. Please briefly explain, here or in the Methods, the rationale for using data from only 3 mice (rather than all 6) for evaluating tracking performance.

We used three mice for this analysis due to the labor-intensive nature of manually annotating 64 ROIs across several days. Given the time constraints of this manual process, we determined that three subjects would provide adequate data to reliably assess tracking performance.

(9) Line 277. Consider clarifying or rephrasing the phrase "across progressively shorter time intervals"? Do you mean across consecutive days?

This has been rephrased as follows: 

Additionally, to assess tracking performance over time, we quantified the proportion of reconstructed ground truth tracks over progressively longer time intervals (first two days, first three days etc. ‘Prop. correct’ in Fig. 4C-F, see Methods). This allowed us to understand how tracking accuracy depends on the number of successive sessions, as well as at which time points the algorithm might fail to successfully track cells.

(10) Line 306. "we also provide additional resources and documentation". Please add a reference or link.

Done, thanks

Track2p  

(11) Lines 342-344. Specify that the raster plots refer to one example mouse, not the entire sample.

Done, thanks.

(12) Lines 996-1002. Please confirm whether only successfully tracked neurons were used to compute the Pearson correlations between all pairs.

Yes of course, this only applies to tracked neurons as it is impossible to compute this for non-tracked pairs.

(13) Line 1003. Add a reference to scikit-learn.

Reference was added to: 

Pedregosa, F., Varoquaux, G., Gramfort, A., Michel, V., Thirion, B., Grisel, O., Blondel, M., Prettenhofer, P., Weiss, R., Dubourg, V., Vanderplas, J., Passos, A., Cournapeau, D., Brucher, M., Perrot, M., & Duchesnay, E. (2011). Scikit-learn: Machine Learning in Python. Journal of Machine Learning Research, 12, 2825–2830. 

(14) Typos.Correct spacing between numeric values and units.

We did not find many typos regarding spacing between the numerical value and the unit symbol (degrees and percent should not be spaced right?).

Reviewer #3 (Recommendations for the authors):

The font size in many of the figures is too small. For example, it is difficult to follow individual ROIs in Figure S3.

Figure font size has been increased, thanks. In Figure S3 there might have been a misunderstanding, since the three FOV images do not correspond to the FOV of the same mouse across three days but rather to the first recording for each of the three mice used in evaluation (the ROIs can thus not be followed across images since they correspond to a different mouse). To avoid confusion we have labelled each of the FOV images with the corresponding mouse identifier (same as in Fig. 4 and 5).

eLife Assessment

This is a valuable study that explores the role of the conserved transcription factor POU4-2 in the maintenance, regeneration, and function of planarian mechanosensory neurons. The authors present convincing evidence provided by gene expression and functional studies to demonstrate that POU4-2 is required for the maintenance and regeneration of mechanosensory neurons and mechanosensory function in planarians. Furthermore, the authors identify conserved genes associated with human auditory and rheosensory neurons as potential targets of this transcription factor.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors explore the role of the conserved transcription factor POU4-2 in planarian maintenance and regeneration of mechanosensory neurons. The authors explore the role of this transcription factor and identify potential targets of this transcription factor. Importantly, many genes discovered in this work are deeply conserved, with roles in mechanosensation and hearing, indicating that planarians may be a useful model with which to study the roles of these key molecules. This work is important within the field of regenerative neurobiology, but also impactful for those studying evolution of the machinery that is important for human hearing.

Strengths:

The paper is rigorous and thorough, with convincing support for the conclusions of the work.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigate the role of the transcription factor Smed-pou4-2 in the maintenance, regeneration and function of mechanosensory neurons in the freshwater planarian Schmidtea mediterranea. First, they characterize the expression of pou4-2 in mechanosensory neurons during both homeostasis and regeneration, and examine how its expression is affected by the knockdown of soxB1, 2, a previously identified transcription factor essential for the maintenance and regeneration of these neurons. Second, the authors assess whether pou4-2 is functionally required for the maintenance and regeneration of mechanosensory neurons.

Strengths:

The study provides some new insights into the regulatory role of pou4-2 in the differentiation, maintenance, and regeneration of ciliated mechanosensory neurons in planarians.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review): 

Summary: 

In this manuscript, the authors explore the role of the conserved transcription factor POU4-2 in planarian maintenance and regeneration of mechanosensory neurons. The authors explore the role of this transcription factor and identify potential targets of this transcription factor. Importantly, many genes discovered in this work are deeply conserved, with roles in mechanosensation and hearing, indicating that planarians may be a useful model with which to study the roles of these key molecules. This work is important within the field of regenerative neurobiology, but also impactful for those studying the evolution of the machinery that is important for human hearing. 

