How were political climates so drastically impacted by the physical changes of their environment? Did communities lose trust in their leaders judgement when the stability of their resources fell short? Or, was there spiritual reasoning behind their political decisions?

This reminds me of how platforms like YouTube have changed over time. Originally a space for independent creators to share their work with the public, with few restrictions or parameters, it has now become a game of pandering to the algorithm. As we've discussed in class, YouTube has become one of the primary tools used for things like the alt-right pipeline. It has become an echo chamber, because of how YouTube has changed its delivery methods. The pressures creators are under to pander to specific streams of consumers has synthesized (especially political) content into two streams, as nuance depletes. Part of this problem began with the need to please advertisers, so as not to get demonetized, then continued as YouTube changed its delivery methods.

This fascinates me. We need a mechanism to regulate AI agents and the 'slop code' they are dropping like manure all over the net. If the international community can develop large-scale organizations to help regulate territorial integrity (the United Nations; though something of a failed model), why can't something similar happen for the Internet in this most fraught AI era? The Wild West was eventually tamed and brought to heel - perhaps the net can be also? I'm probably being too optimistic...

I'd like to think we live in a just democratic society with freedom of speech, but lately those feelings (mostly talking in general from the collective population feelings, not all personal) have been challenged due to the ways in which news channels and social media try to divide people, and the ways that "Big Business" are (for lack of better words) taking over politics. I can see why many people I meet are losing trust in the world and government because of this radicalization. On Meta socials like instagram and facebook, what you can view and post has been more and more censored in recent years. I think it is good and bad to an extent, but also contradictory that, for example, certain sources are limited such as news on meta that are rooted in fact and science (e.g. climate science news/research), but then peoples personal opinions that are rooted in racism, prejudice and sexism are allowed to exist in the comment section or in private groups/websites. Hate crimes and hate speech have statistically increased in recent years due to increased numbers in this content online which is very upsetting. I don't usually rant, but overall I guess my point is that I think it is important to think about censorship in today's society as well as the social-political problems that arise from having too much access to people's opinions.

I think this is becoming a bigger trend in the way people are thinking about technology and the media. Technology is getting so complicated that sometimes it feels like we are creating new things just because we can, not because we actually need them. Eventually, i think people might reach a point where they just don't want/need constant advancements, especially when more research continues to show potential negative effects to our health and well-being. Perhaps at some point having the option to step away from technology might become just as valuable as having access to the newest advancements.

I do not sympathize with AI companies on any front, but there is something to be said about content uploaded on the internet. While I do not agree that AI models taking and regurgitating content is ethical, there seems to be some understanding that once you release any form of content on the internet, you lose your right to how your content will be used, for better or for worse. While in a pre-AI era this would mostly imply other's reusing or paraphrasing your ideas without credit, these are individual users and the overall impact would be low, compared to now, where AI models could be releasing the content to millions everyday. Regardless, although I do not agree it is right, it seems the AI are not acting any differently than how an individual user might act, just on a greater scale.

I agree with the idea that technology itself is not the overall problem. I don't believe technologies are inherently good or bad. Ultimately it comes down to how they are used, who has control over them, and what intentions they are being used for. When considering AI, I think it can can contribute to many problems we see today, but it also has the potential to create opportunities to support a more open and accessible web. It all depends on how it's developed and used.

I find this opinion to be slightly over optimistic with a David versus Goliath sort of stance. The writer is suggesting that we, the common people, have a card to play in this game and power to exert. I would think that the choices and decision made by billionaires and BigTech companies are not easily swayed by the opinions of the working class, especially when they decisions are often supported or possibly funded by political powers. Although the sentiment is nice to read, I am unsure how attainable the end goal is for community of common people when it comes to battling the leading industry. That being said, I do not disagree that we should use any joint power we have to stop decisions that affect our way of life.

