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  1. Oct 2025
    1. A successful research process should go through these steps: Decide on the topic. Narrow the topic in order to narrow search parameters. Consider a question that your research will address. Generate sub-questions from your main question. Determine what kind of sources are best for your argument. Create a bibliography as you gather and reference sources

      best to do a lot of research before you write and essay on a specific topic.

    2. No matter what field of study you are interested in, you will most likely be asked to write a research paper during your academic career

      better to learn the skill.

    3. ou will need to put your thoughts together in a logical, coherent manner. You may want to use the facts you have learned to create a narrative or to support an argument.

      "thesis"

    1. The parkway drive passes through open desert where cattle that graze on neighboring state land occasionally break through fences and stroll onto the road.

      Side note: cattle don't below in creosote flats like this area. That is very much NOT a feature worth keeping in that area.

    2. if the question is having enough water for people to drink and to bathe and to live?

      Sure, new supplies would be great. But also, we can cut out some of the stupid waste we have right now. I'd love to follow Vegas's lead in banning useless grass landscaping around commercial developments, medians/street edges, and so forth. Vegas was the worlds biggest water waster for most of its history, then cut its use quickly by just stopping doing stupid shit.

    3. “I’ll probably stay here,” she said, since anywhere else, her mortgage bill could easily double.

      Not to mention the fact that you probably can't sell. Who is going to buy?

    4. “It’s not about running out. It’s about: Are you willing to pay for what it costs?”

      Yep, exactly. There's no way the nation's fifth largest city is disappearing because of water capacity, especially in a place where solar can power so much.

      Desalination is costly, at least at the moment, but it primarily just requires cheap energy. Well, we have a lot of cheap energy potential here—especially if you cover those canals and pipelines with linear solar.

    5. “They are designed from top to bottom, and everything is beautifully designed for a look, to work well together. It’s very hard to do that in an old farming town.”

      Excellent point

    6. Duane Schooley Jr. bought two houses in Tartesso to rent out at first back in 2018 and 2019 because “we figured that Arizona was going to be a hot spot.” But Schooley, a local Republican party activist, is now openly disdainful of the state’s decision to stop allowing new homes to be built on groundwater supplies. He even doubts the state’s talk of a water shortage.

      A highly speculative move, that's for sure (who wants to rent houses in the middle of nowhere with a low quality of life, especially when those are already the cheapest to buy?). Sorry that you didn't do enough research and that your bet didn't pan out—but that's capitalism for ya.

    7. can discourage lower-cost housing development

      Part of this is migration from more expensive markets. Developers cater to building larger houses than you'd find elsewhere, as someone selling their LA/SF house will look for lots more space than they need since they have more buying power. Every Californian that moves here tells his new neighbor that he can afford almost twice the house for nearly half the price here—so that's what gets built.

    8. “I like the quiet,” she said. “The only things you hear are cars going by, people talking and dogs barking, whereas in cities it was traffic, 24-7.

      This is also my neighborhood, which is still IN THE DAMN CITY. You can do suburban life without moving to the middle of the desert. I'm not sure why I hear this so often from people who got a house on the edge of the city.

    9. The downside is being marooned on a service-less island

      Yup. My ex-wife moved into my Tempe apartment after one month of dating because while she was at work one day, her parents bought a cheap house even further west than the Sun Valley Parkway mentioned above. It was an untenable situation, given that she went to school at ASU almost an hour away, and worked an extra 25 minutes beyond that. I had her move in when she fell asleep driving home one night after work.

    10. WHEN NEWS BROKE OF THE STATE’S 2023 BAN on new groundwater-based subdivisions, sparking apocalyptic national coverage, local and state officials switched into defense mode. “It seems in some ways like there’s criticism for us for doing planning and smart development,” Phoenix Mayor Kate Gallego told the Arizona Republic after the ADWR moratorium was announced. “It is a strength, not a weakness. We are planning ahead. We have a very simple principle: Water first, then development.”

      Haha, yep—that was my point above.

    1. Gaius Julius Caesar (100-44 BCE) was born into an influential patrician family in Rome.

      I find Julius Caesar's life story to be interesting. We watched a movie about it in English class one time. He had a very dramatic ending though.

    2. While the Greeks and Phoenicians had been expanding across the Mediterranean, a city on the west-central coast of Italy was steadily growing in prominence. Founded according to legend in 753 BCE by orphan brothers Romulus and Remus who had been raised by wolves, the early city was ruled by seven kings (some legendary and some real).

      This is interesting. I have seen the statue of the female wolf and the two babies before, but I never knew the possible background story to it.

    3. Zeno saw the universe as a divine entity, with humans sharing its rational spark. His Republic, written as a response to Plato's book of the same name, imagined a stateless community, which shocked Athenian elites

      This is very interesting. The site that they found is super interesting too.

    4. It is harder and holds a sharper edge than copper alone, which makes bronze useful for both practical tools such as knives, axes, plows, and sickles;

      This is interesting that they found this.

    5. Egyptian Book of the Dead: Anubis weighing a dead man's heart against a feather.

      I find these pictures from Egyptian drawings interesting. Especially the fact that most of them have heads of different animals.

    6. Possibly the oldest is called "The Instructions of Shuruppak". Written in Sumerian about 4,600 years ago, it is structured as the advice of a father named Shuruppak, a mythical sage and ruler, to his son Ziusudra (hero of the Sumerian flood myth)

      This would be really cool if it is actually what happened!

    7. . (Note: there is a persistent rumor that Sumerian is a mysterious language that has never been deciphered. This is not true, although it is an isolated language and not part of the Semitic language group, like Akkadian.)

      This is interesting because the writing does not look like any kind that I have seen before, yet somehow, they may have depicted what it says.

    8. One of the interesting mysteries of the Minoans is that none of their surviving art seems to depict a ruler.

      This is interesting because usually back in history and even now, many places have a 'ruler.'

    9. The most famous and recognizable remnants of ancient Egypt, of course, are the pyramids

      I always find pyramids fascinating because I cannot imagine how long it took to build these with just the soil, water, and hardworking people.

    10. By about 9,000 to 7,000 years ago, agricultural techniques including farming and herding had spread from the places they had developed into surrounding regions.

      I find this interesting because even now we do farming. So, in this case, not much changed in different areas except we have tractors and technology to help.

    11. 19th-century American painting of Native American men hunting bison on the Great Plains. Big game hunting in the northeastern woodlands was similarly a male profession.

      I find this interesting and exciting because this would take a lot of courage and strength. Bison are a very tough animal, and they can tear things up.

    12. So if we imagine a culture of people who hunted, fished, gathered, and began cultivating valuable food plants near their camps, we would be thinking of people who supported themselves in a similar way to the woodland Native American tribes of the Northeast.

      I think this is very interesting and a fun way to live (except for the bears and such). I like camping and many outdoor activities so I like to learn more about how these people lived.

    13. One explanation scholars have proposed for the building of monuments like Gobekli Tepe) and Karahan Tepe, and later Stonehenge, is that these sites may have been important meeting places for ancient people.

      I found this interesting to learn more about and how they are some of the oldest sites found.

    1. In the period from the inception of Portuguese migration, 30,645 indentured labourers arrived mainly from Madeira, while smaller groups came from the Azores, Cape Verde and Brazil.

      I wonder if Portuguese came to be in Brazil by similar means.

    2. Discusses the many facets of Portuguese indentureship in Guyana in the mid-to-late-1800s. This ultimately lead to the formation of one of Guyana's six peoples.

    1. The earliest known Maya pyramid may be the Temple of the Masks in Uaxactun.

      I think these temples that are covered in moss look super cool. I would love to visit one but that might never happen.

    1. about the Chinese Empire: it continued (under several dynasties) until 1911. Life-size ceramic sculptures depicting the armies of Qin Shi Huang, the first emperor, buried with him in 210–209 BCE

      This tomb is fascinating and kind of gross at the same time.

    1. The new city (its name literally means New City) was located about fifteen miles from the older settlement at Utica. The Tunisian coast lies directly across the Mediterranean from Sicily, at a narrow spot that all ships traveling from east to west would have to pass. Two large harbors were built; one for the city's powerful navy and the other for commerce. C

      This is impressive considering the lack of technology they had back then. I think it is also a really cool city.

    1. The object of the battle had been control of the Mediterranean coast, which Egypt had lost during the problematic reign of Akhenaten and Nefertiti, who had tried to shift the polytheistic Egyptian religious system to a monotheism focused on Aten (or Amun-Ra), the solar deity.

      This is interesting. The carving is also very interesting.

    1. A thesis is weak when it contains an obvious fact or something that no one can disagree with or provides a dead end.

      dont point out the obvious make an argumentative thesis and have valid points to back it up

    2. A thesis statement must present a relevant and specific argument. A factual statement often is not considered arguable. Be sure your thesis statement contains a point of view that can be supported with evidence.

      a thesis statement is your argumentative sentence.

    3. Asking and then answering this question is vital to forming a thesis that is precise, forceful, and confident.

      asking a question makes the reader intrigued before they fully understand the topic.

    4. he textbook Successful Writing explains that writers need a thesis statement to provide a specific focus for their essay and to organize what they will discuss in the body of their writing

      for the reader to get a better understanding or to create a interest in the writing before reading the full thing.

    1. I think that some of the more complex high agency patterns from the space are behavioral propensities aka kinds of minds. I think that's what minds actually are is that they're they're actually the the the inhabitants of that of that space.

      for - quote - minds occupying platonic space - Michael Levin - I think that some of the more complex high agency patterns from the space are behavioral propensities - aka kinds of minds. - I think that's what minds actually are - they're actually the inhabitants of that of that space. - adjacency - claim - minds in Platonic space - spirituality - Michael Levin

    2. when were the computations done to make zenobots and anthrobots, there's never been any selection pressure to be a good anthropot or a good zenobot.

      for - adjacency - questioning evolution - xenobots - anthrobots - Michael Levin - Is Levin's lab experiements bringing evolution's primacy into question? Is there an even MORE fundamental foundation for life? - Is the platonic form more fundamental than evolution?

    3. I've been thinking about this stuff for decades, and I had not broached the topic of platonic patterns until until this year. And that's because I think it is now actionable.

      for - quote - platonic patterns are now actionable - Michael Levin - I've been thinking about this stuff for decades, and I had not broached the topic of platonic patterns until this year. - And that's because I think it is now actionable. - question - progress trap - moral questions and alarm bells? playing God? - Michael Levin

    4. that's a key part of this. You have to convince the material. This is not you. It it it you know there's ways that it will ignore you. If you do it wrong, it'll ignore you. So you have to be convincing

      for - interlevel communication - Michael Levin - What he's really saying is that we have to find the RIGHT LANGUAGE to speak to the agents at that different level - This is an important lesson for interlevel communication in social systems! - comparison - interlevel comm - cells vs societies

    5. the question is, why didn't that biochemical story get you to this discovery?

      for - quote - Michael Levin - what is a good story? - the question is: Why didn't that biochemical story get you to this (new) discovery? - adjacency - good models - predictive power - good story - a good model is a good language - new words frame the world in new ways, - it allows us to divide reality in different ways - and can lead us to look in places we otherwise might now - and that can lead to new observations

    1. Enkidu confronts Gilgamesh and they fight in the streets of Uruk. The wrestling match ends in a stalemate, since the two are evenly matched.

      This is interesting that they can find evidence of different happenings through carvings from a long time ago.

    1. Language is a symbolic exchange, a means of communication, extending beyond verbal form.

      Something I find interesting about language is how the author discusses it going beyond verbal form. People often only think about verbal communication, but often times body language is just as important. It can show emotion even more than language. This is interesting to apply to UX, as it can show how in language can be applied to influence a user's experience outside of only words.

    2. Communication is an act of conveying meaning. Every behavior, including the absence of action, is a kind of communication therefore, one cannot not communicate (Paul Watzlawick).

      This is probably my favorite part of the article. It doesn't matter if you have blank screens/pages, this ultimately communicates something, whether intentional or not. This is the main underpinning of the entire article. UX and rhetoric are deeply connected, focusing on communication to an audience.

    3. Rhetoric is this intent as rhetoric is universal and architectonic; present everywhere whilst giving structure to all the other arts, sciences, and disciplines. There’s no “wild coincidence” that UX design closely mirrors rhetorical discourse when held at a microscope.

      The author defines rhetoric as a universal framework that shapes all disciplines, not just language.

    4. What is rhetoric? I’m often asked this question upon telling someone that I study the art of rhetoric. Wait… how does that even relate to UX design? It is quite often the follow-up question as I am both a rhetorician and UX designer. The conversation typically then goes a few ways, either I have done my job as a rhetor and shown them the intersections between rhetoric and UX design or I have completely lost them.

      Overall, this article does bring up an interesting Idea between the nature of how regardless of it's advancement there are certain aspects of the human nature that will always bleed through.

    5. Communication is an act of conveying meaning. Every behavior, including the absence of action, is a kind of communication therefore, one cannot not communicate (Paul Watzlawick).Language is a symbolic exchange, a means of communication, extending beyond verbal form. A symbolic exchange can be written, visual, verbal, nonverbal, sonal (pertaining to sound), gestural — anything and everything with the potential to be interpreted by a recipient. Language, in and of itself, is the very result of rhetorical arts as rhetoric famously uses language as one of its instruments for communication.

      All websites communicate a kind of language to each user. This language can help guide users and add accessability for some, but might be less engaging for others. UX design needs to utilize this language to communicate to the user how to use the site or app.

    6. Rhetoric is the art or science that aims to study the capacities needed to inform, persuade, or motivate particular audiences (rhetorician). Furthermore, it is the art or science that seeks to achieve clarity, awaken our sense of beauty, or bring about mutual understanding (rhetor). (James A Herrick).

      This is the first time in the article that mentions the definition of Rhetoric. If I were to paraphrase, Rhetoric is the structured process of bridging your ideas across to another group in the hopes of persuading that other group with your ideas.

    7. Rhetoric is the art or science that aims to study the capacities needed to inform, persuade, or motivate particular audiences (rhetorician). Furthermore, it is the art or science that seeks to achieve clarity, awaken our sense of beauty, or bring about mutual understanding (rhetor). (James A Herrick).

      I never considered that this could be seen as a science. However, that does make sense. You can experiment with rhetoric, test the limits of it, and see results and discover laws that it abides by. However, it is also an art form, which requires practice and nuance. I wonder whether it should be considered more of an art or a science? within science we believe there are universal facts and kernel of truths that we should all believe. In art, there are arguments about whether a singular truth exists, or if the truth varies by perspective and opinion.

    8. I’ve realized it’s much more than just making websites or apps look good. UX design focuses on how people actually experience and interact with technology. It’s about understanding users what they need, what frustrates them, and what makes their experience easier or more enjoyable. A UX designer combines creativity with problem-solving to create designs that are both practical and appealing. In short, UX design is about shaping digital experiences that feel natural and help people achieve their goals without confusion or frustration.

    9. In his Axioms of Communication, Watzlawick listed the first axiom as “one cannot not communicate”. Any mark, sign, sound, or gesture communicates meaning based on social agreement (or in terms of UX Design, social signifiers)

      This was important as the quote means to show that any gesture or sign communicates meaning. In terms of UX every aspect of design speaks for itself.

    10. A discourse on rhetoric & UX design

      That actually makes a lot of sense — UX design really is like modern-day rhetoric. Instead of persuading through speeches, we do it through layouts, visuals, and user flow. It’s about communicating ideas and guiding behavior without saying a word. I never thought about it that way, but it definitely changes how I look at design.

    11. “Rhetoric in the most general sense may perhaps be identified with the energy inherent in communication: the emotional energy that impels the speaker to speak, the physical energy extended in the utterance, the energy level coded in the message, and the energy experienced by the recipient in decoding the message.” — George Kennedy

      UX designers can channel the emotional energy of communication through the subtle visual language of their interfaces, like how some shapes, colors, and other design elements are associated with certain emotions. Some types of designs may promote users to 'communicate' with the product through this emotional design, evoking emotions that compel people to communicate.

    12. Every behavior, including the absence of action, is a kind of communication therefore, one cannot not communicate

      Even silence, (a missing button, unclear/feedback/missing portions of the page) sends a message to the user, everything in design communicates something.

    13. It is our job as designers to align the brand/company’s identity, image, and message to their audience (or users).

      They both trying to get the message out there to the users that will meet the goals of the brand/company. As designers, we must think what will the users want to see and how it will appeal to them to get them to click the link and check out the company.

    14. Film, music, dance, architecture, and even a mobile interface, each involves behavior that creates meaning, meaning for the spectator or recipient to decode and interpret, and thus, rhetoric. This is why it is imperative to always create with intent.

      This idea highlights how communication are embedded in everything humans create. The takeaway is that intentionality matters, creators should always be aware of the messages their work sends and how others might interpret them.

    15. Rhetorical discourse is planned, typically concerned with contingent issues as it is shaped by human motives and responsive to situations, yet dependant on the audience.