Strengths: 

The paper is rigorous and thorough, with convincing support for the conclusions of the work. 

Weaknesses: 

Weaknesses are relatively minor and could be addressed with additional experiments or changes in writing.

Reviewer #2 (Public review): 

Summary: 

In this manuscript, the authors investigate the role of the transcription factor Smed-pou4-2 in the maintenance, regeneration, and function of mechanosensory neurons in the freshwater planarian Schmidtea mediterranea. First, they characterize the expression of pou4-2 in mechanosensory neurons during both homeostasis and regeneration, and examine how its expression is affected by the knockdown of soxB1, 2, a previously identified transcription factor essential for the maintenance and regeneration of these neurons. Second, the authors assess whether pou4-2 is functionally required for the maintenance and regeneration of mechanosensory neurons. 

Strengths: 

The study provides some new insights into the regulatory role of pou4-2 in the differentiation, maintenance, and regeneration of ciliated mechanosensory neurons in planarians. 

Weaknesses: 

The overall scope is relatively limited. The manuscript lacks clear organization, and many of the conclusions would benefit from additional experiments and more rigorous quantification to enhance their strength and impact. 

Reviewing Editor Comments: 

(1) Quantification of pou4-2(+) cells that express (or do not express) hmcn-1-L and/or pkd1L-2(-) is a common suggestion amongst reviewers. It is recognized that Ross et al. (2018) showed that pkd1L-2 and hmcn-1L expression is detected in separate cells by double FISH, and the analysis presented in Supplementary Figure S3 is helpful in showing that some cells expressing pou4-2 (magenta) are not labeled by the combined signal of pkd1L-2 and hmcn-1-L riboprobes (green). However, I am not sure that we can conclude that pkd1L-2 and hmcn-1-L are effectively detected when riboprobes are combined in the analysis. Therefore, quantification of labeled cells as proposed by Reviewers 1 and 2 would help.

Combining riboprobes is a standard approach in the field, and we chose this method as a direct way to determine which cells lack expression of both genes. We agree that providing the raw quantification data would be helpful for readers, and we included this data in Supplementary File S7; the file contains the quantification information for this dFISH experiment represented in Supplementary Figure 3.

(2) It may be helpful to comment on changes (or lack of changes) in atoh gene RNA levels in RNAseq analyses of pou4-2 animals. As mentioned by one of the reviewers, in situs that don't show signal are inconclusive in this regard. 

We fully agree with both reviewers. Two of the planarian atonal homologs are difficult to detect and produce background signals, which we attempted and previously reported in Cowles et al. Development (2013). We conceived performing reciprocal RNAi/in situ experiments, born out of curiosity given the reported role of atonal in the pou4 cascade in other organisms. However, these exploratory experiments lacked a strong rationale for inclusion, particularly given that pou4-2 and the atonal homologs do not share expression patterns, co-expression, or differential expression in our RNA-seq dataset. Therefore, we decided to omit the atonal in situs following pou4-2 RNAi. We retained the experiments showing that knockdown of the atonal genes does not show robust effects on the mechanosensory neuron pattern, as expected. We thank the reviewing editor and reviewers for pinpointing the concern. We agree that additional experiments, such as qPCR experiments, would be needed. We reasoned that while these additional experiments could be informative, they are unlikely to alter the key conclusions of this study substantially.

(3) There seem to be typos at bottom of Figure 10 and top of page 11 when referencing to Figure 4B (should be to 5B instead): "While mechanosensory neuronal patterned expression of Eph1 was downregulated after pou4-2 and soxB1-2 inhibition, low expression in the brain branches of the ventral cephalic ganglia persisted (Figure 4B)." 

Thank you! We have fixed those.

(4) Typo (page 13; kernel?): "...to test to what extent the Pou4 gene regulatory kernel is conserved among these widely divergent animals." 

Regulatory kernels are defined as the minimal sets of interacting genes that drive developmental processes and are the core circuits within a gene regulatory network, but we recognize that this might not be as well known, so we have changed the term to “network” for clarity.

Reviewer #1 (Recommendations for the authors): 

(1) The authors indicate that they are interested in finding out whether POU4-2 is important in the creation of mechanosensory neurons in adulthood as well as in embryogenesis (in other words, whether the mechanism is "reused during adult tissue maintenance and regeneration"). The manuscript clearly shows that planarian POU4 -2 is important in adult neurogenesis in planarians, but there is no evidence presented to show that this is a recapitulation of embryogenesis. Is pou4-2 expressed in the planarian embryo? This might be possible to examine by ISH or through the evaluation of sequencing data that already exists in the literature. 