I agree with this section overall, however I do think the phrase "at one time" feels a little too optimistic. Today, it still seems incredibly difficult and arguably impossible to imagine large enough communities overcoming the extreme wealth and power surrounding these technologies. I think the point of this paragraph is to suggest that what feels impossible today might not always be impossible, however I think we are still very much living in that "impossible" moment, and it will take a huge shift in how technologies are developed and controlled.

This is also applicable to a person's likeness. AI uses images of people on the internet, who may not know that have given consent, to create images of new people. This creates a threat to jobs like acting or modeling. The potential use of a person's image, posted to the internet via Instagram or Facebook, in an AI prompts, without the person's consent is a common fear. It poses a threat to individual people, as well as the future validity of things like photo evidence.

I think creators who are choosing to stay independent and in control of their own content are really refreshing. Wealth and/or recognition would be a convincing reason for most to shift towards open web publishing. However, I believe that maintaining authority over your work and staying true to what belongs to you is more valuable than money.

This is a concern because it allows companies to profit from community-built code without acknowledging the people who created it. When tools are trained on open‑source work and then used to commercialize that same work, it raises serious questions about consent.

I'm curious about what this could look like. Considering the wide variety of factors that have rendered AI a problematic tool, what policies could be put in place to reduce harm enough? Will greed always interfere with good intention, even if a solution is found eventually?

This section reminds of a kerfuffle from a year or so ago. YouTube finally started to crackdown on the use of Ad Blockers. I'm personally a big fan of them. A large section of the community banded together and continually worked on free patches and updates to keep their ad blockers functional -- likewise, the community would repeatedly share when updates were available. Likewise, they would share when certain blockers stopped working, and pointed users towards whichever was currently functional.

This could be a hot take but this could be the end of genuine creativity. If the mass public succumbs to the ease of AI tools, capacity for deep and original thought will dissipate. Having to feed the algorithm and play to what you think others want just to get by will turn into a vicious cycle and mounds of similar content. Scary thought.

I think this can be extremely frustrating, as the internet should be used as a way to have information at your finger tips. The internet is a way for people world wide to educate themselves, and learn information that may not be easily accessible for them in person. Having access to the internet, we as a generation have been fortunate enough to be able to learn things by not having to go to the library and do the research ourselves. By putting a lot of human written information behind paywalls and passwords, makes the information that was previously easily accessible, less affordable for people who may have heavily relied on the internet for free education to expand their knowledge and education in an affordable way.

I think the term "good AI" is interesting. I think in order to achieve a "good AI" is an ongoing process due to the vast unknowns of the future of AI. Therefore I think that the creation of AI needs to be nurtured in the same way as how we nurture our students and children. Humans create these AI's as tools, but it shouldn't just stop there and let them roam free without any guidance. As AI progresses, I think it is important for us to keep it on the right course. Since the future of AI is very unknown, we need to try and raise it in a way that we don't lose control over it. We need to show it how to be good and do good.

I think this is a really interesting point of view. Just like anything on our planet, AI can be used for good and to repair damage just as much as it can be a destructive tool. One thing I do struggle with is having hope that this can happen since it is ultimately up to those in power to decide how these tools will be exploited.

This reminds me of the quote we read in the first class talking about knowledge in the future being a resource like water and food. A very scary thought that I see becoming more real if AI keeps trending in the direction that it is on right now. Putting a price on knowledge is like putting a price on oxygen in my opinion, if knowledge does become a resource, what other human necessities will follow?

I'm intimately familiar with Creative Commons licensing; all of my hobby writing work is posted under CC BY-SA 3.0 so it can be built upon by others in a collaborative fiction project. I love that CC was brought up in class as an example of a "social contract", but it's essential to acknowledge also that it's legally binding. Creative Commons licensing is protected by copyright law in North America and the other nations that observe it, but as the article says: if society is not willing to enforce those legalities, and the government is not incentivized to protect these legal policies, then the law holds no weight. Even in Teacher's College, I have seen all of the principles of academic content reuse ignored by some professors. In Pedagogy of the Arts last year, we were actively required to take educational resources wholesale from the internet and submit them, without any transformative usage of the resources, for a culminating assessment. Teacher candidates are actively being encouraged to violate copyright law by instructors who don't understand what they're doing.