      UX design mirrors this, designs are intentional, user centered/focused, and aimed at influencing user behavior

    16. Rhetorical discourse is planned, typically concerned with contingent issues as it is shaped by human motives and responsive to situations, yet dependant on the audience. And of course, persuasion seeking (six characteristics of rhetorical discourse,

      Rhetoric relates to UX because both involve intentional, persuasive communication shaped by human motives and audience needs. UX designers, like rhetoricians, plan with purpose crafting interfaces that influence user behavior and understanding.

    17. Wait… how does that even relate to UX design?

      The article’s author argues that UX design is not just about crafting interfaces, but also about engaging in a form of rhetorical discourse with users. Rhetoric’s foundations are explained through communication, language, and discourse. Furthermore, the aims of rhetoric (persuasion, clarity, mutual understanding) are applicable to UX design. Because every interaction communicates meaning, designers are using rhetoric, shaping how users interpret and respond to digital products. Studying rhetoric can enrich how designers think about intent, structure, and the ethical dimensions of design.

    18. James A Herrick

      James A Herrick had the captivating thought with rhetoric comprising of persuasion, clarity, beauty, and mutual understanding. This sentiment mirrors the five canons of rhetoric. James A Herrick was a studious member of Hope College and extended his stories in rhetoric along with writing books revolving around rhetoric and augmentation. These books would revolve around history and the enterprise of revolutionary moments in history.

      https://hope.edu/news/2024/campus-life/retired-communication-professor-james-herrick-dies.html

      https://www.wikiwand.com/en/articles/James_A._Herrick#:~:text=He%20has%20written%20extensively%20on%20the%20history%20of,practices%20of%20the%20seventeenth-%20and%20eighteenth-century%20English%20Deists.

    19. A discourse on rhetoric & UX design In UX, we are not only designing a mobile environment but also engaging in a rhetorical discourse with our users.

      srendip ikigai

      Description

    20. Because these acts conveying meaning can be decoded and coded via language, they are in themselves a form of discourse (however, it must be noted that it doesn’t necessarily mean they are rhetorical discourse as in order for discourse to be made rhetorical, it must have the six distinct characteristics of rhetorical discourse).

      This shows how design goes beyond visuals and becomes its own kind of language. I like how it distinguishes between simple communication and true rhetorical discourse, reminding us that meaning in design only becomes rhetorical when it’s intentional, persuasive, and structured with purpose.

    21. Through employing the five steps of the design process: empathize, define, ideate, prototype, and test, we engage in a practice that is planned, adapted to an audience, shaped by human motives, responsive to situations, persuasion seeking and concerned with contingent issues, or in short, rhetorical discourse

      This ties back to the discussions of accessibility by having an accessible design that all users can enjoy and gain from the UX it makes a statement that the designer cares about their topic and cares the same amount or more about their users.

    22. we are not only designing a mobile environment but also engaging in a rhetorical discourse with our users.

      This is a good point. Although some user experiences are not trying to present an argument (like filling out a document), even the way we design these contribute to the reputation or purpose of the company, even if that's just making the user say "that wasn't as hard as I thought it was going to be."

    23. Rhetorical discourse is planned, typically concerned with contingent issues as it is shaped by human motives and responsive to situations, yet dependant on the audience.

      This statement ties in well with the design process for applications. There is a lot of planning that goes on before even thinking about how to code a program. Yet, UX designers are "dependent on the audience" as their design approach is molded by their discussions with the people they're designing for.

    24. “… rhetoric of the present time is a socially-oriented art aimed at promoting healthy and productive human relationships.”

      This shows that communication isn't just about persuasion but about shaping how we relate to each other. It some what reminds us that our words have the power to strengthen the bonds within society.

    25. Rhetoric is universal, that is, present everywhere we turn. Rhetoric is too architectonic in that it is a kind of master discipline that gives orders and lends structure to other disciplines (Richard McKeon).

      I really connected with the idea that rhetoric exists everywhere, even in the things we create like apps, music, or architecture. It shows that design is never neutral because everything we make communicates a message or emotion to someone. This part made me think about how important it is for designers to be intentional since users will always interpret meaning from our work whether we plan it or not.

    26. This exchange between humans and technology has been made possible through UX Design; an exchange between the users and mobile environment

      This made me think about everything we interact with on a daily basis. When you do something like interact with an app for example. That is an exchange of your time and energy. Now multiply this by every user that opens the app. This scenario makes it possible for technology to continue advancing.

    1. Ideally, parents help children become well-integrated, contributing members of society. Of course, family socialization continues throughout the life cycle. Adults change within marriage, and as any parent knows, mothers and fathers learn as much from their children as their children learn from them.

      I agree with this. As much as I’ve learned from my parents, they always tell me they learn from me too. I didn’t really understand that when I was younger because I was just a kid. But later, I realized that raising a child comes with a lot of responsibility. Parents are not just teaching, you’re also helping them grow. As kids grow and learn, parents are learning and growing too, because parenting is something they also experience for the first time.

    2. Because some business and government programs still use this conventional definition, many unmarried but committed partners of the same or opposite sex are excluded from family health care and other benefits. However, our society is gradually coming to recognize as families people with or without legal or blood ties who feel they belong together and define themselves as a family.

      I think it’s interesting that some jobs or government programs don’t give benefits to unmarried couples, even if they live together and love each other. But yet it made me question, What should count as a family in today’s society?

    1. Platonic Space

      for - definition - Platonic Space - a structured, non-physical space of patterns, - such as the properties of mathematical objects, - perhaps other, higher-agency patterns that we detect as forms of - anatomy, - physiology, and - behavior - in the biosphere. - Thus, the contents of this space may inform (in-form) events in our physical world (constraining physics, and enabling biology).

    2. for - source - telegram channel - Michael Lennon - Forms of Life, forms of mind - Michael Levin and Hananel Hazan-led weekly symposium exploring platonic space - from - youtube - interview - Michael Levin - John Vervaeke - https://hyp.is/H727RKOrEfC5IAN-dmo5uw/www.youtube.com/watch?v=iwOJ9PWcPmo

    1. The tactic — shared by job hunters in TikTok videos and across Reddit forums — has become so commonplace in recent months that companies are updating their software to catch it. And some recruiters are taking a tough stance, automatically rejecting those who attempt to trick their A.I. systems.

      I think it's actually really surprising that this is becoming such a normal thing to do that people are making TikTok videos about it to try and help other people do the same thing. I think this also is a very common theme and something that happens a lot with our generation. People will go on TikTok and share information with the world and it actually gets them into a lot of trouble. I imagine people charing how to cheat their way into a job on TikTok most likely won't be able to get a job in the future.

    2. As companies increasingly turn to A.I. to sift through thousands of job applications, candidates are concealing instructions for chatbots within their résumés in hopes of moving to the top of the pile.

      I think that companies using AI to look at resumes is both good and bad. I think that it does remove the bias that can happen when humans are reviewing them but I do also think tha mistakes can happen and the AI might filter through a resume it shouldn't have and then the company looses a really good candidate. I think this is also a downside the fact that candidates realize AI is used and then they cheat their way through the system.

    1. Cassava trees are native to central Brazil, where they were first domesticated between 10,000 and 7,000 years ago. Although the starch of the cassava tree, called manioc, is only familiar to most North Americans as tapioca or in bubble tea,

      I found this very interesting that this is where the tapioca from boba comes from.

    1. The Central Americans created the single-stem, large-eared maize plant we are familiar with, by very gradually improving a native grass called teosinte. Year after year farmers saved seeds from the best plants with the biggest seed heads

      I found this very interesting.

    1. domesticated, animals need to be willing to accept a human as the leader of their herd.

      I find this interesting. I have heard this a different time before but it is interesting to know that even people from history knew that animals needed different things in order to be domesticated.

    1. . While we can’t say for sure that cousins such as Neanderthals did not share this interest and ability to create and to think symbolically, what we do know is that by 35,000 to 20,000 years ago, people were painting the walls of caves in places like France, Spain, Italy, Indonesia, and Africa. In addition, bone flutes dating from 35,000 to possibly 60,000 years old have been found in France, Germany, and Slovenia.

      Wow!! I love to see different things that historians have found.

    1. ao marginalizarem o papel da experiência, da construção de estratégias e as influências situacionais.

      Os estudos quantitativos muitas vezes ignoram dimensões que fazem toda a diferença nos contextos abordados, neste caso no envelhecimento, como a experiência acumulada, a capacidade de criar estratégias e o contexto em que o trabalho é realizado. Ao diminuir tudo a indicadores de "performance" e só olharmos para números, é mais fácil concluir que os mais velhos "declinam" inevitavelmente. Se incluirmos o fator experiência e contexto, por exemplo, já podemos concluir que envelhecer não quer dizer simplesmente que se perdem capacidades, pode traduzir-se sim, em utilizar recursos diferentes para a sua adaptação.

    2. Em 1946 surge, em Cambridge, o primeiro laboratório dedicado aos problemas do envelhecimento, dirigido por Bartlett. Este laboratório, à semelhança dos trabalhos de Miles, centrava-se no estudo da performance humana com o objectivo de definir quais os tipos de trabalho industrial mais adequados para os trabalhadores menos jovens (Welford, 1976, 1986), ou seja, ambos os laboratórios estavam concentrados em avaliar o potencial dos mais velhos para o trabalho, abordando o tema de forma fundamental, isto é, medindo a performance em diferentes idades em tarefas de laboratório supostamente implicadas nas actividades industriais (Welford, 1976, 1985a).

      Achei curioso, o facto de no ano de 1946 já existir a preocupação de avaliar a "performance" dos trabalhadores mais velhos para definir a sua adequabilidade no trabalho industrial. Contudo, parece-me que a abordagem possa ser restrita, restringindo o envelhecimento a testes de laboratório sem considerar o contexto real ou a experiência acumulada desses trabalhadores. Fica a pergunta, se este tipo de estudo não levou ao reforço de estereótipos, em vez de se valorizar o trabalho das pessoas mais velhas no trabalho.

    3. ao marginalizarem o papel da experiência, da construção de estratégias e as influências situacionais.

      Os estudos quantitativos muitas vezes ignoram dimensões que fazem toda a diferença nos contextos abordados, neste caso no envelhecimento, como a experiência acumulada, a capacidade de criar estratégias e o contexto em que o trabalho é realizado. Ao diminuir tudo a indicadores de "performance" e se só olharmos para números, é mais fácil concluir que os mais velhos "declinam" inevitavelmente. Se incluirmos o fator experiência e contexto, por exemplo, podemos concluir que envelhecer não quer dizer simplesmente que se perdem capacidades, pode traduzir-se sim, em utilizar recursos diferentes para a sua adaptação.

    1. Nearly all people whose ancestors were not sub-Saharan Africans got about 2% of their genes from Neanderthals.

      This is interesting that if people were not ancestors of Africans, their genes were part Neanderthals.

    1. Pre-sapiens humans such as Homo erectus left Africa about 1.8 million years ago and spread as far as China and Java.

      I find this interesting that they found the history to go all the way back about 1.8 million years ago.

    1. want their institutions to address academic integrity concerns—albeit via a proactive approach

      Pro-active: tell us the rules instead of punishing us for not following them...give students chances to REVISE their work, if there was a question of unfair use...

    1. nodded heavily

      nodded heavily /ˈnɒdɪd ˈhɛvɪli/

      verba Mengangguk dengan penekanan atau ketidakpastian, sering kali mencerminkan perasaan yang dalam atau berat. Contoh: He nodded heavily, indicating his reluctance to leave. Dia mengangguk berat, menunjukkan ketidakberaniannya untuk pergi.

    1. X frowned at them. “Can’t you tell?” Then X broke into a big, mischievous grin, “It’s a Y!”9

      I think this story is optimistic. it shows that even though people might try to enforce and keep gender norms the same. That does not allow people to grow in the ways they should best. At first that was very difficult for the parents to see, but I think they grew into it. A moment I see optimism is in the paragraph before this, where X says its a Y.

    1. de ma formation artistique académique

      On répète ici une information donnée au premier paragraphe :

      Ni technophile enthousiaste, ni opposant farouche, je les ai abordées avec la réserve dûe à ma formation artistique classique.

      Peut-être vaudrait-il mieux indiquer seulement « sans doute du fait de ma sensibilité » ici.

    1. “tool of thepatriarchy.” It is a tool for anyone who wishes to use it, and who is willing to put the timeand effort into mastering it,

      this ties into the continuous theme of using language as a tool no matter what language that may be it's always used in the same way, to get around and communicate.

    2. substitute “their own language” — or worse, teaching them that our common language issomehow evil — merely sets them up for failure.What’s more, we further erode public confidence in our ability to produce job-readygraduates. (In many surveys over the past 10 years, employers consistently identify poorcommunication skills as one of their chief complaints about new hires.)The responsibility for helping students learn to use standard American English effectively,and insisting that they do so, cannot fall solely on the English department. The purpose offirst-year composition courses should be to introduce students to the basics of goodprofessional communication — grammar, sentence structure, organization, paragraphdevelopment. If subsequent courses do not build upon and reinforce those fundamentals,then students will conclude that such skills must be not all that important. That appears tobe the case, if those employer surveys are any indication.As academics, we rose to positions of privilege and authority based in large part on ourability to “speak the language.” It seems to me the height of arrogance and hypocrisy, if notoutright discrimination, to deny students access to those same opportunities, whether wedo so intentionally or simply through neglect. Our objective as educators ought to be tohelp them attain what we have attained, if not more — and language proficiency is anecessary prerequisite.In short, standard American English is not inherently racist. It is not merely a “tool of thepatriarchy.” It is a tool for anyone who wishes to use it, and who is willing to put the timeand effort into mastering it, regardless of race, gender, ethnicity, cultural background, orsocioeconomic status.Nor will students — once they leave our cushy campuses and enter the professional world— be able to talk and write any way they choose, any more than they will be able to dress orbehave any way they want. Preparing them adequately for life beyond college is arguablyour greatest responsibility — and up to this point, perhaps our biggest failure.

      I think this line ties everything together and shows the authors true feelings towards the topic, and how much it means to him that students are taught ASE, but not because he thinks everyone should know English, because he knows the potential that someone who knows ASE has in the world.

    Annotators

    1. безумно, как люди, живущие обычной жизнью и возможно имея некоторый пробелмы с психикой. может попадали в сонный паралич. верят в то чего нет, и возможно даже сами не до конца в это верят и кое как могут рационаольно об этом подумать.

      то после появляения уореннов, которые окончательно заставили их поверить в то, что у них дома происходит. появялетя тотальная вера в эту реальность, и особо ужасно когда уоренны еще и подыгрывают этому.

  2. docdrop.org docdrop.org
    1. According to some scholars, the school system privileges individuals who comply with dominant culture, like that of middle-class and upper-middle-class teachers, professional staff, and administrators (Bourdieu, 1984, 1986; Bourdieu & Passeron, 1990; Musoba & Baez, 2009). Bourdieu suggests that these privi-leges are likely to be based less on merit or hard work than on the cultural atti-tudes, behaviors, norms, and values of dominant groups. Because Rebecca was assertive and independent, she was penalized. And of course her race made her an especially easy target. It is when these two come together that we see how poverty and race intermingle to marginalize students. Low-income students are more likely to achieve positive educational outcomes (e.g., passing test scores or graduating) once their strengths are recognized, affirmed, and rewarded to the same degree that their middle-class peers' are. Because I was respectful and did not disagree with or challenge other students or educators, teachers accepted me. I was one of the "good ones." My compliance and obedience were rewarded with good grades.

      The values of middle-class culture are regarded as the standard. Students from low-income families and those from minority groups will be punished or marginalized if they do not conform to these values.

    2. As a youth, I was psychologically equipped to confront racism in school. I was taught by my mother to stand up for myself when people used racial slurs. She consistently reminded my brother and me that we should never feel inferior because of the color of our skin. However, I was not adequately prepared to address classism in the education system. There was no pride in being poor. In fact, I did not know anyone who marched in the streets with their fist in the air saying, "Poor is beautiful." I loved being Black, but I hated being poor.

      This reminds me that the oppression within the education system is often intertwined, but the societal response is not balanced. Racial discrimination involves overt confrontation and cultural forces, while class discrimination is more silent and shameful.

    3. My students rarely out themselves as being poor. You could not tell they struggle financially by the papers they turn in to me or by what they say when we discuss things in my sociology classes at the University of St. Thomas. During office hours, however, students reveal to me that they grew up poor, and often they tell me that they are the first person from their family to go to college. They talk about the social distance they feel from their peers who have money. They tell me they often hang out with other poor students to avoid being reminded of what they simply don't have. Many low-income students do not own cars. They are less likely to dine at off-campus restaurants or to have an entire wardrobe of brand-name clothes. They do not go to vacation resorts on spring break. They get tired of being reminded of these differences when they are with wealthier students.

      This passage makes me feel the hidden pressure of economic inequality on university campuses. Although classroom assignments seem to be "equal for all", in daily life, social interactions and consumption, poor students are constantly reminded of what they lack.