We agree that these statements should be precise. We have clarified when we make comparisons to the role of Pou4 in sensory system development in other organisms versus its role in the adult planarian. We examined its expression using the existing database of embryonic gene expression. Thanks for hinting at this idea. We performed BLAST in Planosphere (Davies et al., 2017) to cross-reference our clone matching dd_Smed_v6_30562_0_1, which is identical to SMED30002016. The embryonic gene expression for SMED30002016 indicates this gene is expressed at the expected stages given prior knowledge of the timing of organ development in Schmidtea mediterranea (a positive trend begins at Stage 5, with a marked increase by Stage 6 that remains comparable to the asexual expression levels shown). We thank the reviewer for pointing out this oversight. We have incorporated this result in the paper as a Supplementary Figure and discuss how we can only speculate that it has a similar role as we detect in the adult asexual worms.

(2) Can it be determined whether the punctate pou4-2+ cells outside of the stripes are progenitors or other neural cell types? Are there pou4-2+ neurons that are not mechanosensory cell types? Could there be other roles for POU4-2 in the neurogenesis of other cell types? It might help to show percentages of overlap in Figure 4A and discuss whether the two populations add up to 100% of cells. 

These are good questions that arise in part from other statements that need clarification in the text (pointed out by Reviewer 2). We think some of the dorsal pou4-2⁺ might represent progenitor cells undergoing terminal differentiation (see Supplementary Figure 4). We attempted BrdU pulse chase experiments but were not successful in consistently detecting pou4-2 at sufficient levels with our protocol. In response to this helpful comment, we have included this question as a future direction in the revised Discussion. Finally, we have edited our description of the expression pattern. We already pointed out that there are other cells on the ventral side that are not affected when soxB1-2 is knocked down. We attempted to resolve the potential identity of those cells working with existing scRNA-seq data in collaboration with colleagues, but their low abundance made it difficult to distinguish other populations. While we acknowledge this interesting possibility, we have chosen to focus this report on the role of pou4-2 downstream of soxB1-2, as this represents the most well-supported aspect of the dataset and was positively highlighted by both the reviewer and editor.

(3) The authors discuss many genes from their analysis that play conserved roles in mechanosensation and hearing. Were there any conserved genes that came up in the analysis of pou4-2(RNAi) planarians that have not yet been studied in human hearing and neurodevelopment? I am wondering the extent to which planarians could be used as a discovery system for mechanosensory neuron function and development, and discussion of this point might increase the impact of this paper or provide critical rationale for expanding work on planarian mechanosensation. 

Indeed, we agree that planarians could be used to identify conserved genes with roles in mechanosensation and have included this point in the Discussion. In this study, we have focused on demonstrating the conservation of gene regulation. While this study was initially based on a graduate thesis project, we have since generated a more comprehensive dataset from isolated heads, which we are currently analyzing. This has been emphasized in the revised Discussion.

Minor: 

(1) For Figure 6E, the authors could consider showing data along a negative axis to indicate a decrease in length in response to vibration and to more clearly show that this decrease doesn't occur as strongly after pou4-2(RNAi). 

We displayed this behavior as the percent change, as this is a standard way to represent this data. As the percent change is a positive value, we represent the data as these positive values.

(2) The authors should consider quantifying the decrease of pou4-2 mRNA after atonal(RNAi) conditions, either by RT-qPCR or cell quantification. Visually, the signal in the stripes after atoh8-2(RNAi) seems lower, particularly in the tail. The punctate pattern outside the stripes may also be decreased after atoh8-1(RNAi). But quantification might strengthen the argument. 

We agree with the reviewer and acknowledge that we should have been more cautious in interpreting these results. Those two genes are difficult to detect and did not show specific patterns in Cowles et al. (2013). The reviewer is correct that additional experiments are necessary before reaching conclusions, but we do not think as discussed earlier we do not think new experiments would provide insights for the major conclusions. These experiments were exploratory in nature and tangential to our main conclusions, especially in the absence of reciprocal evidence (e.g., shared expression patterns, co-expression, or differential expression in our RNA-seq data. Therefore, we decided to eliminate the atonal in situs following pou4-2 RNAi.

Reviewer #2 (Recommendations for the authors): 

A. Expression of pou4-2 in ciliated mechanosensory neurons: 

(1) The conclusion that pou4-2 is expressed in ciliated mechanosensory neurons is primarily based on co-expression analysis using a published single-cell dataset. Although the authors later show that a subset of pou4-2 cells also express pkd1L-2 (Figure 4A), a known marker of ciliated mechanosensory neurons, this finding is not properly quantified. I recommend moving Figure 4A to earlier in the manuscript (e.g., to Figure 2) and expanding the analysis to include additional known markers of this cell type. Proper quantification of the extent of co-localization is necessary to support the claim robustly. 