Seeing the way the internet has changed throughout my life has been somewhat alarming. Although the majority of changes have been positive and very useful, there are some ways that it has changed for the worse. AI is a very useful tool is used ethically but it seems that there has been a plethora of harmful side effects since it's exponential growth.

I can speak to this personally, seeing this happen IRL. A business I worked with used an open software to help streamline their warehouse management systems. When automated agents and LLM submissions came into play the amount of sifting, managing correct submissions, and TIME was immense which leads to an intense amount of collateral damage. Specifically HUMAN time spent on corrections is completely disabling and makes these platforms, and software unusable for others.

If we are comparing this to education; It feels similar to sifting through the noise with your students. Helping students using their critical thinking skills and what could be categorized as hallow and what is genuine to knowledge building.

I'm not fully familiar with all the players that operate in these spaces, but as far as I know, many of the players in the open ecosystem are not heavily funded. Majority of the players in the open ecosystem are small groups or individuals , which means they will not have the funding to compete with the corporations that are funding the LLMs and profiting greatly off of them. Moreover, due to the nature of an open ecosystem, many of the players may even be at odds with each other or carry differing opinions on how to tackle this issue. This suggests that any sort of collective effort to combat this will be difficult in the open internet ecosystem due to lack of cohesion

I find AI Slop is becoming a much more prominent problem on YouTube. I'm old enough to remember YouTube being launched in 2005. Watching the progression of personality made content to AI generated content has been sad and terrifying to say the least. Though, I'm unsure, I think there are also some monetary connotations to this with things like Ad Sense. What exactly is the protocol in place for users using AI to create content and collect ad revenue?

During my grade 10 civics and careers placement I had the opportunity to look more into large language models (LLM's). I think it is very sad that these bots are able to take someones content that they posted for free and in matter of seconds the bots take a summary for the information. I think it is sad that these days many consumers of the content just rely on the AI bots to give them the information. I remember when we didn't have the AI summary when we first searched something up and had to actually search for our answers and now it just pops out a summary.

This comment really stood out to me because I agree that we often take the open internet for granted without thinking about how much we rely on it every day... Honestly, I never really think about how easily I access things like Google and other independent websites, and I rarely stop to think about the people and communities that helped make those resources available in the first place... So, after reading the earlier examples in the article, I found myself wondering what the internet might look like if those open spaces continue to be replaced or controlled by large companies who evidently make millions of dollars off of the work and information that others have made freely available for the benefit of others.

I have found that Wikipedia is something that I used to use in high school and through my first university degree a lot. Even if you couldn't use it as a source you could always use to create an idea of what you wanted and use it's sources as your own. AI are creating shortcuts by training its AI on this vital source of information. Wikipedia is a free website that relies on users and donations to stay afloat. Less traffic means less donation which means less donations (as the article states) which threatens Wikipedia as a whole.

When these AI models are learning information, but not keeping a connection back to the original content where they learnt it from it can be incredibly harmful, especially for AI novices, to be able to discern whether the information they are getting from AI is reliable. Especially in a classroom setting, students are often looking for the quick answer, which AI does provide, but if there are no links to the credible sources where they have got this information from it can cause students to just pull that information without a second thought. It is incredibly important as teachers that we try to relay the importance of looking for information from reliable and credible sources.

I think this goes far beyond money. The end goal of AI in my opinion is ultimately to supplant the need for a working class. To get there, the investors have no qualms about further devastating our natural world and quality of life. Perhaps I am a pessimist, but I cannot see our current trajectory leading somewhere which ends in the mass enlightenment many AI advocates profess.

An intriguing hyperbole, but one must interrogate the reasoning behind the uber-rich fixating and lobbying for AI integration. Altman's OpenAI is far from profitable, literally hemorrhaging money, and many other companies are in a constant state of free fall, only staying afloat with expensive stipends from these rich parties.