    4. This insistence on compliance is also one aspect of schooling that keeps some students from feeling they can challenge the very structures that repress them

      This line reveals how schools often value obedience over critical thinking, especially for students from marginalized backgrounds. By teaching compliance as the path to success, the system discourages questioning and resistance, the very skills needed to challenge inequality. What seems like discipline or respect can actually become a tool of social control, training students to fit into systems of power rather than transform them.

    5. f we do not intentionally unveil the hidden advantages that middle-class and upper-class students have over their low-income peers, we run the risk of indirectly reinforcing these inequalities in our classrooms.

      This line underscores how inaction sustains inequality. When educators ignore the invisible privileges that benefit wealthier students, like prior academic preparation, confidence, or access to resources, they allow those advantages to persist unchallenged. True equity requires conscious effort: teachers must not only support low-income students but also critically examine the systems and assumptions that favor others. Equality in education isn’t passive—it demands awareness and action.

    6. wonder whether one of the more privileged students would have been celebrated as assertive for raising the same concerns that resulted in me being seen as a troublemaker.

      This line exposes the double standard in how schools interpret student behavior through the lens of privilege. When marginalized students speak up, their actions are often labeled as disrespectful or disruptive, while similar behavior from privileged peers is praised as confidence or leadership. It shows how bias operates subtly in classrooms, rewarding conformity from some students and punishing it from others, revealing that equity in education requires not just fairness in grading, but fairness in how voices are heard.

    7. According to some scholars, the school system privileges individuals who comply with dominant culture, like that of middle-class and upper-middle-class teachers, professional staff, and administrators

      This line reveals that schools don’t just teach academics; they teach conformity to the norms of those in power. Students who already fit the dominant culture’s expectations are rewarded, while those who express different cultural identities or resist assimilation are punished or overlooked. It shows how “success” in education often depends less on talent or effort and more on how well a student mirrors the values of those running the system.

    8. schools rarely reward poor students for the quali-ties they bring to their schools: their perseverance, compassion, flexibility, patience, and creativity,

      This line exposes how education systems privilege the cultural traits of wealthier students while overlooking the resilience and emotional intelligence that often come from growing up with less. It challenges the idea of “merit” by showing that schools don’t operate as neutral spaces. They value certain behaviors and backgrounds over others. True equity in education would mean recognizing and rewarding the diverse strengths that students from low-income backgrounds already possess.

  3. docdrop.org docdrop.org
    1. Social scientists have identified significant resources, or forms of capital, th::tr play a role in influencing student academic out-comes. Research has shown that economic capital, that is, the w~alch and income of parents, is one of the primary factors influ-ep.cing student achieveme11t (Coleman and others, 1966; Roth-stein, 2004; Farkas, 2004 ). Student achievement is also influenced _l,y more subtle resources sud; as social capital-the benefits derived from c<;mnections to networks and individuals with power and influence (Coleman, 1988; Stanton-Salazar, 1997, 2001; Noguera, 2003 )-and cultural capital (Bourdieu and Wacquant, 1992)-the t~sces, styles, habits, language, behaviors, appearance, and customs c.hat serve as indicators of status anJ privilege. All three forms of c?pital-e';onomic, social, and cultural-play a role in perpetuat-ing disparate educational experiences anJ differential access to edu-cational opportunities. However, they do so in interaction with seemingly neutral structures that operate within schools and society. Chantelle's comments reveal how easily a student who lacks economic, social, and cultural capital can become lost within Berkeley High's large and impersonal bureaucratic structure. She

      This passage combines the theory of capital with Chantelle's actual situation, allowing me to have a clearer understanding of how structural inequality operates in schools.

    2. Jennifer: Much easier. I'm in geometry, :rnd it's like "Oh, okay. I know how to do that." I have a [private] tutor now, and she's planning to be a math teacher at Berkeley High, and rhe [geome-try] books she's like an exjpert at going through because her school created them. So she's, like, "I understand how they think about this." So she understands the books ... and she helps me with that. So I'm getting a lot better, and I'm understanding things a lot better now, but it's only because of her.

      Jennifer's experience highlights the inequality of educational resources: students with private tutors tend to make greater academic progress and gain more confidence, while those without additional resources may struggle.

    3. hantelle: Yeah, because last year I had prealgebra and this year I'm going to take one semester of prealgebra, and then maybe I'll be ready for algebra, hut ifl'm nor, I'm going to take prealgebrn again so I really know what I'm doing. Because, see, my brother, when he came [to Berkeley High], he didn't go to prealgebra. He went to prealgebra in middle school, and then he went to algebra here, and he never went to prealgebra here, so he needed to go to prealgebra this year because it's his last year.

      The conversation highlights the different paces at which students progress in their mathematics learning: some students need to repeatedly study basic algebra to lay a solid foundation, while others have to return to lower-level courses in high school due to course scheduling or衔接 issues.

    4. Ninth-grade students noted qualitative differences between their advanced and "regular" courses (Talbert, 1990).

      This line shows how inequality in education isn’t just about course titles, it’s about the quality of teaching and expectations students experience. Advanced classes often receive more engaged instruction, motivated peers, and higher academic standards, while “regular” courses can unintentionally reinforce low expectations. This separation shapes how students see themselves and their potential, illustrating how tracking subtly trains some students for success and others for disengagement.

    5. But a close look at the other course assignments of ninth-grade students reveals how dif-ferences related to race, class, and language establish patterns that have profound ramifications for students' subsequent opportunities.

      This line exposes how inequality becomes institutionalized early in education. Even when schools appear fair, by mixing students in some classes, subtle tracking systems in subjects like math or science sort students by background in ways that shape their future opportunities. It shows that inequality doesn’t always stem from explicit bias but from structures that quietly reinforce privilege and limit mobility for marginalized students.

    6. ennifer's parents relied on their economic capital to hire a private tutor. It turned out that her tutor also had quite a bit of social capital because this particular tutor was planning to become a math teacher at Berkeley High and was familiar with the textbook and ways of thinking used in the geometry class.

      This line highlights how privilege often multiplies through the intersection of wealth and connections. Jennifer’s family’s money (economic capital) bought not just academic help but insider knowledge (social capital) that aligned perfectly with the school’s system. This shows how inequality can persist even in seemingly merit-based environments. Students like Jennifer benefit from invisible advantages that the system itself rewards, while others like Chantelle are left outside those networks of support.

    7. There is relatively little that the school can do to address the inequalities in the backgrounds of students like Jennifer and Chantelle. However, it is possible to address school conditions that contribute to disparities in achievement,

      This line shows how schools can either reproduce or resist inequality depending on their internal structures. While educators can’t change a student’s socioeconomic background, they can reform tracking systems, counseling access, and resource distribution to create fairer opportunities. It emphasizes that inequality isn’t inevitable. Schools themselves can either maintain privilege or become tools for equity, depending on how intentionally they are structured.

    8. I have a [private] tutor now, and she's planning to be a math teacher at Berkeley High, and rhe [geome-try] books she's like an exjpert at going through because her school created them.

      This line exposes how access to private tutoring and insider knowledge reinforces educational inequality. Jennifer’s success isn’t just due to effort, but rather it’s supported by resources that connect her to the very system evaluating her. Her tutor’s familiarity with the school’s materials gives Jennifer an advantage that many students, like Chantelle, simply can’t access. This shows how inequality is subtly structured within schools, where privilege often operates through networks, familiarity, and access to guidance rather than overt discrimination.

  4. docdrop.org docdrop.org
    1. Why might growing gaps in family income cause an increasing gap between the school success of low-income and higher-income children? According to economic theory, families with higher incomes are better able to purchase or produce important "inputs" into their young chil-dren's development-for example, nutritious meals, enriched home learn-ing environments and child-care settings outside the home, and safe and stimulating neighborhood environments.4 Alternatively, psychologists and sociologists focus on how economic disadvantage impairs the quality of family relationships. 5

      This explains how the income gap translates into the education gap: From an economic perspective, it focuses on material conditions (food, education, housing, community), while from a psychological/sociological perspective, it emphasizes the quality of family relationships.

    2. The study first assessed the children shortly after they began kinder-garten, providing a picture of their skills at the starting line of their for-mal schooling. It shows that children from families in the top 20 percent of the income distribution already outscore children from the bottom 20 percent by 106 points in early literacy. This difference is nearly twice the size of the gap between the average reading skills of white and both black and Hispanic children at that age, and nearly equal to the amount that the typical child learns during kindergarten. Moreover, the reading gap was even larger when the same children were tested in fifth grade. Gaps in mathematics achievement are also substantial. 2

      This passage highlights the significant impact of family income on academic performance: At the beginning of their education, children from wealthy families already have significantly higher reading and writing scores, with the gap being so large that it is comparable to an entire year's worth of learning outcomes

    3. Which of these factors are most powerful in determining a child's s Uc-cess in school? While Annette Lareau and her team did not monitor school progress or behavioral development for the children in her study, includ-ing Anthony and Alexander, many national studies have investigated gaps in school performance among children from similarly disparate back-grounds. As shown in chapter 2, math and reading gaps between high-and low-income children have grown substantially over the past three decades. Data from a recent national study of children who entered kindergarten in the fall of 1998 allow for a more detailed look at income-based gaps as chil-dren progress through school (figure 3.1).1 As before, a 100-point difference in figure 3.1 corresponds to one standard deviation. Each bar shows the relative size of the gap between high-and low-income children.

      National data indicates that income disparity is a key factor influencing children's academic performance.

    4. A SNAPSHOT OF Alexander Williams and Anthony Mears at age twenty finds them on strikingly different educational and, in all likelihood, career trajectories. Alexander appears well on his way to an Ivy League degree and medical school. Anthony has a job, but the recent violent deaths of two friends have him just hoping that he will still be alive in five years.

      This passage, by comparing Alexander and Anthony, highlights how education and social environment profoundly influence the life paths of young people.

    5. Parental education levels probably matter even more than family struc-ture and income.2

      This line reveals that knowledge and awareness passed down from educated parents can be as powerful as financial resources in shaping a child’s future. Educated parents often know how to navigate institutions, advocate for their children, and provide guidance that builds confidence and opportunity. It shows how privilege is not only economic but also cultural: access to information, networks, and expectations about success perpetuate inequality across generations.

    6. Depression and other forms of psychological distress can profoundly affect parents' interactions with their children.1

      This line highlights how poverty’s impact goes beyond material hardship as it reaches into the emotional lives of families. When parents face chronic stress or depression from financial strain, their ability to nurture and engage with their children can be compromised. This shows that breaking cycles of poverty isn’t just about providing money, but also supporting mental health and emotional stability within families. Economic stress and psychological distress form a feedback loop that affects both generations.

    7. Exposure to lead paint affects children's nervous systems, resulting in hyperactivity and irritability, with long-term con-sequences for both intellectual and emotional development.

      This line powerfully illustrates how poverty can physically manifest in a child’s body and brain. Environmental hazards like lead exposure aren’t simply bad luck; they’re symptoms of structural inequality that trap families in cycles of disadvantage. Children’s developmental setbacks, then, are not the result of personal failure but of social neglect, showing how deeply poverty and environment intertwine to shape opportunity and success.

    8. hildren from more affluent families are more engaged than their low-income peers. Also, children from low-income families are more likely to engage in antisocial behavior and to have mental health problems.

      This line reveals how economic inequality extends beyond academics to affect emotional and social development. It suggests that wealth provides not only material advantages but also psychological stability and a sense of security that helps children focus and participate positively in school. Poverty, on the other hand, can create stressors that interfere with learning and behavior, showing that education gaps are deeply tied to social and mental well-being, not just access to resources.

    9. With an income of more than $300,000, Alexander's family was able to spend far more money on Alexander's education, lessons, and other enrichment activities than Anthony's parents could devote to their son's needs.

      This line highlights how economic privilege directly influences educational opportunity. Alexander’s access to enrichment activities, like tutoring, music lessons, and extracurriculars, reflects how wealth can buy not only academic resources but also cultural and social advantages that compound over time. The contrast to Anthony’s situation shows that academic success is not simply a matter of effort or intelligence, but also of the unequal distribution of resources that shape a child’s environment from an early age.

  5. docdrop.org docdrop.org
    1. Clara and her twin brother, Francisco, were born and raised in a small Mexican village. Their father had come to California on his own as a bracero railway worker during World War II, and when Clara and Francisco were eight, he brought his entire family (the twins, their mother, and two older siblings) as legal immigrants to Los Angeles. Be-cause family resources were scant, they settled first in Watts, a poverty-stricken, gang-ridden, mostly black neighborhood.

      This passage describes the immigration experience of Clara's family: from their village in Mexico to the United States, they initially settled in the impoverished Watts neighborhood in Los Angeles.

    2. We meet in the comfortable living room, facing glass doors opening onto a patio, a serene blue pool, and a colorful garden. Pictures of Isa-bella in dance costumes accent a grand piano. The adjacent dining room provides a calm space for conversation and homework, an environment that served as a refuge from Troy High School, which all three children attended and where, Clara reports, you can feel quivers of anxiety, as kids compete for the highest SAT scores and spots at Harvard, Stanford, and NYU. Her kids have had a very different experience growing up here than Clara and Ricardo had growing up a generation ago in South Central LA.

      This passage depicts the living conditions of a typical middle-upper class family, while also highlighting the differences across generations.

    3. hat is decidedly not similar about these two schools, however, are their student populations, as measured by poverty rates, ethnic backgrounds, English proficiency, and even physical fitness. Santa Ana students are overwhelmingly poor and Latino and heavily Spanish-speaking, whereas Troy students come from ethnically diverse, eco-nomically upscale backgrounds. More striking still are the contrasts in the "output" measures of the two schools-graduation rates, statewide academic and SAT test scores, truancy and suspension rates. Students at Santa Ana are four times more likely than students at Troy to drop out, roughly ten rimes more likely to be truant or suspended, and only one third as likely to take the SAT. If they do take the SAT, on average they score in the bottom quartile nationwide, whereas the average SAT taker at Troy scores in the top 10-15 percent

      This passage highlights the significant disparity between the two schools: students at Santa Ana are mostly poor Latinos with poor academic performance and high dropout rates; while students at Troy are more affluent and ethnically diverse, with outstanding academic achievements that rank among the best in the country. This reflects educational inequality.

    4. Orange County includes 34 incorporated cities, many of them worlds apart. As one local demographer puts it, "You have areas of pov-erty and areas of great affluence and less of a middle." 3 Laguna Beach, for example, is 91 percent non-Hispanic white, with a per capita income of $84,000, whereas Santa Ana, the county seat, just 20 miles away, is 95 percent Hispanic (50 percent foreign-born), with a per capita income of$17,000.

      There is a significant disparity within Orange County, with a clear social and economic divide.

    5. That image has, however, been gradually altered by large-scale de-mographic changes over the last 40 years. Since 1970 the population of Orange County has more than doubled to over 3 million people.

      This statement highlights that Orange County has undergone significant changes over the past 40 years due to a dramatic increase in population, and its original image no longer aligns with the current reality.

    1. Bailes AH, Masood S, Jehl N, Davis A, Giberson J, Aghaeepour N, Shu CH, Chang A, McGinnis RS, McGinnis EW, Cashman C, Hill A, Gill J, Simons LE

      The correct order is: Anna H. Bailes, Chi-Hung Shu, Alan Chang, Sahrish Masood, Nicole Jehl, Aliyah Davis, Jeremy Giberson, Casey Cashman, Allison Hill, Javed Gill, Ryan S. McGinnis, Ellen W. McGinnis, Nima Aghaeepour, Laura E. Simons

    2. Anna Heller Bailes;  Sahrish Masood;  Nicole Jehl;  Aliyah Davis;  Jeremy Giberson;  Nima Aghaeepour;  Chi-Hung Shu;  Alan Chang;  Ryan S McGinnis;  Ellen W McGinnis;  Casey Cashman;  Allison Hill;  Javed Gill;  Laura E Simons

      The correct order is: Anna H. Bailes, Chi-Hung Shu, Alan Chang, Sahrish Masood, Nicole Jehl, Aliyah Davis, Jeremy Giberson, Casey Cashman, Allison Hill, Javed Gill, Ryan S. McGinnis, Ellen W. McGinnis, Nima Aghaeepour, Laura E. Simons

  6. drive.google.com drive.google.com
    1. Andthank God she was there, for I was filled with that icy dreadagain. Everything I did seemed awkward to me, and every-thing I said sounded freighted with hidden meaning. I wastrying to remember everything I'd heard about dope addictionand I couldn't help watching Sonny for signs. I wasn't doing itout of malice. I was trying to find out something about mybrother. I was dying to hear him tell me he was safe.

      From this passage we can see the narrator keeps looking after his brother for fear of him being trapped by drugs again ;the theme of the obligation toward brotherly love.