As pointed out by the reviewer, there is substantive evidence from our lab and other reports. King et al. also showed pou4-2 and pkd1L-2 ‘regulation’ by their scRNA-seq data, and this function is conserved in the acoel Hofstenia miamia (Hulett et al., PNAS 2024 ). Our analysis shows convincing co-localization by scRNA-seq and expression of soxB1-2 and neural markers in the respective populations. Furthermore, we included colocalization of pou4-2 with mechanosensory genes using fluorescence in situ hybridization (Figure 3B, Supplementary Figure 4, and Supplementary File S7). We are confident the data conclusively show pou4-2 regulates pkd1L-2 expression in a subset of mechanosensory neurons. Given the strength of existing observations and previously published data, we believe that additional staining experiments are not essential to support this conclusion. 

(2) There appears to be a conceptual inconsistency in the interpretation of pou4-2 expression dynamics. On one hand, the authors suggest that delayed pou4-2 expression indicates a role in late-stage differentiation (p.6). On the other hand, they propose that pou4-2 may be expressed in undifferentiated progenitors to initiate downstream transcriptional programs (p.8). These interpretations should be reconciled. Additionally, claims regarding pou4-2 expression in progenitor populations should be supported by co-localization with established stem cell or progenitor markers, rather than inferred from signal intensity alone. 

This is an excellent point, and we agree with the reviewer that this section requires editing. As described in response to Reviewer 1, we attempted BrdU pulse chase experiments but were not successful in consistently detecting pou4-2 at sufficient levels with our protocol. Furthermore, we could not obtain strong signals in double labeling experiments in pou4-2 in situs combined with piwi-1 or PIWI-1 antibodies. We will include those experiments as a future direction and amend our conclusions accordingly.

(3) The expression pattern shown in Figure 1B raises questions about the precise anatomical localization of pou4-2 cells. It is unclear whether these cells reside in the subepidermal plexus or the deeper submuscular plexus, which represent distinct neuronal layers (Ross et al., 2017). The observed signals near the ventral nerve cords could suggest submuscular localization. To clarify this, higher-resolution imaging and co-staining with region-specific neural markers are recommended. 

In Ross et al. (2018), we showed that the pkd1L-2⁺ cells are located submuscularly. The pkd1L-2 cells express pou4-2, thus the pou4-2⁺ cells are located in the same location. Based on co-expression data and co-expression with PKD genes, we are confident it is submuscular.

B. The functional requirements of pou4-2 in the maintenance of mechanosensory neurons: 

(1) To evaluate the functional role of pou4-2 in maintaining mechanosensory neurons, the authors performed whole-animal RNA-seq on pou4-2(RNAi) and control animals, identifying a significant downregulation of genes associated with mechanosensory neuron expression. However, the presentation of these findings is fragmented across Figures 3, 4, and 5. I recommend consolidating the RNA-seq results (Figure 3) and the subsequent validation of downregulated genes (Figures 4 and 5) into a single, cohesive figure. This would improve the logical flow and clarity of the manuscript. 

As suggested by the reviewer, we have combined Figures 3 and 4 (new Figure 3), which we believe improves the flow. We decided to keep Figure 5 (new Figure 4) as a standalone because it focuses on the characterization of new genes revealed by RNAseq and scRNA-seq data mining that were not previously reported in Ross et al. 2018 and

2024.

(2) In pou4-2(RNAi) animals, pkd1L-2 expression appears to be entirely lost, while hmcn-1-L shows faint expression in scattered peripheral regions. The authors suggest that an extended RNAi treatment might be necessary to fully eliminate hmcn-1-L expression. However, an alternative explanation is that pou4-2 is not essential for maintaining all hmcn-1-L cells, particularly if pou4-2 expression does not fully overlap with that of hmcn-1-L. This possibility should be acknowledged and discussed. 

We agree and have acknowledged this point in the revised text.

(3) On page 9, the section title claims that "Smed-pou4-2 regulates genes involved in ciliated cell structure organization, cell adhesion, and nervous system development." While some differentially expressed genes are indeed annotated with these functions based on homology, the manuscript does not provide experimental evidence supporting their roles in these biological processes in planarians. The title should be revised to avoid overstatement, and the limitations of extrapolating a function solely from gene annotation should be acknowledged. 

Excellent point. We have edited the text to indicate that the genes were annotated or implicated.

(4) The cilia staining presented in Figure 6B to support the claim that pou4-2 is required for ciliated cell structure organization is unconvincing. Improved imaging and more targeted analysis (e.g., co-labeling with mechanosensory markers) are needed to support this conclusion. 