At risk of sounding political, it seems that this focus on developing LLMs is more focused on control rather than profit. With Anthropic chopping up rare books to feed to Claude, it's pretty cut and dry. The fiery furnace runs on information and water, but will any gods intervene to save us from the blaze?

I find this point very interesting. It reminds me of the earlier days of the internet when I was younger, when the internet was used more as a positive tool and less of the negative impacts were seen. I like the idea of future generations getting to use the internet like it once was used before the negatives outweighed the positives.

I find this an interesting thought because the whole idea of the open web is to allow anybody to publish information for others to see and contribute to. Creating rival GenAI to fight against "Big Tech" would just be feeding into the problem and destroying the open web further. I'm interested to know what strategies are available for open ecosystem players to use against big tech and GenAI.

As websites where AI trains its content begin to disappear from the web, where will AI get its training from? I feel like this could become an ouroboros situation of AI training on AI content (which has already occurred). Information may become increasingly distorted as the years go by. Its more imperative now to make sure these websites stay alive in the upcoming years where information was created/sourced by real people

found this comment on power being the driving force behind AI very interesting. It is evident that the power provided by LLMs is extremely attractive given how it has been shoehorned into almost all aspects of modern technology. With AI currently in a bubble I wonder how i large authoritarian players like whether it be tech companies or governments will continue to implement AI in the future. It seems more and more that LLMs are meant to be used as a tool that can be used to potentially withhold or control public knowledge. The quote from Sam Altman that we were shown in class where he mentions trying to make intelligence a service fee similar to electricity was Alarming!

So: I've been meaning to donate to Wikipedia for a long time. I have used it frequently for nearly twenty years and consider it one of the last and most crucial bastions of unbiased and well-written truth in the current societal moment. While I have seen Wikipedia's messaging asking for donations many times, I am often too preoccupied by my own research pursuits to get around to donating. Reading this passage was a real call to action for me; learning that Wikipedia isn't just struggling to acquire donations to the extent that any non-profit does, but that it is actually being targeted for destruction by corporations in the information age, spurred me to pause my reading of this article and immediately make a donation. Wikimedia Commons provides a wealth of essential resources, including CC and Public Domain assets I have used in academic and creative projects. To learn that its use is being supplanted by LLMs to a harmful extent is truly disheartening.

I think with widespread AI we tend to only think of the larger industry displacement. AI hurts individuals who make a living off of niche jobs. The ability to code websites, pull data, and automate entry of data in seconds will put hardworking people out of business. I wonder how job markets will shift, or if it will largely cease to exist.

The Internet Archive is particularly useful in keeping individuals accountable for their actions. For example, in the upcoming US midterm elections, candidates are changing their websites with new stances completely different from before. We need these kinds of websites. But, I am a bit skeptical of the volunteering/donation route. These feel like short term solutions to the fight. Without proper political/institutional support, these websites will continue to struggle by themselves.

A concerning trend I have been seeing in my students and friends is that they typically Google search something and immediately rely on the Gemini AI-generated response without validating the information from actual websites or other sources. This article discusses how AI platforms train their content on Wikipedia—which, classically, we have been told in school, is not a reliable source, due to its reliance on volunteer contributions—and thus, I think that if these AI-bots don't distinguish between reliable sources and non-reliable sources on the internet, AI content will remain not entirely trustworthy. I wonder how/if AI companies are training their AI to ensure that they only train from reliable content? I know that some companies such as Outlier have been hiring AI-trainers to correct the AI responses in niche subjects.

This is an excellent observation and a hugely problematic element of the AI-beleaguered internet. The risk of a biased AI generated consensus across any and all fields of inquiry is one of the most frightening elements of the AI era. How are the tech companies training AI to decipher true from apocryphal? Who decides what is misinformation when history is clearly authored by the winners?