    2. Creolebegan to tell us what the blues were all about. They were notabout anything very new.

      When Creole says that ' blues weren't anything new' ,is it because the music they play all derived from their past feelings and emotions? or is it telling us that sonny's expressing his life? ( But I think both are things he wanted to tell us)

    3. And he was giving it back, as every-thing must be given back, so that, passing through death, itcan live forever. I saw my mother's face again, and felt, for thefirst time, how the stones of the road she had walked on musthave bruised her feet. I saw the moonlit road where my fa-ther's brother died. And it brought something else back to me,and carried me past it, I saw my little girl again and feltIsabel's tears again, and I felt my own tears begin to rise.

      the narrator's reaction to Sonny's music shows howdleeply it moves him. I think it's also a turning point where he finally begins to understand Sonny.

    4. '1 couldn't tell you when Mama died-but the reason I wantedto leave Harlem so bad was to get away from drugs. And then,when I ran away, that's what I was running from-really.When I came back, nothing had changed, I hadn't changed, Iwas just-older." And he stopped, drumming with his fingerson the windowpane. The sun had vanished, soon darknesswould fall. I watched his face. "It can come again," he said,almost as though speaking to himself. Then he turned to me."It can come again," he repeated. "I just want you to knowthat.""All right," I said, at last. "So it can come again, All right."He smiled, but the smile was sorrowful. "I had to try to tellyou," he said.

      It shows how trapped he felt by his environment. Harlem represents pain , poverty , and a cycle he wants to escape. This highlights a main theme of the story - how hard it is to break free from suffering.

    5. "Imean, I'll have a lot of studying to do, and I'll have to studyeverything, but, I mean, I want to play with-jazz musicians."He stopped. "I want to play jazz," he said.

      It's clear that Black people have a really deep connection with music, and jazz is a big part of Black culture. This idea plays a significant role in Sonny's Blues, highlights how music becomes a way to deal with pain and as a form of expression.

    6. "You got to hold on to your brother,"she said, and don't let him fall, no matter what it looks likeis happ~ning to him and no matter how evil you gets with him.You gomg to be evil with him many a time. But don't youforget what I told you, you hear?"

      The narrator's mother, by charging him watching over Sonny, is asking him to serve as his brother's keeper. Another passage about the obligation toward brotherly love.

    7. The way I always see her isthe way she used to be on a Sunday afternoon, say, when th eold folks were talking after the big Sunday dinner. I always seeher wearing pale blue. She'd be sitting on the sofa. And myfather would be sitting in the easy chair, not far from her. Andthe living room would be full of church folks and relative s.

      'Sunday dinner' and 'church' are strongly associate with the Black culture in the US, representing a traditional rooted in the resilience of African Americans during and after slavery.

    8. "why does he want to die? He mustwant to die, he 's killing himself, why does he want to die?"He looked at me in surprise. He licked his lips. "He don 'twant to die. He wants to live. Don't nobody want to die,ever."

      The speaker, as someone who has tried heroin before , must have a remarkably stable mind to avoid being controlled by the drug. I strongly agree with the line,'He wants to live. Don't nobody want to die, ever.' It reveals the underlying reason why many people become addicted; they long to escape a reality that they cannot bare living.

    1. More recently, curriculum integration is often based on the premise that curriculum should be based on issues, concerns, and problems from real life and then organizing units or themes that extend beyond separate subject areas.

      When giving an understanding of curriculum integration, how doses educators develop the themes to bring a connection to their students? Does curriculum integration mean to pair subjects of learning with each other? Is there a school system that was created by education integration?

    1. Another way to understand archives, by contrast, is as “desire settings,” to use art historian Romi Crawford’s phrase for urban sites that invite “myriad scenarios of learning, labor, and conviviality.”

      This term "desire setting" is so interesting to me. Archives as paces shaped by longing, imagination, and human action. The word "desire" immediately opens up a more emotional, even poetic dimension. It reminds me of the Gumby chapter, where his scrapbooks functioned in a similar way. His scrapbooks were creative and imaginative, as well as political and queer. He archived what mattered to him, what he felt should be remembered. In that way, his scrapbooks became a kind of desire setting, they reflected both a yearning for representation and a refusal to let certain stories disappear.

    Annotators

    1. ecades' worth of demographic data reveals that the American Dream and its promise of upward mobility is largely a myth. In truth, the economic class a person is born into is the primary determinant of how he or she will fair economically as an adult.

      This is so important to note. Throughout our life we were promised hard work pays off. In my life I've seen so many people work so hard and still be stuck in the same place for the rest of their life. It is a false reality to keep people working for the people exploiting them for profit.

    2. Individualistic rather than collective in nature,

      This is capitalism. It is supposed to make people assume that they have options because they have "free individuality" but it comes at a bigger, more expensive and oppressive cost than assumed. (environmental, 1% of wealthy people having majority of the wealth in the nation, redlining, etc)

    3. He reasoned that the system only functioned as long as the workers did not recognize their unity as a class of laborers, their shared economic and political interests, and the power inherent in their numbers.

      This makes so much sense. We have seen, especially in the current administration that has been making people pit against each other, distracting them from the fact the biggest war they are in, is the people in power.

    4. Marx saw false consciousness as a product of an unequal social system controlled by a powerful minority of elites. The false consciousness among workers, which prevented them from seeing their collective interests and power, was created by the material relations and conditions of the capitalist system, by the ideology (the dominant worldview and values) of those who control the system, and by social institutions and how they function in society.

      He saw a higher power that plays out in the shadows and oversees the power and capitalist system that the world has.

    5. In the U.S. this belief is encapsulated in the ideal of "the American Dream." Viewing society and one's place within it based on the set of assumptions derived from "common sense" thinking results in a perception of being an individual rather than part of a collective. Economic success and failure rest squarely on the shoulders of the individual and do not take into account the totality of the social, economic, and political systems that shape our lives.

      They use the title of American dream as like a dream statement to say when you live happily but for a lot of people in the world nowadays the "American Dream" ain't realistic at this point.

    6. Class consciousness refers to the awareness by a social or economic class of their position and interests within the structure of the economic order and social system in which they live.

      They live in a economic pyramid, which sounds like if they don't make enough they aren't as "valuable" as someone who makes a lot of money.

    1. A loud, flashy-handed salesman type who has more important things to do than to sit on a jury.He is quick to show temper, quick to form opinions on things about w

      Arly

    1. Author response:

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

      Reviewer #1 (Public Review):

      The authors sought to test whether anterior insular cortex neurons increase or decrease firing during fear behavior and freezing, bi-directionally control fear via separate, anatomically defined outputs. Using a fairly simple behavior where mice were exposed to tone-shock pairings, they found roughly equal populations that do indeed either increase or decrease firing during freezing. Next, they sought to test whether these distinct populations may also have distinct outputs. Using retrograde tracers they found that the anterior insular cortex contains non-overlapping neurons which project to the mediodorsal thalamus or amygdala. Mediodorsal thalamus-projecting neurons tended to cluster in deep cortical layers while amygdala-projecting neurons were primarily in more superficial layers. Stimulation of insula-thalamus projection decreased freezing behavior, and stimulation of insula-amygdala projections increased fear behavior. Given that the neurons that increased firing were located in deep layers, that thalamus projections occurred in deep layers, and that stimulation of insula-thalamus neurons decreased freezing, the authors concluded that the increased firing neurons may be thalamus projections. Similarly, given that decreased-firing neurons tended to occur in more superficial layers, that insula-amygdala projections were primarily superficial, and that insula-amygdala stimulation increased freezing behavior, authors concluded that the decreased firing cells may be amygdala projections. The study has several strengths though also some caveats.

      Strengths:

      The potential link between physiological activity, anatomy, and behavior is well laid out and is an interesting question. The activity contrast between the units that increase/decrease firing during freezing is clear.

      It is nice to see the recording of extracellular spiking activity, which provides a clear measure of neural output, whereas similar studies often use bulk calcium imaging, a signal that rarely matches real neural activity even when anatomy suggests it might (see London et al 2018 J Neuro - there are increased/decreased spiking striatal populations, but both D1 and D2 striatal neurons increase bulk calcium).

      Weaknesses:

      The link between spiking, anatomy, and behavior requires assumptions/inferences: the anatomically/genetically defined neurons which had distinct outputs and opposite behavioral effects can only be assumed the increased/decreased spiking neurons, based on the rough area of the cortical layer they were recorded.

      Yes, we are aware that we could not provide a direct link between spiking, anatomy and behavior. We have specifically noted this in the discussion section and added a possible experiment that could be carried out to provide a more direct link in a future study.

      [Lines 371-375] We would like to provide a more direct evidence between the neuronal response types and projection patterns in future studies by electrophysiologically identifying freezing-excited and freezing-inhibited aIC neurons and testing whether those neurons activates to optogenetic activation of amygdala or medial thalamus projecting aIC neurons.

      The behavior would require more control to fully support claims about the associative nature of the fear response (see Trott et al 2022 eLife) - freezing, in this case, could just as well be nonassociative. In a similar vein, fixed intertrial intervals, though common practice in the fear literature, pose a problem for neurophysiological studies. The first is that animals learn the timing of events, and the second is that neural activity is dynamic and changes over time. Thus it is very difficult to determine whether changes in neural activity are due to learning about the tone-shock contingency, timing of the task, simply occur because of time and independently of external events, or some combination of the above.

      Trott et al. (2022) stated that "...freezing was the purest reflection of associative learning." The nonassociative processes mentioned in the study were related to running and darting behaviors, which the authors argue are suppressed by associative learning. Moreover, considerable evidence from immediate postshock freezing and immediate postshock context shift studies all indicate that the freezing response is an associative (and not nonassociative) response (Fanselow, 1980 and 1986; and Landeira-Fernandez et al., 2006). Thus, our animals' freezing response to the tone CS presentation in a novel context, following three tone CS-footshock US pairings, most likely reflects associative learning. 

      Concerning the issue of fixed inter-trial intervals (ITIs), which are standard in fear conditioning studies, particularly those with few CS-US paired trials, we acknowledge the challenge in interpreting the neural correlates of behavior. However, the ITIs in our extinction study was variable and we still found neural activities that had significant correlation with freezing. The results of our extinction study, carried out with variable it is, suggest that the aIC neural activity changes measured in this study is likely due to freezing behavior associated with fear learning, not due to learning the contingencies of fixed ITIs.

      Reviewer #2 (Public Review):

      In this study, the authors aim to understand how neurons in the anterior insular cortex (insula) modulate fear behaviors. They report that the activity of a subpopulation of insula neurons is positively correlated with freezing behaviors, while the activity of another subpopulation of neurons is negatively correlated to the same freezing episodes. They then used optogenetics and showed that activation of anterior insula excitatory neurons during tones predicting a footshock increases the amount of freezing outside the tone presentation, while optogenetic inhibition had no effect. Finally, they found that two neuronal projections of the anterior insula, one to the amygdala and another to the medial thalamus, are increasing and decreasing freezing behaviors respectively. While the study contains interesting and timely findings for our understanding of the mechanisms underlying fear, some points remain to be addressed.

      We are thankful for the detailed and constructive comments by the reviewer and addressed the points. Specifically, we included possible limitations of using only male mice in the study, included two more studies about the insula as references, specified the L-ratio and isolated distance used in our study, added the ratio of putative-excitatory and putative-inhibitory neurons obtained from our study, changed the terms used to describe neuronal activity changes (freezing-excited and freezing-inhibited cells), added new analysis (Figure 2H), rearranged Figure 2 for clarity, added new histology images, and added atlas maps with viral expressions (three figure supplements).

      Reviewer #1 (Recommendations For The Authors):

      - I would suggest keeping the same y-axis for all figures that display the same data type - Figure 5D, for example.

      Thank you for the detailed suggestion. We corrected the y-axis that display the same data type to be the same for all figures.

      - In the methods, it says 30s bins were used for neural analysis (line 435). I cannot imagine doing this, and looking at the other figures, it does not look like this is the case so could you please clarify what bins, averages, etc were used for neural and behavioral analysis?

      Bin size for neural analysis varied; 30s, 5s, 1s bins were used depending on the analysis. We corrected this and specified what time bin was used for which figure in the methods.

      Bin size for neural and freezing behavior was 30s and we also added this to the methods.

      - I would not make any claims about the fear response here being associative/conditional. This would require a control group that received an equal number of tone and shock exposures, whether explicitly unpaired or random.

      The unpaired fear conditioning paradigm, unpaired tone and shock, suggested by the reviewer is well characterized not to induce fear behavior by CS (Moita et al., 2003 and Kochli et al., 2015). In addition, considerable evidence from immediate post-shock freezing and immediate post-shock context shift studies all indicate that the freezing response is an associative (and not nonassociative) response (Fanselow, 1980 and 1986; and Landeira-Fernandez et al., 2006). Thus, our animals' freezing response to the tone CS presentation in a novel context, following three tone CS-footshock US pairings, most likely reflects associative learning.

      - I appreciate the discussion about requiring some inference to conclude that anatomically defined neurons are the physiologically defined ones. This is a caveat that is fully disclosed, however, I might suggest adding to the discussion that future experiments could address this by tagging insula-thalamus or insula-amygdala neurons with antidromic (opto or even plain old electric!) stimulation. These experiments are tricky to perform, of course, but this would be required to fully close all the links between behavior, physiology, and anatomy.

      As suggested, we have included that, in a future study, we would like to elucidate a more direct link between physiology, anatomy and behaviors by optogenetically tagging the insula-thalamus/insula-amygdala neurons and identifying whether it may be a positive or a negative cell (now named the freezing-excited and freezing-inhibited cells, respectively) in the discussion.

      [Lines 371-375] We would like to provide a more direct evidence between the neuronal response types and projection patterns in future studies by electrophysiologically identifying freezing-excited and freezing-inhibited aIC neurons and testing whether those neurons activates to optogenetic activation of amygdala or medial thalamus projecting aIC neurons.

      Reviewer #2 (Recommendations For The Authors):

      Major comments:

      (1) As all experiments have been performed only in male mice, the authors need to clearly state this limit in the introduction, abstract, and title of the manuscript.

      With increasing number of readers becoming interested in the biological sex used in preclinical studies, we also feel that it should be mentioned in the beginning of the manuscript. As suggested, we explicitly wrote that we only used male mice in the title, abstract, and introduction. In addition, we discussed possible limitations of only using male mice in the discussion section as follows:

      [Lines 381-386] Another factor to consider is that we have only used male mice in this study. Although many studies report that there is no biological sex difference in cued fear conditioning (42), the main experimental paradigm used in this study, it does not mean that the underlying brain circuit mechanism would also be similar. The bidirectional fear modulation by aIC→medial thalamus or the aIC→amygdala projections may be different in female mice, as some studies report reduced cued fear extinction in females (42).

      (2) The authors are missing important publications reporting findings on the insular cortex in fear and anxiety. For example, the authors should cite studies showing that anterior insula VIP+ interneurons inhibition reduces fear memory retrieval (Ramos-Prats et al., 2022) and that posterior insula neurons are a state-dependent regulator of fear (Klein et al., 2021). Also, regarding the anterior insula to basolateral amygdala projection (aIC-BLA), the author should include recent work showing that this population encodes both negative valence and anxiogenic spaces (Nicolas et al., 2023). 

      We appreciate the detailed suggestions and we added appropriate publications in the discussion section. The anterior insula VIP+ interneuron study (Ramos-Prats et al., 2022) is interesting, but based on the evidence provided in the paper, we felt that the role of aIC VIP+ interneuron in fear conditioning is low. VIP+ interneurons in the aIC seem to be important in coding sensory stimuli, however, it’s relevance to conditioned stimuli seems to be low; overall VIP intracellular calcium activity to CS was low and did not differ between acquisition and retrieval. Also, inhibition of VIP did not influence fear acquisition. VIP inhibition during fear acquisition did reduce fear retrieval (CS only, no light stimulation), but this does not necessarily mean that VIP activity will be involved in fear memory storage or retrieval, especially because intracellular calcium activity of VIP+ neurons was low during fear conditioning and retrieval.

      Studies by Klein et al. (2021) and Nicolas et al. (2023) are integrated in the discussion section as follows.

      [Lines 297-301] Group activity of neurons in the pIC measured with fiberphotometry, interestingly, exhibited fear state dependent activity changes—decreased activity with high fear behavior and increased activity with lower fear behavior (29)—suggesting that group activity of the pIC may be involves in maintain appropriate level of fear behavior.

      [Lines 316-319] Another distinction between the aIC and pIC may be related with anxiety, as a recent study showed that group activity of aIC neurons, but not that of the pIC, increased when mice explored anxiogenic space (open arms in an elevated plus maze, center of an open field box) (32).

      (3) The authors should specify how many neurons they excluded after controlling the L-ratio and isolation distance. It is also important to specify the percentage of putative excitatory and inhibitory interneurons recorded among the 11 mice based on their classification (the number of putative inhibitory interneurons in Figure 1D seems too low to be accurate).