We have addressed this concern by adjusting the language to be more precise and indicate that the stereotypical banded pattern is disrupted with decreased cilia labeling along the dorsal ciliated stripe. Indeed, our conclusion overstated the observations made with the staining and imaging resolution. Thank you.

C. The functional requirements of pou4-2 in the regeneration of mechanosensory neurons: 

To evaluate the role of pou4-2 in the regeneration of mechanosensory neurons, the authors performed amputations on pou4-2(RNAi) and control(RNAi) animals and assessed the expression of mechanosensory markers (pkd1L-2, hmcn-1-L) alongside a functional assay. However, the results shown in Figure 4B indicate the presence of numerous pkd1L-2 and hmcn-1-L cells in the blastema of pou4-2(RNAi) animals. This observation raises the possibility that pou4-2 may not be essential for the regeneration of these mechanosensory neurons. The authors should address this alternative interpretation. 

Our interpretation is that there were very few cells expressing the markers compared to controls. The pattern was predominantly lost, which is consistent with other experiments shown in the paper. However, we have added the additional caveat suggested by the reviewer.

Minor points: 

(1) On p.8, the authors wrote "every 12 hours post-irradiation". However, this is not consistent with the figure, which only shows 0, 3, 4, 4.5, 5, and 5.5 dpi. 

We corrected this. Thank you for catching the mistake!

(2) On p.12, the authors wrote "Analysis of pou4-2 RNAi data revealed differentially expressed genes with known roles in mechanosensory functions, such as loxhd-1, cdh23, and myo7a. Mutations in these genes can cause a loss of mechanosensation/transduction". This is misleading because, to my knowledge, the role of these genes in planarians is unknown. If the authors meant other model systems, they should clearly state this in the text and include proper references. 

The reviewer is correct that we are referencing findings from other organisms. We have clarified this point in the revised text. The appropriate references were included and cited in the first version.

(3) On p.7, the authors wrote, "conversely, the expression of atonal genes was unaffected in pou4-2 RNAi-treated regenerates (Supplementary Figure S2B)". However, it is unclear whether the Atoh8-1 and Atoh8-2 signals are real, as the quality of the in situ results is too low to distinguish between real signals and background noise/non-specific staining. 

This valid concern was addressed in our response to Reviewer 1. We have adjusted the figure and the text accordingly.

(4) On p.6 the authors wrote "pinpointed time points wherein the pou4-2 transcripts were robustly downregulated". However, the current version of the manuscript does not provide data explaining why Pou4-2 transcripts are robustly downregulated on day 12. 

Yes, we determined the appropriate time points using qPCR for all sample extractions. As an example, see the figure for qPCR validation at day 12 showing that pou4-2 and pkd1L2 are down.

Author response image 1.

In this graph, samples labeled “G” represent four biological controls of gfp(RNAi) control animals, and samples labeled “P” represent four biological controls of pou4-2(RNAi)animals at day 12 in the RNAi protocol.

(5) On p.13, the authors wrote "collecting RNA from how animals." Is this a typo? 

Thanks for catching the typo. It should read “whole” animals. We have corrected this.

(6) On p.14, the authors wrote "but the expression patterns of planarian atonal genes indicated that they represent completely different cell populations from pou4-2-regulated mechanosensory neurons". However, this is unclear from the images, as the in situ staining of Atoh8-1 and Atoh82 are potentially failed stainings. 

We agree. We have edited accordingly.

eLife Assessment

This valuable manuscript presents an open-source and low-cost acoustic system for quantifying biting and chewing in mice. The approach is carefully validated against human observers, demonstrating strong methodological reliability and enabling high-resolution analysis of feeding microstructure. The tool has broad relevance for studies of appetite circuits and pharmacological interventions. A significant contribution is the identification of previously unrecognized "meal-related" neurons in the lateral hypothalamus, providing novel biological insight into food consumption. While the support for the methodological advances is compelling and robust, some circuit-level conclusions are preliminary or incomplete, relying on small pilot samples and manual classification, and should be interpreted with caution. This paper will be of interest to those interested in ingestive behavior and/or hypothalamus.

Reviewer #1 (Public review):

This is an interesting and valuable paper by Gil-Lievana, Arroyo et al. that presents an open-source method (the "Crunchometer") for quantifying biting and chewing behavior in mice using audio detection. The work addresses an important and unmet need in the field: quantitative measures of feeding behavior with solid foods, since most prior approaches have been limited to liquids. The authors make a clear and compelling case for why this problem is important, and I fully agree with their motivation.