I liken this to how the robber barons operated in the US after the turn of the 20th century. Back then, the free market was so free that it operated as a "wild west" with little regulation. This allowed for richer and larger individuals and companies to dominate the market, buying out and swallowing all competition with sheers market share and economic power and pressure. In this context I believe that the companies backing these LLMs operate in a similar manner. There are little to no regulations in the online space, and these corperations are taking advantage of that to squeeze out or buy up competition. For example, as a small animator, it is increasingly hard to compete with AI animation, and when combined with the monetary drive for many people to create AI animations quickly compared to manually animating, it will slowly choke out the market for traditional animators. With no regulation, the "robber baron" econoy we saw historically will return to take over the online space

I think it is very unfortunate that the generosity and hardwork of those keeping the web open is exploited and disregarded by others. Although this article refers to the open web, I believe that this fact represents some facets of society in general. Living in a capitalistic society does sadly mean that the kindness and selflessness of others is often capitalized upon for the bettering of someone else's life, company, income and social status.

Whomever is in control of an AI model ultimately dictates what it can say and what content it can produce. What really frightens me is the implementation of AI in weaponry. Computers are meant to be efficient problem solvers. The thought that at the press of a button, an explosive payload could be homing in your position simply because you disagree with the position of those with access to said weaponry. Once more, allocating the responsibility of murder to an unthinking algorithm helps to sanitize the complicity of those who make the kill order.

I think that this point is really interesting because it is true that the reason the web can be such a useful tool is because it can be accessible to a large amount of people, and also connects those people who wouldn't be able to typically interact. It creates a line that everyone, both consumers and producers of these open web platforms, must balance of when is it too much? If these businesses continue to create these open web sources for a monetary return without thought for the impacts on the consumers, it can lead to a slippery slope where there is no more line and these platforms can be used for harm.

This is an exceedingly evocative image, but not entirely innacurate. Altman has been pretty clear on wanting to privatize intelligence and make information a commodity on the internet. He stands directly in the way of an open-access internet through pushing AI slop responses and reducing human agency in the internet space in the name of "efficiency".

This is one of many examples where the article does not pull its punches. It uses politically charged and highly opinionated language, which will likely seem compelling to readers who align politically with what has been read so far, but will also likely push away readers who are differently opinionated. This isn't necessarily a critique, as this is an opinion piece which I personally feel is right to use provocative statements of this nature. I believe that the Internet Archive is, on the whole, a public good that is essential for historical research, and for ensuring accountability of the corporations and other powerful institutions increasingly in control of these platforms.

This is an interesting thought. While I do agree that it would be possible to effectively push back if the masses work towards this one similar goal, I find it to be an unrealistic thought experiment. While the open web came about through community involvement, it was not in an ecosystem that had an active adversary fighting in the opposite direction. Moreover, I would posit that the co-operative spirit amongst the general populace online is generally weaker now than it was in the 90s and 2000s with how combative online spaces have become. I imagine it is a taller task in the modern day to get people to rally behind one cause, which would be necessary for a collective effort like fighting back against LLMs

Another point is that other creators increasingly use subscription newsletters to connect directly with their audience. Because algorithms can shadow ban their videos or posts, newsletters have become another option for connection with an audience. A newsletter I'm subscribed to Hank and John Green's.

I feel as though this aspect of the web is being stripped away. As platforms of public discourse are vacuumed up by private groups or individuals, their agendas or personal grievances begin to dictate what is and isn't permissible. For example, the change that has occurred on twitter after Elon's acquisition.

It really is amazing that humans managed to agree long enough to create the commons of the open web

Gender roles don't always impact children and families in a positive way.

With the current state of our country where younger generations have access to the media and adults around them constantly talk about the horrible things going on in the world, I wonder if it causes a child's chronosystem to change quicker than it normally would?

How does a child's brain and sense of self develop differently based on the level of support, love, nurture/care, etc they receive from their microsystem?

Intrapersonal Communication Interpersonal Communication Intercultural Communication Group Communication<br /> Public Communication

Neutral and blameless

I feel like I need to read the book, because I cannot see how they propose to achieve this.