      We use manual cluster cutting and only cut clusters that are visually well isolated. So we hardly have any neurons that are excluded after controlling for L-ratio and isolation distance. The criterion we used was L-ratio<0.3 and isolation distance>15, and we specified this in the methods as follows.

      [Lines 454-458] We only used well-isolated units (L-ratio<0.3, isolation distance>15) that were confirmed to be recorded in the aIC (conditioned group: n = 116 neurons, 11 mice; control group: n = 14 neurons, 3 mice) for the analysis (46). The mean of units used in our analysis are as follows: L-ratio = 0.09 ± 0.012, isolation distance = 44.97 ± 5.26 (expressed as mean ± standard deviation).

      As suggested, we also specified the percentage of putative excitatory and inhibitory interneurons recorded from our study in the results and methods section. The relative percentage of putative excitatory and inhibitory interneurons were similar for both the conditioned and the control groups (conditioned putative-excitatory: 93.1%, putative-inhibitory: 6.9%; control putative-excitatory: 92.9%, putative-inhibitory: 7.1%). Although the number of putative-interneurons isolated from our recordings is low that is what we obtained. Putative inhibitory neurons, probably because of their relatively smaller size, has a tendency to be underrepresented than the putative excitatory cells.

      [Lines 83-87] Of the recorded neurons, we analyzed the activity of 108 putative pyramidal neurons (93% of total isolated neurons) from 11 mice, which were distinguished from putative interneurons (n = 8 cells, 7% of total isolated neurons) based on the characteristics of their recorded action potentials (Figure 1D; see methods for details).

      [Lines 464-467] The percentage of putative excitatory neurons and putative inhibitory interneurons obtained from both groups were similar (conditioned putative-excitatory: 93.1%, putative-inhibitory: 6.9%; control putative-excitatory: 92.9%, putative-inhibitory: 7.1%).

      (4) While the use of correlation of single-unit firing frequency with freezing is interesting, classically, studies analyze the firing in comparison to the auditory cues. If the authors want to keep the correlation analysis with freezing, rather than correlations to the cues, they should rename the cells as "freezing excited" and "freezing inhibited" cells instead of positive and negative cells.

      As suggested, we used the terms “freezing-excited” and “freezing-inhibited” cells instead of positive and negative cells.

      (5) To improve clarity, Figure 2 should be reorganized to start with the representative examples before including the average of population data. Thus Panel D should be the first one. The authors should also consider including the trace of the firing rate of these representative units over time, on top of the freezing trace, as well as Pearson's r and p values for both of them. Then, the next panels should be ordered as follows: F, G, H, C, A, B, I, and finally E.

      We have rearranged Figure 2 based on the suggestions.

      (6) It is unclear why the freezing response in Figure 2 is different in current panels F, G, and H. Please clarify this point.

      It was because the freezing behaviors of slightly different population of animals were averaged. Some animals did not have positive/negative (or both) cells and only the behavior of animals with the specified cell-type were used for calculating the mean freezing response. With rearrangement of Figure 2, now we do not have plots with juxtaposed mean neuronal response-types and behavior.

      (7) Even though the peak of tone-induced firing rate change between negative and positive cells is 10s later for positive cells, the conclusion that this 'difference suggests differential circuits may regulate the activities of different neuron types in response to fear' is overstating the observation. This statement should be rephrased. Indeed, it could be the same circuits that are regulated by different inputs (glutamatergic, GABA, or neuromodulatory inputs).

      We agree and delete the statement from the manuscript.

      (8) The authors mention they did not find tone onset nor tone offset-induced responses of anterior insula neurons. It would be helpful to represent this finding in a Figure, especially, which were the criteria for a cell to be tone onset or tone offset responding.

      We added how tone-onset and tone-offset were analyzed in the methods section and added a plot of the analysis in Figure 2H.

      (9) Based on the spread of the viral expression shown in Figure 3B, it appears that the authors are activating/inhibiting insula neurons in the GI layer, whereas single-unit recordings report the electrodes were located in DI, AID, and AIV layers. The authors should provide histology maps of the viral spread for ChR2, NpHR3, and eYFP expression.

      Thank you for the excellent suggestion. Now the histological sample in Figure 3B is a sample with expression in the GI/DI/AID layers and it also has an image taken at higher resolution (x40) to show that viral vectors are expressed inside neurons. We also added histological maps with overlay of viral expression patterns of the ChR2, eYFP, and NpHR3 groups in Figure 3—figure supplement 1.

      (10) In Figure 5B, the distribution of terminals expressing ChR2 appears much denser in CM than in MD. This should be quantified across mice and if consistent with the representative image, the authors should refer to aIC-CM rather than aIC-MD terminals.

      Overall, we referred to the connection as aIC-medial thalamus, which collectively includes both the CM and the MD. Microscopes we have cannot determine whether terminals end at the CM or MD, but the aIC projections seems to pass through the CM to reach the MD. The Allen Brain Institute’s Mouse brain connectivity map (https://connectivity.brain-map.org/projection/experiment/272737914) of a B6 mouse, the mouse strain we used in our study, with tracers injected in similar location as our study also supports our speculation and shows that aIC neuronal projections terminate more in the MD than in the CM. In addition, the power of light delivered for optogenetic manipulation is greatly reduced over distance, and therefore, the MD projecting terminals which is closer to the optic fiber will be more likely to be activated than the CM projecting terminals. However, since we could not determine whether the aIC terminate at the CM or the MD, we collectively referred to the connection as the aIC-medial thalamus throughout the manuscript.

      Author response image 1.

      (11) Histological verifications for each in vivo electrophysiology, optogenetic, and tracing experiments need to include a representative image of the implantation/injection site, as well as a 40x zoom-in image focusing on the cell bodies or terminals right below the optic fiber (for optogenetic experiments). Moreover, an atlas map including all injection locations with the spread of the virus and fiber placement should be added in the Supplement Figures for each experiment (see Figure S1 Klein et al., 2021). Similarly, the authors need to add a representation of the spread of the retrograde tracers for each mouse used for this tracing experiment.

      As suggested, we added a histology sample showing electrode recording location for in-vivo electrophysiology in Figure 1 and added atlas maps for the optogenetic and tracing experiments in supplementary figures. We also provide a 40x zoom-in image of the expression pattern for the optogenetic experiments (Figure 3B).

      (12) To target anterior insula neurons, authors mention coordinates that do not reach the insula on the Paxinos atlas (AP: +1.2 mm, ML: -3.4 mm, DV: -1.8 mm). If the DV was taken from the brain surface, this has to be specified, and if the other coordinates are from Bregma, this also needs to be specified. Finally, the authors cite a review from Maren & Fanselow (1996), for the anterior insula coordinates, but it remains unclear why.

      AP and ML coordinates are measurement made in reference to the bregma. DV was calculated from the brain surface. We specified these in the Methods. We did not cite a review from Maren & Fenselow for the aIC coordinates.

      Minor comments:

      (1) A schematic of the microdrive and tetrodes, including the distance of each tetrode would also be helpful.

      We used a handcrafted Microdrives with four tetrodes. Since they were handcrafted, the relative orientation of the tetrodes varies and tetrode recording locations has to be verified histologically. We, however, made sure that the distance between tetrodes to be more than 200 μm apart so that distinct single-units will be obtained from different tetrodes. We added this to the methods as follows.

      [Lines 430-431] The distance between the tetrodes were greater than 200 μm to ensure that distinct single-units will be obtained from different tetrodes.

      (2) Figure 2E: representation of the baseline firing (3-min period before the tone presentation) is missing.

      Figure 2E is the 3 min period before tone presentation

      (3) Figure 2: Averages Pearson's correlation r and p values should be stated on panels F, G, and H (positive cell r = 0.81, P < 0.05; negative cell r = -0.68, P < 0.05).

      They were all originally stated in the figures. But with reorganization of Figure 2, we now have a plot of the Pearson’s Correlation with r and p values in Figure 2F.

      (4) Figure 2I: Representation of the absolute value of the normalized firing is highly confusing. Indeed, as the 'negative cells' are inhibited to freezing, firing should be represented as normalized, and negative for the inhibited cells.

      To avoid confusion, we did not take an absolute value of the “negative cells”, which are now called the “freezing-inhibited cells”.

      (5) Figure 4E (retrograde tracing): representation of individual values is missing.

      Figure 4E now has individual values.

      References:

      London, T. D., Licholai, J. A., Szczot, I., Ali, M. A., LeBlanc, K. H., Fobbs, W. C., & Kravitz, A. V. (2018). Coordinated ramping of dorsal striatal pathways preceding food approach and consumption. Journal of Neuroscience, 38(14), 3547-3558.

      Trott, J. M., Hoffman, A. N., Zhuravka, I., & Fanselow, M. S. (2022). Conditional and unconditional components of aversively motivated freezing, flight and darting in mice. Elife, 11, e75663.

      Fanselow, M. S. (1980). Conditional and unconditional components of post-shock freezing. The Pavlovian journal of biological science: Official Journal of the Pavlovian, 15(4), 177-182.

      Fanselow, M. S. (1986). Associative vs topographical accounts of the immediate shock-freezing deficit in rats: implications for the response selection rules governing species-specific defensive reactions. Learning and Motivation, 17(1), 16-39.

      Landeira-Fernandez, J., DeCola, J. P., Kim, J. J., & Fanselow, M. S. (2006). Immediate shock deficit in fear conditioning: effects of shock manipulations. Behavioral neuroscience, 120(4), 873.

      Moita, M. A., Rosis, S., Zhou, Y., LeDoux, J. E., & Blair, H. T. (2003). Hippocampal place cells acquire location-specific responses to the conditioned stimulus during auditory fear conditioning. Neuron, 37(3), 485-497.

      Kochli, D. E., Thompson, E. C., Fricke, E. A., Postle, A. F., & Quinn, J. J. (2015). The amygdala is critical for trace, delay, and contextual fear conditioning. Learning & memory, 22(2), 92-100.

      Ramos-Prats, A., Paradiso, E., Castaldi, F., Sadeghi, M., Mir, M. Y., Hörtnagl, H., ... & Ferraguti, F. (2022). VIP-expressing interneurons in the anterior insular cortex contribute to sensory processing to regulate adaptive behavior. Cell Reports, 39(9).

      Klein, A. S., Dolensek, N., Weiand, C., & Gogolla, N. (2021). Fear balance is maintained by bodily feedback to the insular cortex in mice. Science, 374(6570), 1010-1015.

      Nicolas, C., Ju, A., Wu, Y., Eldirdiri, H., Delcasso, S., Couderc, Y., ... & Beyeler, A. (2023). Linking emotional valence and anxiety in a mouse insula-amygdala circuit. Nature Communications, 14(1), 5073.

      Maren, S., & Fanselow, M. S. (1996). The amygdala and fear conditioning : Has the nut been cracked? Neuron, 16(2), 237‑240. https://doi.org/10.1016/s0896-6273(00)80041-0

    1. Author response:

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

      Reviewer #1 (Public review):

      (1) The primary weakness of the paper concerns its conclusion of having generated "homogenous mature microglia", partly based on the RNAseq analysis. However, the comparison of gene profiles was carried out only between "hiPSC-derived mature microglia" and the proliferating myeloid progenitors. While the transcriptome profiles revealed a trend of enrichment of microglia-like gene expression in "hiPSC-derived mature microglia" compared to proliferating myeloid progenitors, this is not sufficient to claim they are "mature microglia". It is important that one carries out a comparative analysis of the RNAseq data with those of primary human microglia, which may be done by leveraging the public database. To convincingly claim these cells are mature microglia, questions need to be addressed including how similar the molecular signatures of these cells are compared with the fully differentiated primary microglia cell or if they remain progenitor-like or take on mosaic properties, and how they distinguish from macrophages.

      We greatly appreciate the insightful comments and suggestions from the reviewers, which were instrumental in enhancing our data analysis and organization. In response to the feedback, we have updated the terminology from “mature microglia” to simply “microglia” while clarifying in our text that these are fully differentiated microglia under single-type cell culture conditions.

      Guided by the reviewer's advice, we incorporated RNA-seq data from human brain microglia studies conducted by Dr. Poon and Dr. Blurton-Jones' Lab (Abud et al., Neuron, 2017) and Dr. Huitinga's Lab (van der Poel et al., Nat Commun, 2019). We then conducted a comparative analysis of the gene expression profiles between our fully differentiated hiPSC-derived microglia and those from fetal/adult brain microglia (see Fig.2. Suppl. B, C and D; Suppl. table 1 and table 2). The correlation analysis revealed that our hiPSC-derived microglia closely resemble fetal and adult brain microglia, distinguishing them significantly from monocytes and inflammatory monocytes.

      (2) While the authors attempted to demonstrate the functional property of "hiPSC-derived mature microglia" in culture, they used LPS challenge, which is an inappropriate assay. This is because human microglia respond poorly to LPS alone but need to be activated by a combination of LPS with other factors, such as IFNγ. Their data that "hiPSC-derived mature microglia" showed robust responses to LPS indeed implicates that these cells do not behave like mature human microglia.

      We appreciate the feedback received. In response, we cultured hiPSC-derived microglia cells and subjected them to treatments with IFNγ, LPS, and a combination of both IFNγ+LPS, as illustrated in Figure 3 suppl. Our findings revealed that the IFNγ+LPS combination notably enhanced the expression of IL1a, IL1b, TNFa, CCL8, and CXCL10, whereas IL6 and CCL2 levels remained unchanged. Treatment with IFNγ alone significantly elevated the expression of TNFa, CCL8, CXCL10, and CCL2. These outcomes align with the findings reported by Rustenhoven et al. (Sci Rep, 2016), suggesting that the functionality of our hiPSC-derived microglia cells closely mirrors that of primary human adult microglia cells.

      (3) The resolution of Figs. 4 - 6 is so low that even some of the text and labels are hardly readable. Based on the morphology shown in Fig. 4 and the statement in line 147, these hiPSC-derived "cells altered their morphology to a rounded shape within an hour of incubation and rapidly internalized the fluorescent-labeled particles". This is a peculiar response. Usually, microglia do not respond to fluorescent-labeled zymosan by turning into a rounded shaped within an hour when they internalize them. Such a behavior usually implicates weak phagocytotic capacity.

      Thank you for your insightful comments. During submission, the main text's PDF version was converted online, resulting in low-quality output. We have since updated this with a high-resolution version. The observed alterations in cell morphology following zymosan phagocytosis may be attributed to the high zymosan concentration used (2mg/ml). We conducted an assessment to understand the impact of zymosan concentration on the morphology of hiPSC-derived microglial cells, as shown in Figure 4 suppl B. Our findings indicate that microglia cells adopt an amoeboid, rounded shape at zymosan concentrations exceeding 20ug/ml. To clarify this point, we have amended the text to read: "The cells altered their morphology and rapidly internalized the fluorescent-labeled particles."

      (4) Data presented in Fig. 5 are not very convincing to support that transplanted cells were immunopositive for "human CD11b (Fig.5C), as well as microglia signature markers P2ry12 and TMEM119 (Fig.5D)" (line 167). The resolution and magnification of Fig. 5D is too low to tell the colocalization of tdT and human microglial marker immunolabeling. In the flat-mount images (C, I), hCD11b immunolabeling is not visible in the GCL or barely visible in the IPL. This should be discussed.

      We are grateful for the reviewer's comments. As previously mentioned, the low quality of the images was due to the online conversion of the PDF version. We have now submitted both high-quality PDF and Word versions for the reviewer's assessment. In these high-quality versions, the colocalization of tdT with human P2ry12 and TMEM119 is distinctly visible. Additionally, we have updated the hTMEM119 staining images in Figure 5D. The results from hCD11b staining align with those observed in mouse CD11b staining, notably showing more effective staining in the outer plexiform layer (OPL) microglia cells. The reason for this—whether it pertains to a staining issue, a variance in CD11b expression among microglia cells in the OPL and ganglion layer (GL), or differences in the samples due to varying conditions—is not yet clear and warrants further investigation.

      (5) Microglia respond to injury by becoming active and lose their expression of the resting state microglial marker, such as P2ry12, which is used in Fig. 6 for detection of migrated microglia. To confirm that these cells indeed respond to injury like native microglia, one should check for activated microglial markers and induction of pro-inflammatory cytokines in the sodium iodate-injury model.

      The reviewer's insights are spot-on. We utilized preserved retinas to extract mRNA, which was then reverse-transcribed to cDNA for conducting qRT-PCR using human-specific primers, as detailed in the updated Table 5. The findings revealed that following retinal pigment epithelium (RPE) injury for 3 days, the transplanted hiPSC-derived microglial cells exhibited an increase in the production of inflammatory cytokines and upregulated genes related to phagocytosis, migration, and adhesion. Conversely, there was a decrease in the expression of microglia-specific signature genes and neurotrophic factors, as demonstrated in Figure 7 suppl.