The system is carefully validated against human-scored video data and is shown to be at least as accurate, and in some cases more accurate, than human observers. This is a major strength of the study. I also particularly appreciate the demonstration of the technology in the context of LHA circuitry, which nicely illustrates its utility and importance for mechanistic studies of feeding. I also appreciate the ability to readily time-lock neural data to individual crunches. Overall, the manuscript is well-executed and represents a useful contribution to the field.

The comments I have are largely minor and should be straightforward to address:

(1) The authors should report sample sizes for all mouse cohorts, either alongside the statistics or in the figure legends for mean data.

(2) Clarification is needed as to whether crunch detection fidelity is influenced by the hardness or softness of the food. The focus here is on standard pellets, with some additional high-fat pellet data, but it would be useful to know how generalizable the method is across different textures.

(3) The authors should comment on how susceptible the Crunchometer is to background noise. For example, how well does it perform in the presence of white noise, experimenter movement, or other task-related sounds?

(4) Chemogenetic activation of LHA GABAergic neurons is used. DREADD-based activation may strongly drive these neurons in a way that is not directly comparable to optogenetic or more physiological manipulations. While I do not think additional experiments are required, it would strengthen the discussion to briefly acknowledge this limitation.

Reviewer #2 (Public review):

Summary:

This manuscript introduces the Crunchometer, a low-cost, open-source acoustic platform for monitoring the microstructure of solid food intake in mice. The Crunchometer is designed to overcome the limitations of existing methods for studying feeding behavior in rodents. The goal was to provide a tool that could precisely capture the microstructure of solid food intake, something often overlooked in favor of liquid-based assays, while being affordable, scalable, and compatible with neural recording techniques. By doing so, the authors aimed to enable detailed analysis of how physiological states, drugs, and specific neural circuits shape naturalistic feeding behaviors.

Strengths:

The study's strengths lie in its clear innovation, methodological rigor in validation against human annotation, and demonstration of broad utility across behavioral and neuroscience paradigms. The approach addresses a significant methodological gap in the field by moving beyond liquid-based feeding assays and provides an accessible tool for precisely dissecting ingestive behavior. The system is validated across multiple contexts, including physiological state (fed vs. fasted), pharmacological manipulation (semaglutide), and circuit-level interventions (chemogenetic activation of LH neurons), and is further shown to integrate seamlessly with both electrophysiology and calcium imaging.

(1) Introduces a low-cost, open-source acoustic tool for measuring solid food intake, filling a critical gap left by expensive and proprietary systems.

(2) Makes the method easily adoptable across labs with detailed setup instructions and shared benchmark datasets.

(3) Provides high temporal precision for detecting bite events compared to human observers.

(4) Successfully distinguishes feeding microstructure (bites, bouts, IBIs, gnawing vs. consumption) with greater objectivity than manual annotation.

(5) Demonstrates compatibility with electrophysiology and calcium imaging, enabling fine-scale alignment of neural activity with feeding behavior.

(6) Effectively discriminates between fed vs. fasted states, validating physiological sensitivity.

(7) Captures the pharmacological effects of semaglutide, although this is really just reduced feeding and associated readouts (bouts, latency, etc).

(8) Has potential to distinguish consummatory vs. non-consummatory behaviors (e.g., food spillage, gnawing); however, the current SVM model struggles to separate biting from gnawing due to similar acoustic profiles, and manual validation is still required.

(9) Provides potential for closed-loop experiments.

Weaknesses:

Several limitations temper the strength of the conclusions: the supervised classifier still requires manual correction for gnawing, generalizability across different setups is limited, and the neuroscience findings, particularly calcium imaging of GABAergic and glutamatergic neurons, are based on small pilot samples. These issues do not undermine the value of the tool, but mean that the neural circuit findings should be interpreted as preliminary.

(1) Some neuroscience findings (calcium imaging of GABAergic vs. glutamatergic neurons) are based on small pilot samples (n=2 mice per condition), limiting generalizability.

(2) Chemogenetic and pharmacological experiments used small cohorts, raising statistical power concerns.

(3) Correlation with actual food intake is modest and sometimes less accurate than human observers.

(4) Sensitive to hoarding behavior, which can reduce detection accuracy and requires manual correction for misclassifications (e.g., tail movements, non-food noises). However, these limitations are discussed and not ignored.

Conclusion:

Overall, this is an exciting and impactful methodological advance that will likely be widely adopted in the field. I recommend minor revisions to clarify the limits of classifier generalizability, better contextualize the small-sample neuroscience findings as pilot data, and discuss future directions (e.g., real-time closed-loop applications).

Reviewer #3 (Public review):

Summary:

The manuscript provides detailed information on the construction of open-source systems to monitor ingestive behavior with low-cost equipment. Overall, this is a welcome addition to the arsenal of equipment that could be used to make measurements. The authors show interesting applications with data that reveal important neurophysiological properties of neurons in the lateral hypothalamus. The identification of previously unknown "meal-related" neurons in the LH highlights the utility of the device and is a novel insight that should spark further investigation on the LH. This manuscript and videos provide a wealth of useful information that should be a must-read for anyone in the ingestive behavior or hypothalamus fields.

A scholarly introduction to the history and utility of various ways feeding is measured in rodents is provided. One point - the microstructure of eating solid food - has been studied extensively (for one of many studies, see https://doi.org/10.1371/journal.pone.0246569 ). However, I agree that the crunchometer will allow for more people to access recordings during food intake and temporally lock consummatory behavior to neural activity.

Questions on results:

(1) It is unclear why 10% sucrose solution was used as a liquid instead of water, given that the study is focusing on the solid food source.

(2) It is unclear how essential the human verification is in the pipeline - results for Figure 1 keep referring to the verification as essential. Is that dispensable once the ML algorithms have been trained?

(3) The ability to extrapolate food quantity consumed is limited, with high variability. This limitation does not undercut the utility of the crunchometer, but should be highlighted as one of the parameters that are not suitable for this system. This limitation should be added to the limitations section.

(4) The ability to discriminate between gnawing and consummatory behavior is a strength (Figure 5), and these findings are important. However, it is unclear what can be made of mice that have 'gnawing' behavior in the fasted state (like in Figure 3). It seems they would need to be eliminated from the analysis with this tool?

(5) Why is there a post-semaglutide fed group and not a fasted group in Figure 4? It seems both would have been interesting, as one could expect an effect on feeding even 24h after semaglutide treatment. This would help parse the preference better because the animals eat such a small amount on semaglutide, that it is hard to compare to the fasted condition with saline treatment.

(6) The identification of 'meal-related' neurons in the LH is another strength of the manuscript. Although there is currently insufficient data, could similar recordings be used to give a neurophysiological definition of a 'meal' duration/size? Typically, these were somewhat arbitrarily defined behaviorally. Having a neural correlate to a 'meal' would be a powerful tool for understanding how meals are involved in overall caloric intake.

(7) The conclusion in the title of Figure 8 is premature, given the pilot nature and small number of neurons and mice sampled.

Conclusion:

Overall, this report on the Crunchometer is well done and provides a valuable tool for all who study food intake and the behaviors around food intake. Clarification or answers to the points above will only further the utility and understanding of the tool for the research community. I am excited to see the future utility of this tool in emerging research.

Yes - the oral history project incorporated the 5Rs. I particularly think the "reach" element was present. By recording the interviews and sharing them on a website - it allows students in class (and beyond) to hear these stories.

Yes - definitely. The oral history project helped to share stories of people and their lived experiences. This was an innovative way to share those stories with a broader audience. I enjoyed (and learned) from listening to some.

eLife Assessment

This paper is an important overview of the currently published literature on low-intensity focused ultrasound stimulation (TUS) in humans, providing a meta-analysis of this literature that explores which stimulation parameters might predict the directionality of the physiological stimulation effects. The overall synthesis is convincing. The database proposed by the paper has the potential to become a key community resource if carefully curated and developed.

Reviewer #1 (Public review):

This paper is a relevant overview of the currently published literature on low-intensity focused ultrasound stimulation (TUS) in humans, with a meta-analysis of this literature that explores which stimulation parameters might predict the directionality of the physiological stimulation effects.

The pool of papers to draw from is small, which is not surprising given the nascent technology. It seems, nevertheless, relevant to summarise the current field in the way done here, not least to mitigate and prevent some of the mistakes that other non-invasive brain stimulation techniques have suffered from, most notably the theory- and data free permutation of the parameter space.

A database summarising the literature and allowing for quantitative assessment of these studies is a key contribution of the paper. If curated well, it can become a valuable community resource.

Comments on revisions:

The paper is much improved. There remain a few caveats the authors may want to address.

I'm not going to dwell on this if the authors don't agree, but remain critical about the inclusion of TPS in the discussion. It's comparing apples and oranges, and unless there's a personal interest the authors have in TPS, it remains puzzling why it is included in the first place. As per my previous review, the literature on TPS, and especially the main example cited, has been highly criticised, including national patient and medical associations. A mere disclaimer that more work is needed isn't enough, in this reviewer's opinion - I simply don't understand why the authors go out on a limb here when the rest of the paper is done so well and thoroughly.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

Summary:

This paper is a relevant overview of the currently published literature on lowintensity focused ultrasound stimulation (TUS) in humans, with a meta-analysis of this literature that explores which stimulation parameters might predict the directionality of the physiological stimulation effects.