      Reviewer #1 (Recommendations For The Authors):

      Line 52: "Microglia cell repopulation research suggests that: 1) if no injury or infection occurs, retinal microglia cells can sustain their homeostasis indefinitely" - this statement is too strong or delivers a confusing message; it needs clarification or to be backed up by evidence. Recent single cell RNA sequencing analyses suggest that even under a normal condition, residential microglia do not present as a single homeostatic cell cluster, rather a subpopulation of activated inflammatory microglia are constantly detectable in the normal retina. This is likely because normal retinal neurons can be stressed due to various reasons, such as the temporal accumulation of misfolded proteins, exposed to strong light, or ageing, etc.

      We appreciate the comments. We changed the sentence to read, "Microglia cell repopulation research suggests that: 1) retinal resident microglia cells can sustain their population with the local dividing and migration if any perturbations do not exceed the threshold of the recovery speed by local neighbor microglia cells."

      Line 83: "we applied an appropriate protocol for culturing human iPSC-derived microglia cells" - it would be more appropriate if the word "appropriate" can be replaced by either "unique" or a phrase like "we adopted a (previously published) protocol...".

      Thanks! We changed it to “We modified a previously published protocol to culture human iPSC-derived microglia cells.".

      Fig. 1F,G: A method of flow cytometry will provide more comprehensive cell quantification for percentages of positively labeled cells than cell counts under high magnification confocal images.

      Thanks for the comments! We agreed with the reviewer. Given the experimental resources available, the quantifications of confocal images did provide a reasonable assessment. We will perform flow cytometry analysis in future experiments.

      Reviewer #2 (Public review):

      Weaknesses:

      Gene expression analysis of mature microglia cells should be better interpreted and it would be beneficial to compare the iPSC-derived microglia gene set to a human microglial cell line (for example, HMC3) instead of myeloid progenitor cells.<br /> The way that the manuscript has been written, unfortunately, is not optimal. I recommend that the entire manuscript be edited and proofread in English. The text contains spelling and grammar mistakes, and the manuscript is inconsistent in several parts. The manuscript should also be revised for a scientific paper format.

      We appreciate the reviewer's comments and have taken them into consideration along with similar inquiries from Reviewer 1. Following the suggestions, we conducted a comparison of gene expression profiles between our hiPSC-derived microglia and those from fetal/adult brain microglia, as depicted in the updated Fig.2. Suppl. B, C and D; as well as in the Suppl. table 1 and table 2. The correlation analysis demonstrated that the hiPSC-derived microglia cells closely resemble fetal and adult brain microglia, significantly differing from monocytes and inflammatory monocytes. Additionally, we have revised the manuscript to adhere more closely to the conventional scientific format.

      Reviewer #2 (Recommendations For The Authors):

      Specific suggestions for improvement:

      - Regarding the characterization of human iPSC-derived microglia, P2RY12 is a general hematopoietic cell marker. One cannot judge the maturity of microglia only by P2RY12 expression (for example, line 261). The expression of more specific markers such as TMEM119 and PROS1 should be studied and discussed.

      We are thankful for the reviewer's valuable feedback. In response:

      We have removed the term "mature" and clarified that the hiPSC-derived microglia we studied are fully differentiated within single-type cell culture conditions.

      We performed a comparative analysis of the gene expression profiles between our hiPSC-derived microglia and microglia from human brains, as illustrated in the updated Fig.2. Suppl. B, C and D. The results affirm that hiPSC-derived microglia closely resemble human fetal and adult microglia.

      We noted that the expression of TMEM119 in hiPSC-derived microglia under in vitro single-type cell culture conditions is notably low, as shown in the below A. This suggests that the stimulatory factors in our single-type cell culture might not sufficiently induce TMEM119 expression in microglia. The necessity for a retinal environment or interaction with neuronal and/or other glial cells for TMEM119 expression mirrors the behavior of infiltrating peripheral monocytes in pathological conditions, which initially lack TMEM119 but later differentiate into microglial-like macrophages that express TMEM119, as reported by Ma et al. in Sci Rep (2017).

      Additionally, our findings suggest that PROS1 is not uniquely characteristic of microglia but is expressed across a variety of cell types. Within our specific culture conditions, we noted a higher expression of PROS1 in microglial progenitor cells, as shown in Author response image 1B and C.

      Author response image 1.

      - In Figure 2, Part E, the names of the genes or pathways in the figure are not clear, and are these genes the set that are the most differentially expressed between iPSCs-derived microglia and MPC? The analysis needs more explanation.

      We regret any confusion caused by our previous explanation. To clarify, we compiled a list of microglia-enriched genes from the research conducted by Barres BA Lab (Bennett et al., Proc Natl Acad Sci U S A, 2016) and from our own RNA sequencing data of mouse retinal microglia, identifying a total of 130 genes predominantly expressed in microglia (Suppl. Table 3). We then applied this gene list to analyze our hiPSC-derived microglia RNA sequencing data, resulting in the identification of 71 microglia-specific genes. These 71 genes were subjected to Ingenuity Pathway Analysis (IPA) to visualize the signaling pathways involved. The details of these microglia genes can be found in the updated suppl. table 3.

      - Lines 124 to 128 mention that high expression of Stat3, IL1b, and IL6 and their central role in pathway analysis emphasize the efficiency of the maturation protocol. Regarding the fact that Stat3, IL1b, and IL6 are contributors to proinflammatory pathways, it is not convincing that the high expression of these genes in iPSC-derived microglia demonstrates the efficiency of the maturation protocol, given that microglia are not stimulated.

      Thanks for the comments! We added the sentences about the comparison results between hiPSC-derived microglia and human brain microglia. We have also replaced the “mature” with “functional.” The sentence reads, “Thus, our method of obtaining differentiated microglia is a reliable method to generate a large number of homogenous functional microglia cells.”

      - Statistical analysis is missing for some graphs, for example, figures 1-3 and 5.

      We appreciate the comments. We have added the statistical results in the revised version.

      - The legend for Figure 3 needs to be rewritten. The graphs or applied assays should be explained in the legend, not the interpretation of the data.

      The legend was rewritten.

      - There is no Figure 3 in the supplement figures file.

      We added Figure 3. Suppl.

      - hTMEM119 staining in Figure 5, Part D, is mostly background. Please provide another image.

      The images were unclear after on-line converting due to the low number of pixels. We replaced them with new hTMEM119 staining images in Figure 5D.

      - In line 176, figure 5I has been forgotten to be mentioned.

      Thank you very much! We added 5I.

      - Lines 241 to 244 state that more than 50% of the AMD-associated genes are highly expressed in retinal microglia according to Fig. discussion suppl A & B. It is not clear that the gene set that was used for analysis is from a healthy retinal microglia or AMD-related ones. Please explain precisely.

      Thank you for your feedback. The gene list we referenced originates from a Genome-Wide Association Study (GWAS) that compared patients with Age-related Macular Degeneration (AMD) to healthy cohorts. We did not directly utilize this list in our experiments but referred to it to underscore the importance of microglia cells in the context of AMD.

      Some of the English proofreading and manuscript format comments:

      Line 805: Iba1 is written in lowercase. Is it human IBA1? It is not consistent with the way it is written in the text (in line 117, for example).

      Thank you for pointing out the error. We reformed all Iba1 as “Iba1”. The Iba1 we used here are all from Wako (#019–19741), which labels both mouse and human microglial cells.

      Line 814: microglia-enriched gene expression instead of microglia-enrich gene expression

      Thank you! We changed it.

      Line 345: Starting a sentence with lower case letter.

      Thank you! We changed it.

      Line 342: Myeloid lineage instead of myeloid cell linage.

      Thank you! We changed it.

      Line 815: What does FPKM stand for? The abbreviations should be explained.

      The FPKM is the abbreviation of Fragments Per Kilobase of transcript per Million mapped reads. We added it in the text.

      Line 309: The manuscript has occasionally referred to PLX-5622 without a minus. Please follow a uniform format.

      We changed all “PLX5622” to “PLX-5622”.

      Lines 327-331: should be rewritten.

      The mentioned paragraph was rewritten.

      Lines 335-340: should be rewritten.

      The mentioned sentence was rewritten.

      Line 135: qRT-PCR instead of QPCR," as it is also mentioned in the methods and material. The correction also applies to all the QPCRs in the text.

      We changed “QPCR” with “qRT-PCR”

      Figure 3: Graph B should be right side of graph A

      Images description: It is better to have the images description in the left side of the image, for example, figure 5 part B, GL, IPL and OPL

      Thanks for the suggestion. We changed the image organization as per the reviewer’s advice.

      Lines 258 to 260 in the discussion have also been repeated with the same words in the introduction.

      The mentioned paragraph was rewritten.

      Lines 327-331 should be rewritten.

      The mentioned paragraph was rewritten.

      Lines 335-340 should be rewritten.

      The mentioned paragraph was rewritten.

    1. Author Response

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

      Public Reviews:

      Reviewer #1 (Public Review):

      Qin et al. set out to investigate the role of mechanosensory feedback during swallowing and identify neural circuits that generate ingestion rhythms. They use Drosophila melanogaster swallowing as a model system, focusing their study on the neural mechanisms that control cibarium filling and emptying in vivo. They find that pump frequency is decreased in mutants of three mechanotransduction genes (nompC, piezo, and Tmc), and conclude that mechanosensation mainly contributes to the emptying phase of swallowing. Furthermore, they find that double mutants of nompC and Tmc have more pronounced cibarium pumping defects than either single mutants or Tmc/piezo double mutants. They discover that the expression patterns of nompC and Tmc overlap in two classes of neurons, md-C and md-L neurons. The dendrites of md-C neurons warp the cibarium and project their axons to the subesophageal zone of the brain. Silencing neurons that express both nompC and Tmc leads to severe ingestion defects, with decreased cibarium emptying. Optogenetic activation of the same population of neurons inhibited filling of the cibarium and accelerated cibarium emptying. In the brain, the axons of nompC∩Tmc cell types respond during ingestion of sugar but do not respond when the entire fly head is passively exposed to sucrose. Finally, the authors show that nompC∩Tmc cell types arborize close to the dendrites of motor neurons that are required for swallowing, and that swallowing motor neurons respond to the activation of the entire Tmc-GAL4 pattern.

      Strengths:

      • The authors rigorously quantify ingestion behavior to convincingly demonstrate the importance of mechanosensory genes in the control of swallowing rhythms and cibarium filling and emptying

      • The authors demonstrate that a small population of neurons that express both nompC and Tmc oppositely regulate cibarium emptying and filling when inhibited or activated, respectively

      • They provide evidence that the action of multiple mechanotransduction genes may converge in common cell types

      Thank you for your insightful and detailed assessment of our work. Your constructive feedback will help to improve our manuscript.

      Weaknesses:

      • A major weakness of the paper is that the authors use reagents that are expressed in both md-C and md-L but describe the results as though only md-C is manipulated-Severing the labellum will not prevent optogenetic activation of md-L from triggering neural responses downstream of md-L. Optogenetic activation is strong enough to trigger action potentials in the remaining axons. Therefore, Qin et al. do not present convincing evidence that the defects they see in pumping can be specifically attributed to md-C.

      Thank you for your comments. This is important point that we did not adequately address in the original preprint. We have obtained imaging and behavioral results that strongly suggest md-C, rather than md-L, are essential for swallowing behavior.

      36 hours after the ablation of the labellum, the signals of md-L were hardly observable when GFP expression was driven by the intersection between Tmc-GAL4 & nompC-QF (see F Figure 3—figure supplement 1A). This observation indicates that the axons of md-L likely degenerated after 36 hours, and were unlikely to influence swallowing. Moreover, the projecting pattern of Tmc-GAL4 & nompC-QF>>GFP exhibited no significant changes in the brain post labellum ablation.

      Furthermore, even after labellum ablation for 36 hours, flies exhibited responses to light stimulation (see Figure 3—figure supplement 1B-C, Video 5) when ReaChR was expressed in md-C. We thus reasoned that md-C but not md-L, plays a crucial role in the swallowing process.

      • GRASP is known to be non-specific and prone to false positives when neurons are in close proximity but not synaptically connected. A positive GRASP signal supports but does not confirm direct synaptic connectivity between md-C/md-L axons and MN11/MN12.

      In this study, we employed the nSyb-GRASP, wherein the GRASP is expressed at the presynaptic terminals by fusion with the synaptic marker nSyb. This method demonstrates an enhanced specificity compared to the original GRASP approach.

      Additionally, we utilized +/ UAS-nSyb-spGFP1-10, lexAop-CD4-spGFP11 ; + / MN-LexA fruit flies as a negative control to mitigate potential false signals originating from the tool itself (Author response image 1, scale bar = 50μm). Beside the genotype Tmc-Gal4, Tub(FRT. Gal80) / UAS-nSyb-spGFP1-10, lexAop-CD4-spGFP11 ; nompC-QF, QUAS-FLP / MN-LexA fruit flies discussed in this manuscript, we also incorporated genotype Tmc-Gal4, Tub(FRT. Gal80) / lexAop-nSyb-spGFP1-10, UAS-CD4-spGFP11 ; nompC-QF, QUAS-FLP / MN-LexA fruit flies as a reverse control (Author response image 2). Unexpectedly, similar positive signals were observed, indicating that, positive signals may emerge due to close proximity between neurons even with nSyb-GRASP.

      Author response image 1.

      It should be noted that the existence of synaptic projections from motor neurons (MN) to md-C cannot be definitively confirmed at this juncture. At present, we can only posit the potential for synaptic connections between md-C and motor neurons. A more conclusive conclusion may be attainable with the utilization of comprehensive whole-brain connectome data in future studies.

      Author response image 2.

      • As seen in Figure 2—figure supplement 1, the expression pattern of Tmc-GAL4 is broader than md-C alone. Therefore, the functional connectivity the authors observe between Tmc expressing neurons and MN11 and 12 cannot be traced to md-C alone

      It is true that the expression pattern of Tmc-GAL4 is broader than that of md-C alone. Our experiments, including those flies expressing TNT in Tmc+ neurons, demonstrated difficulties in emptying (Figure 2A, 2D). Notably, we encountered challenges in finding fly stocks bearing UAS>FRT-STOP-P2X2. Consequently, we opted to utilize Tmc-GAL4 to drive UAS-P2X2 instead. We believe that the results further support our hypothesis on the role of md-C in the observed behavioral change in emptying.

      Overall, this work convincingly shows that swallowing and swallowing rhythms are dependent on several mechanosensory genes. Qin et al. also characterize a candidate neuron, md-C, that is likely to provide mechanosensory feedback to pumping motor neurons, but the results they present here are not sufficient to assign this function to md-C alone. This work will have a positive impact on the field by demonstrating the importance of mechanosensory feedback to swallowing rhythms and providing a potential entry point for future investigation of the identity and mechanisms of swallowing central pattern generators.

      Reviewer #2 (Public Review):

      In this manuscript, the authors describe the role of cibarial mechanosensory neurons in fly ingestion. They demonstrate that pumping of the cibarium is subtly disrupted in mutants for piezo, TMC, and nomp-C. Evidence is presented that these three genes are co-expressed in a set of cibarial mechanosensory neurons named md-C. Silencing of md-C neurons results in disrupted cibarial emptying, while activation promotes faster pumping and/or difficulty filling. GRASP and chemogenetic activation of the md-C neurons is used to argue that they may be directly connected to motor neurons that control cibarial emptying.

      The manuscript makes several convincing and useful contributions. First, identifying the md-C neurons and demonstrating their essential role for cibarium emptying provides reagents for further studying this circuit and also demonstrates the important of mechanosensation in driving pumping rhythms in the pharynx. Second, the suggestion that these mechanosensory neurons are directly connected to motor neurons controlling pumping stands in contrast to other sensory circuits identified in fly feeding and is an interesting idea that can be more rigorously tested in the future.

      At the same time, there are several shortcomings that limit the scope of the paper and the confidence in some claims. These include:

      a) the MN-LexA lines used for GRASP experiments are not characterized in any other way to demonstrate specificity. These were generated for this study using Phack methods, and their expression should be shown to be specific for MN11 and MN12 in order to interpret the GRASP experiments.

      Thanks for the suggestion. We have checked the expression pattern of MN-LexA, which is similar to MN-GAL4 used in previous work (Manzo et al., PNAS., 2012, PMID:22474379) . Here is the expression pattern:

      Author response image 3.

      b) There is also insufficient detail for the P2X2 experiment to evaluate its results. Is this an in vivo or ex vivo prep? Is ATP added to the brain, or ingested? If it is ingested, how is ATP coming into contact with md-C neuron if it is not a chemosensory neuron and therefore not exposed to the contents of the cibarium?

      The P2X2 experimental preparation was done ex vivo. We immersed the fly in the imaging buffer, as described in the Methods section under Functional Imaging. Following dissection and identification of the subesophageal zone (SEZ) area under fluorescent microscopy, we introduced ATP slowly into the buffer, positioned at a distance from the brain

      c) In Figure 3C, the authors claim that ablating the labellum will remove the optogenetic stimulation of the md-L neuron (mechanosensory neuron of the labellum), but this manipulation would presumably leave an intact md-L axon that would still be capable of being optogenetically activated by Chrimson.

      Please refer to the corresponding answers for reviewer 1 and Figure 3—figure supplement 1.

      d) Average GCaMP traces are not shown for md-C during ingestion, and therefore it is impossible to gauge the dynamics of md-C neuron activation during swallowing. Seeing activation with a similar frequency to pumping would support the suggested role for these neurons, although GCaMP6s may be too slow for these purposes.

      Profiling the dynamics of md-C neuron activation during swallowing is crucial for unraveling the operational model of md-C and validating our proposed hypothesis. Unfortunately, our assay faces challenges in detecting probable 6Hz fluorescent changes with GCaMP6s.

      In general, we observed an increase of fluorescent signals during swallowing, but movement of alive flies during swallowing influenced the imaging recording, so we could not depict a decent tracing for calcium imaging for md-C neurons. To enhance the robustness of our findings, patching the md-C neurons would be a more convincing approach. As illustrated in Figure 2, the somata of md-C neurons are situated in the cibarium rather than the brain. patching of the md-C neuron somata in flies during ingestion is difficult.

      e) The negative result in Figure 4K that is meant to rule out taste stimulation of md-C is not useful without a positive control for pharyngeal taste neuron activation in this same preparation.

      We followed methods used in the previous work (Chen et al., Cell Rep., 2019, PMID:31644916), which we believe could confirm that md-C do not respond to sugars.

      In addition to the experimental limitations described above, the manuscript could be organized in a way that is easier to read (for example, not jumping back and forth in figure order).

      Thanks for your suggestion and the manuscript has been reorganized.

      Reviewer #3 (Public Review):

      Swallowing is an essential daily activity for survival, and pharyngo-laryngeal sensory function is critical for safe swallowing. In Drosophila, it has been reported that the mechanical property of food (e.g. Viscosity) can modulate swallowing. However, how mechanical expansion of the pharynx or fluid content sense and control swallowing was elusive. Qin et al. showed that a group of pharyngeal mechanosensory neurons, as well as mechanosensory channels (nompC, Tmc, and Piezo), respond to these mechanical forces for regulation of swallowing in Drosophila melanogaster.

      Strengths:

      There are many reports on the effect of chemical properties of foods on feeding in fruit flies, but only limited studies reported how physical properties of food affect feeding especially pharyngeal mechanosensory neurons. First, they found that mechanosensory mutants, including nompC, Tmc, and Piezo, showed impaired swallowing, mainly the emptying process. Next, they identified cibarium multidendritic mechanosensory neurons (md-C) are responsible for controlling swallowing by regulating motor neuron (MN) 12 and 11, which control filling and emptying, respectively.

      Weaknesses:

      While the involvement of md-C and mechanosensory channels in controlling swallowing is convincing, it is not yet clear which stimuli activate md-C. Can it be an expansion of cibarium or food viscosity, or both? In addition, if rhythmic and coordinated contraction of muscles 11 and 12 is essential for swallowing, how can simultaneous activation of MN 11 and 12 by md-C achieve this? Finally, previous reports showed that food viscosity mainly affects the filling rather than the emptying process, which seems different from their finding.

      We have confirmed that swallowing sucrose water solution activated md-C neurons, while sucrose water solution alone could not (Figure 4J-K). We hypothesized that the viscosity of the food might influence this expansion process.

      While we were unable to delineate the activation dynamics of md-C neurons, our proposal posits that these neurons could be activated in a single pump cycle, sequentially stimulating MN12 and MN11. Another possibility is that the activation of md-C neurons acts as a switch, altering the oscillation pattern of the swallowing central pattern generator (CPG) from a resting state to a working state.

      In the experiments with w1118 flies fed with MC (methylcellulose) water, we observed that viscosity predominantly affects the filling process rather than the emptying process, consistent with previous findings. This raises an intriguing question. Our investigation into the mutation of mechanosensitive ion channels revealed a significant impact on the emptying process. We believe this is due to the loss of mechanosensation affecting the vibration of swallowing circuits, thereby influencing both the emptying and filling processes. In contrast, viscosity appears to make it more challenging for the fly to fill the cibarium with food, primarily attributable to the inherent properties of the food itself.

      Reviewer #4 (Public Review):

      A combination of optogenetic behavioral experiments and functional imaging are employed to identify the role of mechanosensory neurons in food swallowing in adult Drosophila. While some of the findings are intriguing and the overall goal of mapping a sensory to motor circuit for this rhythmic movement are admirable, the data presented could be improved.

      The circuit proposed (and supported by GRASP contact data) shows these multi-dendritic neurons connecting to pharyngeal motor neurons. This is pretty direct - there is no evidence that they affect the hypothetical central pattern generator - just the execution of its rhythm. The optogenetic activation and inhibition experiments are constitutive, not patterned light, and they seem to disrupt the timing of pumping, not impose a new one. A slight slowing of the rhythm is not consistent with the proposed function.

      Motor neurons implicated in patterned motions can be considered effectors of Central Pattern Generators (CPGs)(Marder et al., Curr Biol., 2001, PMID: 11728329; Hurkey et al., Nature., 2023, PMID:37225999). Given our observation of the connection between md-C neurons and motor neurons, it is reasonable to speculate that md-C neurons influence CPGs. Compared to the patterned light (0.1s light on and 0.1s light off) used in our optogenetic experiments, we noted no significant changes in their responses to continuous light stimulation. We think that optogenetic methods may lead to overstimulation of md-C neurons, failing to accurately mimic the expansion of the cibarium during feeding.

      Dysfunction in mechanosensitive ion channels or mechanosensory neurons not only disrupts the timing of pumping but also results in decreased intake efficiency (Figure 1E). The water-swallowing rhythm is generally stable in flies, and swallowing is a vital process that may involve redundant ion channels to ensure its stability.

      The mechanosensory channel mutants nompC, piezo, and TMC have a range of defects. The role of these channels in swallowing may not be sufficiently specific to support the interpretation presented. Their other defects are not described here and their overall locomotor function is not measured. If the flies have trouble consuming sufficient food throughout their development, how healthy are they at the time of assay? The level of starvation or water deprivation can affect different properties of feeding - meal size and frequency. There is no description of how starvation state was standardized or measured in these experiments.

      Defects in mechanosensory channel mutants nompC, piezo, and TMC, have been extensively investigated (Hehlert et al., Trends Neurosci., 2021, PMID:332570000). Mutations in these channels exhibit multifaceted effects, as illustrated in our RNAi experiments (see Figure 2E). Deprivation of water and food was performed in empty fly vials. It's important to note that the duration of starvation determines the fly's willingness to feed but not the pump frequency (Manzo et al., PNAS., 2012, PMID:22474379).

      In most cases, female flies were deprived water and food in empty vials for 24 hours because after that most flies would be willing to drink water. The deprivation time is 12 hours for flies with nompC and Tmc mutated or flies with Kir2.1 expressed in md-C neurons, as some of these flies cannot survive 24h deprivation.

      The brain is likely to move considerably during swallow, so the GCaMP signal change may be a motion artifact. Sometimes this can be calculated by comparing GCaMP signal to that of a co-expressed fluorescent protein, but there is no mention that this is done here. Therefore, the GCaMP data cannot be interpreted.

      We did not co-express a fluorescent protein with GCaMP for md-C. The head of the fly was mounted onto a glass slide, and we did not observe significant signal changes before feeding.

      Recommendations for the authors:

      Reviewer #1 (Recommendations For The Authors):

      .>Abstract: I disagree that swallow is the first step of ingestion. The first paragraph also mentions the final checkpoint before food ingestion. Perhaps sufficient to say that swallow is a critical step of ingestion.

      Indeed, it is not rigorous enough to say “first step”. This has been replaced by “early step”.

      Introduction:

      Line 59: "Silence" should be "Silencing"

      This has been replaced.

      Results:

      Lines 91-92: I am not clear about what this means. 20% of nompC and 20% of wild-type flies exhibit incomplete filling? So nompC is not different from wild-type?

      Sorry for the mistake. Viscous foods led to incomplete emptying (not incomplete filling), as displayed in Video 4. The swallowing behavior differs between nompC mutants and wild-type flies, as illustrated in Figure 1C, Figure 1—figure supplement 1A-C and video 1&5.

      When fed with 1% MC water solution (Figure 1—figure supplement 1E-H). We found that when fed with 1% MC watere solution, Tmc or piezo mutants displayed incomplete emptying, which could constitute a long time proportion of swallowing behavior; while only 20% of nompC flies and 20% of wild-type flies sporadically exhibit incomplete emptying, which is significantly different. Though the percent of flies displaying incomplete pump is similar between nompC mutant and wild-type files, you can find it quite different in video 1 and 5.

      Line 94: Should read: “while for foods with certain viscosity, the pump of Tmc or piezo mutants might"

      What evidence is there for weakened muscle motion? The phenotypes of all three mutants is quite similar, so concluding that they have roles in initiation versus swallowing strength is not well supported -this would be better moved to the discussion since it is speculative.

      Muscles are responsible for pumping the bolus from the mouth to the crop. In the case of Tmc or piezo mutants, as evidenced by incomplete filling for viscous foods (see Video 4), we speculate that the loss of sensory stimuli leads to inadequate muscle contraction. The phenotypes observed in Tmc and piezo mutants are similar yet distinct from those of the wild-type or nompC mutant, as shown in Video 1 and 4. The phrase "due to weakened muscle motion" has been removed for clarity.

      Line 146: If md-L neurons are also labeled by this intersection, then you are not able to know whether the axons seen in the brain are from md-L or md-C neurons. Line 148: cutting the labellum is not sufficient to ablate md-L neurons. The projections will still enter the brain and can be activated with optogenetics, even after severing the processes that reside in the labellum.

      Please refer to the responses for reviewer #1 (Public Review):” A major weakness of the paper…” and Figure 4.

      Line 162: If the fly head alone is in saline, do you know that the sucrose enters the esophagus? The more relevant question here is whether the md-C neurons respond to mechanical force. If you could artificially inflate the cibarium with air and see the md-C neurons respond that would be a more convincing result. So far you only know that these are activated during ingestion, but have not shown that they are activated specifically by filling or emptying. In addition, you are not only imaging md-C (md-L is also labeled). This caveat should be mentioned.

      We followed the methods outlined in the previous work (Chen et al., Cell Rep., 2019, PMID:31644916), which suggested that md-C neurons do not respond to sugars. While we aimed to mechanically stimulate md-C neurons, detecting signal changes during different steps of swallowing is challenging. This aspect could be further investigated in subsequent research with the application of adequate patch recording or two-photon microscopy (TPM).

      Figure 3: It is not clear what the pie charts in Figure 3 A refer to. What are the three different rows, and what does blue versus red indicate?

      Figure 3A illustrates three distinct states driven by CsChrimson light stimulation of md-C neurons, with the proportions of flies exhibiting each state. During light activation, flies may display difficulty in filling, incomplete filling, or a normal range of pumping. The blue and red bars represent the proportions of flies showing the corresponding state, as indicated by the black line.

      Figure 4: Where are the example traces for J? The comparison in K should be average dF/F before ingestion compared with average dF/F during ingestion. Comparing the in vitro response to sucrose to the in vivo response during ingestion is not a useful comparison.

      Please refer to the answers for reviewer #2 question d).

      Reviewer #2 (Recommendations For The Authors):

      Suggested experiments that would address some of my concerns listed in the public review include:

      a) high resolution SEZ images of MN-LexA lines crossed to LexAop-GFP to demonstrate their specificity

      b) more detail on the P2X2 experiment. It is hard to make suggestions beyond that without first seeing the details.

      c) presenting average GCaMP traces for all calcium imaging results

      d) to rule out taste stimulation of md-C (Figure 4K) I would suggest performing more extensive calcium imaging experiments with different stimuli. For example, sugar, water, and increasing concentrations of a neutral osmolyte (e.g. PEG) to suppress the water response. I think that this is more feasible than trying to get an in vitro taste prep to be convincing.

      Please refer to the responses for public review of reviewer #2.

      Reviewer #3 (Recommendations For The Authors):

      Below I list my suggestions as well as criticisms.

      (1) It would be excellent if the authors could demonstrate whether varying levels of food viscosity affect md-C activation.

      That is a good point, and could be studied in future work.

      (2) It is not clear whether an intersectional approach using TMC-GAL4 and nompC-QF abolishes labelling of the labellar multidendritic neurons. If this is the case, please show labellar multidendritic neurons in TMC-GAL4 only flies and flies using the intersectional approach. Along with this question, I am concerned that labellum-removed flies could be used for feeding assay.

      Intersectional labelling using TMC-GAL4 and nompC-QF could not abolish labelling of the labellar multidendritic neurons (Author response image 4). Labellum-removed flies could be used for feeding assay (Figure 3—figure supplement 1B-C, video 5), but once LSO or cibarium of fly was damaged, swallowing behavior would be affected. Removing labellum should be very careful.

      Author response image 4.

      (3) Please provide the detailed methods for GRASP and include proper control.

      Please refer to the responses for public review of reviewer #1.

      (4) The authors hypothesized that md-C sequentially activates MN11 and 12. Is the time gap between applying ATP on md-C and activation of MN11 or MN12 different? Please refer to the responses for public review of reviewer #3. The time gap between applying ATP on md-C and activation of MN11 or MN12 didn’t show significant differences, and we think the reason is that the ex vivo conditions could not completely mimic in vivo process.

      I found the manuscript includes many errors, which need to be corrected.

      (1) The reference formatting needs to be rechecked, for example, lines 37, 42, and 43.

      (2) Line 44-46: There is some misunderstanding. The role of pharyngeal mechanosensory neurons is not known compared with chemosensory neurons.

      (3) Line 49: Please specify which type of quality of food. Chemical or physical?

      (4) Line 80 and Figure 1B-D Authors need to put filling and emptying time data in the main figure rather than in the supplementary figure. Otherwise, please cite the relevant figures in the text(S1A-C).

      (5) Line 84-85; Is "the mutant animals" indicating only nompC? Please specify it.

      (6) Figure 1a: It is hard to determine the difference between the series of images. And also label filling and emptying under the time.

      (7) S1E-H: It is unclear what "Time proportion of incomplete pump" means. Please define it.

      (8) Please reorganize the figures to follow the order of the text, for example, figures 2 and 4

      (9) Figure 4A. There is mislabelling in Figure 4A. It is supposed to be phalloidin not nc82.

      (10) Figure 4K: It does not match the figure legend and main text.

      (11) Figure 4D and G: Please indicate ATP application time point.

      Thanks for your correction and all the points mentioned were revised.

      Reviewer #4 (Recommendations For The Authors):

      The figures need improvement. 1A has tiny circles showing pharynx and any differences are unclear.

      The expression pattern of some of these drivers (Supplement) seems quite broad. The tmc nompC intersection image in Figure 1F is nice but the cibarium images are hard to interpret: does this one show muscle expression? What are "brain" motor neurons? Where are the labellar multi-dendritic neurons?

      Tmc nompC intersection image show no expression in muscles. Somata of motor neurons 12 or 11 situated at SEZ area of brain, while somata of md-C neurons are in the cibarium. Image of md-L neurons was posted in response for reviewer #3 (Recommendations For The Authors):

      Why do the assays alternate between swallowing food and swallowing water?

      Thank for your suggestion, figure 1A has been zoomed-in. The Tmc nompC intersection image in Figure 2F displayed the position of md-C neurons in a ventral perspective, and muscles were not labelled. We stained muscles in cibarium by phalloidin and the image is illustrated in Figure 4A, while we didn’t find overlap between md-C neurons and muscles. Image of md-L neurons were posted as Author response image 4.

      In the majority of our experiments, we employed water to test swallowing behavior, while we used methylcellulose water solution to test swallowing behavior of mechanoreceptor mutants, and sucrose solution for flies with md-C neurons expressing GCaMP since they hardly drank water when their head capsules were open.

      How starved or water-deprived were the flies?

      One day prior to the behavioral assays, flies were transferred to empty vials (without water or food) for 24 hours for water deprivation. Flies who could not survive 24h deprivation would be deprived for 12h.

      How exactly was the pumping frequency (shown in Fig 1B) measured? There is no description in the methods at all. If the pump frequency is scored by changes in blue food intensity (arbitrary units?), this seems very subjective and maybe image angle dependent. What was camera frame rate? Can it capture this pumping speed adequately? Given the wealth of more quantitative methods for measuring food intake (eg. CAFE, flyPAD), it seems that better data could be obtained.

      How was the total volume of the cibarium measured? What do the pie charts in Figure 3A represent?