The pool of papers to draw from is small, which is not surprising given the nascent technology. It seems nevertheless relevant to summarize the current field in the way done here, not least to mitigate and prevent some of the mistakes that other non-invasive brain stimulation techniques have suffered from, most notably the theory- and data-free permutation of the parameter space.

The meta-analysis concludes that there are, at best, weak trends toward specific parameters predicting the direction of the stimulation effects. The data have been incorporated into an open database that will ideally continue to be populated by the community and thereby become a helpful resource as the field moves forward.

Strengths:

The current state of human TUS is concisely and well summarized. The methods of the meta-analysis are appropriate. The database is a valuable resource.

We thank the reviewer for their positive assessment of the revised manuscript and the potential importance of the resource to the TUS community. 

Suggestions:

The paper remains lengthy and somewhat unfocused, to the detriment of readability. One can understand that the authors wish to include as much information as possible, but this reviewer is sceptical that this will aid the use of the databank, or help broaden the readership. For one, there is a good chunk of repetition throughout. The intro is also somewhat oscillating between TMS, tDCS and TUS. While the former two help contextualizing the issue, it doesn't seem necessary. In the section on clinical applications of TUs and possible outcomes of TUS, there's an imbalance of the content across examples. That's in part because of the difference in knowledge base but some sections could probably be shortened, eg stroke. In any case, the authors may want to consider whether it is worth making some additional effort in pruning the paper

We thank the reviewer for these suggestions. We have checked for redundancy and that the clinical review section is more balanced, although some of the sections have more TUS studies than others, therefore some imbalance is unavoidable. As some examples, we have condensed the “Stroke and neuroprotection in brain injury” section (lines 624-647). This helps to improve the clarity and readability of the manuscript.

The terms or concept of enhancement and suppression warrant a clearer definition and usage. In most cases, the authors refer to E/S of neural activity. Perhaps using terms such as "neural enhancement" etc helps distinguish these from eg behavioural or clinical effects. Crucially, how one maps onto the other is not clear. But in any case, a clear statement that the changes outlined on lines 277ff do not

We thank the reviewer for this point and agree that it is important to distinguish neural E/S, as we had intended, from behavioral effects. In the first instance and in several places we add ‘neural’ before enhancement/suppression.  Also see Lines 276-279: “Probable net neural enhancement versus suppression was characterised as follows. Note that our use of the terms enhancement and suppression refers exclusively to the increase or decrease of neural activity, respectively, as measured by, neurophysiological methods (EEG-ERPs, BOLD fMRI, etc.) and does not imply equivalent changes in behavioural responses” 

Please see also lines 108-116.

Re tb-TUS (lines 382ff), it is worth acknowledging here that independent replication is very limited (eg Bao et al 2024; Fong et al bioRxiv 2024) and seems to indicate rather different effects

We have updated this section by referencing Bao et al. and Fong et al., as examples of the limited independent replication of tbTUS results. Please see lines 392-396. “However, independent replication of these findings remains limited. For example, Bao, found reduced motor cortex excitability – measured as decreased TMS-MEP amplitude in M1 -- that lasted up to 30 minutes post-sonication (Bao et al., 2024). Whereas Fong reported no significant effects between tbTUS and sham conditions in M1 excitability (Fong et al., 2024).”

The comparison with TPS is troublesome. For one, that original study was incredibly poorly controlled and designed. Cherry-picking individual (badly conducted) proof-of-principle studies doesn't seem a great way to go about as one can find a match for any desired use or outcome. Moreover, other than the concept of "pulsed" stimulation, it is not clear why that original study would motivate the use of TUS in the way the authors propose; both types of stimulation act in very different ways (if TPS "acts" at all). But surely the cited TPS study does not "demonstrate the capability for TUS for pre-operative cognitive mapping". As an aside, why the authors feel the need to state the "potential for TPS... to enhance cognitive function" is unclear, but it is certainly a non-sequitur. This review feels quite strongly that simplistic analogies such as the one here are unnecessary and misleading, and don't reflect the thoughtful discussion of the rest of the paper. In the other clinical examples, the authors build their suggestions on other TUS studies, which seems more sensible.

This is an excellent point, and we have removed that statement replacing it with: “However, TPS effects studies remain highly limited and would require further study and comparison to effects with other TUS protocols.”. Please see lines 561-562. We thank the reviewer for the supportive comments on the rest of the review.

Notice that the way the equation solves that with the solved value. If 10^pH-pKA is greater than 1, the Conjugate Base or [A-] is in higher concentration than the lewis acid [HA], alternatively an increased HA concentration means a larger denominator --> 10^pH-pKA closer to 0; Conjugate Acid is higher in concentration.