      The pump frequency was computed as the number of pumps divided by the time scale, following the methodology outlined in Manzo et al., 2012. Swallowing curves were plotted using the inverse of the blue food intensity in the cibarium. In this representation, ascending lines signify filling, while descending lines indicate emptying (see Figure 2D, 3B). We maintain objectivity in our approach since, during the recording of swallowing behavior, the fly was fixed, and we exclusively used data for analysis when the Region of Interest (ROI) was in the cibarium. This ensures that the intensity values accurately reflect the filling and emptying processes. Furthermore, we conducted manual frame-by-frame checks of pump frequency, and the results align with those generated by the time series analyzer V3 of ImageJ.

      For the assessment of total volume of ingestion, we referred the methods of CAFE, utilizing a measurable glass capillary. We then calculated the ingestion rate (nL/s) by dividing the total volume of ingestion by the feeding time.

      The changes seem small, in spite of the claim of statistical significance.

      The observed stability in pump frequency within a given genotype underscores the significance of even seemingly small changes, which is statistically significant. We speculate that the stability in swallowing frequency suggests the existence of a redundant mechanism to ensure the robustness of the process. Disruption of one channel might potentially be partially compensated for by others, highlighting the vital nature of the swallowing mechanism.

      How is this change in pump frequency consistent with defects in one aspect of the cycle - either ingestion (activation) or expulsion (inhibition)?

      Please refer to Figure 2, 3. Both filling and emptying process were affects, while inhibition mainly influences emptying time (Figure 1—figure supplement 1).

      for the authors:

      Line 48: extensively

      Line 62 - undiscovered.

      Line 107, 463: multi

      Line 124: What is "dysphagia?" This is an unusual word and should be defined.

      Line 446: severe

      Line 466: in the cibarium or not?

      Thanks for your correction and all the places mentioned were revised.

    1. Reviewer #2 (Public Review):

      The goal of the present study is to better understand the 'control objectives' that subjects adopt in a video-game-like virtual-balancing task. In this task, the hand must move in the opposite direction from a cursor. For example, if the cursor is 2 cm to the right, the subject must move their hand 2 cm to the left to 'balance' the cursor. Any imperfection in that opposition causes the cursor to move. E.g., if the subject were to move only 1.8 cm, that would be insufficient, and the cursor would continue to move to the right. If they were to move 2.2 cm, the cursor would move back toward the center of the screen. This return to center might actually be 'good' from the subject's perspective, depending on whether their objective is to keep the cursor still or keep it near the screen's center. Both are reasonable 'objectives' because the trial fails if the cursor moves too far from the screen's center during each six-second trial.

      This task was recently developed for use in monkeys (Quick et al., 2018), with the intention of being used for the study of the cortical control of movement, and also as a task that might be used to evaluate BMI control algorithms. The purpose of the present study is to better characterize how this task is performed. What sort of control policies are used. Perhaps more deeply, what kind of errors are those policies trying to minimize? To address these questions, the authors simulate control-theory style models and compare with behavior. They do in both in monkeys and in humans.

      These goals make sense as a precursor to future recording or BMI experiments. The primate motor-control field has long been dominated by variants of reaching tasks, so introducing this new task will likely be beneficial. This is not the first non-reaching task, but it is an interesting one and it makes sense to expand the presently limited repertoire of tasks. The present task is very different from any prior task I know of. Thus, it makes sense to quantify behavior as thoroughly as possible in advance of recordings. Understanding how behavior is controlled is, as the authors note, likely to be critical to interpreting neural data.

      From this perspective - providing a basis for interpreting future neural results - the present study is fairly successful. Monkeys seem to understand the task properly, and to use control policies that are not dissimilar from humans. Also reassuring is the fact that behavior remains sensible even when task-difficulty become high. By 'sensible' I simply mean that behavior can be understood as seeking to minimize error: position, velocity, or (possibly) both, and that this remains true across a broad range of task difficulties. The authors document why minimizing position and minimizing velocity are both reasonable objectives. Minimizing velocity is reasonable, because a near-stationary cursor can't move far in six seconds. Minimizing position error is reasonable, because the trial won't fail if the cursor doesn't stray far from the center. This is formally demonstrated by simulating control policies: both objectives lead to control policies that can perform the task and produce realistic single-trial behavior. The authors also demonstrate that, via verbal instruction, they can induce human subjects to favor one objective over the other. These all seem like things that are on the 'need to know' list, and it is commendable that this amount of care is being taken before recordings begin, as it will surely aid interpretation.

      Yet as a stand-alone study, the contribution to our understanding of motor control is more limited. The task allows two different objectives (minimize velocity, minimize position) to be equally compatible with the overall goal (don't fail the trial). Or more precisely, there exists a range of objectives with those two at the extreme. So it makes sense that different subjects might choose to favor different objectives, and also that they can do so when instructed. But has this taught us something about motor control, or simply that there is a natural ambiguity built into the task? If I ask you to play a game, but don't fully specify the rules, should I be surprised that different people think the rules are slightly different?

      The most interesting scientific claim of this study is not the subject-to-subject variability; the task design makes that quite likely and natural. Rather, the central scientific result is the claim that individual subjects are constantly switching objectives (and thus control policies), such that the policy guiding behavior differs dramatically even on a single-trial basis. This scientific claim is supported by a technical claim: that the authors' methods can distinguish which objective is in use, even on single trials. I am uncertain of both claims.

      Consider Figure 8B, which reprises a point made in Figure 1&3 and gives the best evidence for trial-to-trial variability in objective/policy. For every subject, there are two example trials. The top row of trials shows oscillations around the center, which could be consistent with position-error minimization. The bottom row shows tolerance of position errors so long as drift is slow, which could be consistent with velocity-error minimization. But is this really evidence that subjects were switching objectives (and thus control policies) from trial to trial? A simpler alternative would be a single control policy that does not switch, but still generates this range of behaviors. The authors don't really consider this possibility, and I'm not sure why. One can think of a variety of ways in which a unified policy could produce this variation, given noise and the natural instability of the system.

      Indeed, I found that it was remarkably easy to produce a range of reasonably realistic behaviors, including the patterns that the authors interpret as evidence for switching objectives, based on a simple fixed controller. To run the simulations, I made the simple assumption that subjects simply attempt to match their hand position to oppose the cursor position. Because subjects cannot see their hand, I assumed modest variability in the gain, with a range from -1 to -1.05. I assumed a small amount of motor noise in the outgoing motor command. The resulting (very simple) controller naturally displayed the basic range of behaviors observed across trials (see Image 1)

      Peer review image 1.

      Some trials had oscillations around the screen center (zero), which is the pattern the authors suggest reflects position control. In other trials the cursor was allowed to drift slowly away from the center, which is the pattern the authors suggest reflects velocity control. This is true even though the controller was the same on every trial. Trial-to-trial differences were driven both by motor noise and by the modest variability in gain. In an unstable system, small differences can lead to (seemingly) qualitatively different behavior on different trials.

      This simple controller is also compatible with the ability of subjects to adapt their strategy when instructed. Anyone experienced with this task likely understands (or has learned) that moving the hand slightly more than 'one should' will tend to shepherd the cursor back to center, at the cost of briefly high velocity. Using this strategy more sparingly will tend to minimize velocity even if position errors persist. Thus, any subject using this control policy would be able to adapt their strategy via a modest change in gain (the gain linking visible cursor position to intended hand position).

      This model is simple, and there may be reasons to dislike it. But it is presumably a reasonable model. The nature of the task is that you should move your hand opposite where the cursor is. Because you can't see your hand, you will make small mistakes. Due to the instability of the system, those small mistakes have large and variable effects. This feature is likely common to other controllers as well; many may explicitly or implicitly blend position and velocity control, with different trials appearing more dominated by one versus the other. Given this, I think the study presents only weak evidence that individual subjects are switching their objective on individual trials. Indeed, the more parsimonious explanation may be that they aren't. While the study certainly does demonstrate that the control policy can be influenced by verbal instructions, this might be a small adjustment as noted above.

      I thus don't feel convinced that the authors can conclusively tell us the true control policy being used by human and monkey subjects, nor whether that policy is mostly fixed or constantly switching. The data are potentially compatible with any of these interpretations, depending on which control-style model one prefers.

      I see a few paths that the authors might take if they chose.<br /> --First, my reasoning above might be faulty, or there might be additional analyses that could rule out the possibility of a unified policy underlying variable behavior. If so, the authors may be able to reject the above concerns and retain the present conclusions. The main scientifically novel conclusion of the present study is that subjects are using a highly variable control policy, and switching on individual trials. If this is indeed the case, there may be additional analyses that could reveal that.<br /> --Second, additional trial types (e.g., with various perturbations) might be used as a probe of the control policy. As noted below, there is a long history of doing this in the pursuit system. That additional data might better disambiguate control policies both in general, and across trials.<br /> --Third, the authors might find that a unified controller is actually a good (and more parsimonious) explanation. Which might actually be a good thing from the standpoint of future experiments. Interpretation of neural data is likely to be much easier if the control policy being instantiated isn't in constant flux.

      In any case, I would recommend altering the strength of some conclusions, particularly the conclusion that the presented methods can reliably discriminate amongst objectives/policies on individual trials. This is mentioned as a major motivation on multiple occasions, but in most of these instances, the subsequent analysis infers the objective only across trial (e.g., one must observe a scatterplot of many trials). By Figure 7, they do introduce a method for inferring the control policy on individual trials, and while this seems to work considerably better than chance, it hardly appears reliable.

      In this same vein I would suggest toning down aspects of the Introduction and Discussion. The Introduction in particular is overly long, and tries to position the present study as unique in ways that seem strained. Other studies have built links between human behavior, monkey behavior, and monkey neural data (for just one example, consider the corpus of work from the Scott lab that includes Pruszynski et al. 2008 and 2011). Other studies have used highly quantitative methods to infer the objective function used by subjects (e.g. Kording and Wolpert 2004). The very issue that is of interest in the present study - velocity-error-minimization versus position-error-minimization - has been extensively addressed in the smooth pursuit system. That field has long combined quantitative analyses of behavior in humans and monkeys, along with neural recordings. Many pursuit experiments used strategies that could be fruitfully employed to address the central questions of the present study. For example, error stabilization was important for dissecting the control policy used by the pursuit system. By artificially stabilizing the error (position or velocity) at zero, or at some other value, one can determine the system's response. The classic Rashbass step (1961) put position and velocity errors in opposition, to see which dominates the response. Step and sinusoidal perturbations were useful in distinguishing between models, as was the imposition of artificially imposed delays. The authors note the 'richness' of the behavior in the present task, and while one could say the same of pursuit, it was still the case that specific and well-thought through experimental manipulations were pretty critical. It would be better if the Introduction considered at least some of the above-mentioned work (or other work in a similar vein). While most would agree with the motivations outlined by the authors - they are logical and make sense - the present Introduction runs the risk of overselling the present conclusions while underselling prior work.

    1. Author Response

      Reviewer #1 (Public Review):

      The manuscript, "A versatile high-throughput assay based on 3D ring-shaped cardiac tissues generated from human induced pluripotent stem cell-derived cardiomyocytes" developed a unique culture platform with PEG hydrogel that facilitates the in-situ measurement of contractile dynamics of the engineered cardiac rings. The authors optimized the tissue seeding conditions, demonstrated tissue morphology with expressions of cardiac and fibroblast markers, mathematically modeled the equation to derive contractile forces and other parameters based on imaging analysis, and ended by testing several compounds with known cardiac responses.

      To strengthen the paper, the following comments should be considered:

      1) This paper provided an intriguing platform that creates miniature cardiac rings with merely thousands of CMs per tissue in a 96-well plate format. The shape of the ring and the squeezing motion can recapitulate the contraction of the cardiac chamber to a certain degree. However, Thavandiran et al (PNAS 2013) created a larger version of the cardiac ring and found the electrical propagation revealed spontaneous infinite loop-like cycles of activation propagation traversing the ring. This model was used to mimic a reentrant wave during arrhythmia. Therefore, it presents great concerns if a large number of cardiac tissues experience arrhythmia by geometry-induced re-entry current and cannot be used as a healthy tissue model. It would be interesting to see the impulse propagation/calcium transient on these miniature cardiac rings and evaluate the % of arrhythmia occurrence.

      The size is a key factor impacting the electrical propagation within the generated tissues. Our ring-shaped cardiac tissues have a diameter of 360µm, which is largely smaller than other tissues proposed so far, including in Thavandiran et al (PNAS 2013) where circular tissues had a reported size > 1mm. As shown in Figure 4E (and highlighted below in Author response image 1), tissues under basal conditions display regular beating rates without spontaneous arrhythmias. Videos also show that the tissue contraction is homogeneous around the pillar, suggesting that the smaller size favors the electrical propagation and limits the occurrence of spontaneous reentrant waves. Optical mapping measurements will be performed in the future to assess the occurrence of reentrant waves.

      **Author response image 1. **

      Poincaré plot showing the plots between successive RR intervals (Data from Figure 4E in basal conditions). Linear regression with 95% confidence interval indicates identity.

      2) The platform can produce 21 cardiac rings per well in 96-well plates. The throughput has been the highest among competing platforms. The resulting tissues have good sarcomere striation due to the strain from the pillars. Now the emerging questions are culture longevity and reproducibility among tissues. According to Figure 1E, there was uneven ring formation around the pillar, which leads to the tissue thinning and breaking off. There is only 50% survival after 20 days of culture in the optimized seeding group. Is there any way to improve it? The tissues had two compartments, cardiac and fibroblast-rich regions, where fibroblasts are responsible for maintaining the attachment to the glass slides. Do the cardiac rings detach from the glass slides and roll up? The SD of the force measurement is a quarter of the value, which is not ideal with such a high replicate number. As the platform utilizes imaging analysis to derive contractile dynamics, calibration should be done based on the angle and the distance of the camera lens to the individual tissues to reduce the error. On the other hand, how reproducible of the pillars? It is highly recommended to mechanically evaluate the consistency of the hydrogel-based pillars across different wells and within the wells to understand the variance. Figure 2B reports the early results obtained as the system was tested and developed. Since then, we have tested different iPSC lines and confirm that the overall yield is higher (up to 20 tissues at D14 for some cell lines), however dependent of cell lines.

      The tissues do not detach from the glass slides. It is very rare to see tissues roll up on the central pillar. As shown in Figure 1B, the pillars have a specific shape to avoid tissues to roll up as they develop and contract.

      3) Does the platform allow the observation of non-synchronized beating when testing with compounds? This can be extremely important as the intended applications of this platform are drug testing and cardiac disease modeling. The author should elaborate on the method in the manuscript and explain the obtained results in detail. The arrhythmogenic effect of a drug can be derived from the regularity of the beat-to-beat time. Indeed, we show that dofetilide increases the variability in the beat-to-beat time by plotting for each beat, the beat-to-beat time with the next beat as a function of the beat-to-beat time with the previous beat.

      4) The results of drug testing are interesting. Isoproterenol is typically causing positive chronotropic and positive inotropic responses, where inotropic responses are difficult to obtain due to low tissue maturity. It is inconsistent with other reported results that cardiac rings do not exhibit increased beating frequency, but slightly increased forces only. Zhao et al were using electrical pacing at a defined rate during force measurement, whereas the ring constructs are not.

      We agree. The difference in the response to isoproterenol with previous papers may be explained by different incubation timing with the drug. In our case, the tissues were incubated for 5 minutes at 37•C before being recorded.

      Overall, the manuscript is well written and the designed platform presented the unique advantages of high throughput cardiac tissue culture. Besides the contractile dynamics and IHC images, the paper lacks other cardiac functional evaluations, such as calcium handling, impulse propagation, and/or electrophysiology. The culture reproducibility (high SD) and longevity (<20 days) still remain unsolved.

      Since the submission, we have managed to keep some tissues and analyze them up to 32 days. At that time point the tissues are still beating. Nevertheless, a specific study concerning tissue longevity has not been carried out as the tissues were usually fixed after 14 days to be stained and analyze their structure.

      Reviewer #2 (Public Review):

      The authors should be commended for developing a high throughput platform for the formation and study of human cardiac tissues, and for discussing its potential, advantages and limitations. The study is addressing some of the key needs in the use of engineered cardiac tissues for pharmacological studies: ease of use, reproducible preparation of tissues, and high throughput.

      There are also some areas where the manuscript should be improved. The design of the platform and the experimental design should be described in more detail.

      It would be of interest to comprehensively document the progression of tissue formation. To this end, it would be helpful to show the changes in tissue structure through a series of images that would correspond to the progression of contractile properties shown in Figure 3.

      Our results indicate that the fibroblasts/cardiomyocytes segregation likely happens as soon as the tissue is formed, as the fibroblasts are critical for tissue generation. The change with time in the shape of the contractile ring is reported in Figure 1E, with a series of images which correspond to the timepoints of Figure 3.

      The very interesting tissue morphology (separation into the two regions) that was observed in this study is inviting more discussion.

      Finally, the reader would benefit from more specific comparisons of the contractile function of cardiac tissues measured in this study with data reported for other cardiac tissue models.

    1. All praise is due to Allah, the Powerful, the King, the Almighty, theInvisible, the Creator of day and night and the orbiting spheres, theRestorer of the land, the Bountiful.

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