The Royal Companion was a trim line name for Royal portables in the 40s and 50s along with variations like the Aristocrat and the Quiet De Luxe. For some of its life it's essentially a cheaper Quiet De Luxe which may be missing features like a tabulator, Magic Margins, card fingers, or a touch control. You'd have to cross check the manual and the typewriter database to see how yours may differ from the QDL at the time it was made. Often it was a $5-10 price differential at the time for the difference in features, but with inflation this would be the equivalent of $140 now.

You could essentially use the list of QDL users from Polt's site and say that they all used a typewriter similar to your Companion. (Depending on the photos and angles, some of the listed Quiet De Luxe users may have really been using either a Companion or an Aristocrat, but it's incredibly difficult to discern unless you can see the specific feature differences between them.)

See also: - https://boffosocko.com/2024/06/25/users-of-the-early-henry-dreyfuss-royal-quiet-de-luxe-portable-typewriters/<br /> - https://typewriterdatabase.com/royal.72.typewriter-serial-number-database<br /> - https://typewriterdatabase.com/Royal.Companion.72.bmys - Manuals at: https://site.xavier.edu/polt/typewriters/tw-manuals.html

Reply to u/chocolate-sheep at https://old.reddit.com/r/typewriters/comments/1wi0jd6/writers_using_royal_companions/

Wrong answer. None of A,B,C are strictly diagonally dominant matrices. But the answer shows A as strictly diagonally dominant matrix.

For me personally, I can sometimes feel discouraged to share my perspective on a topic when it does not quite align with what is normally brought up. I have to remind myself that it is important for us to share our perspectives in order to achieve a better understanding of worlds outside of our own.

Do's (Dos looks like the Spanish world for two and could be confusing to Spanish speakers)

Should it be (ABA, 2021) to follow MLA

Furthermore is another option to use instead of additionally

during incarceration and after their release

revise for clarity- Following in-prison drug treatment with continued treatment after release has been shown to reduce drug use and the likelihood of returning to prison (recidivism).

What are the functions of the peripheral nervous system and and Central nervous system

These few sentences really shocked me due to the fact that 1000 years is a relatively short time and if we continue with the population growth as it is, we are in big trouble. If 500 years is all it takes for people to form a human carpet over the whole continent, then society as a whole has to decide how to go forward. It is an interesting dilemma as I can't imagine the human population ever decreasing with our technological advancements, unless something wiped out a chunk of the human race (natural disasters, pandemic, war, etc.). I wonder what the most humane way to decrease the population would be?

Active site for nicotine and acetylcholine

This is about the ligand binding site

these proteins bind Acetylcholine

Knowledge from the historical perspective is essential in order to make today and the rest of the future better. It is important to understand how history everyday can be learned from to improve and acknowledge that the world is constantly evolving, but there are ways to make progress for the better.

History can be viewed as only past occurrences, but we are living in history now and I think that it is interesting that everyday, people are creating history.

People have been and still to this day are upset with the fact the world is continuing to build and take down nature to make more room for the people that are moving into the area. This is upsetting people to the point that they move to a new environment that would satisfy their needs and interests.

Humans are initiating change within the world and environment. Humans are wanting change and creating new opportunities to make the planet different whether it’s for the good or bad. There are thoughts and ideas that are being put to use which is then changing the world, which is then going to be looked at as history.

Surely a false dichotomy

It needs to be more powerful than the UN, there has to be a way to leverage trade deals with China but that must come from Trump

I need to listen to the Ezra Klein china talk

Paper clips

Make it monetarily beneficial to be ethical

Fear mongering

Strongest section so far, invoke this physical presence

Hinting at this narrative of smelly CS coders with no sense of the real world

I mean, AI is the latest iteration of putting information overload back into pandoras box

Feels achievable but insufficient

Omission of global agreement

Lack the necessary expertise

And also we are close, but maybe good cognitively too, also politically unachievable

Again, I wonder what nuclear scientists were saying

Strict father?

And yet the president says no, china is driving the race

expository even

I mean the jacob piece is interesting here but also who is the audience? everyone? very neutral in tone

The region differs from the docs under CloudFormation, which uses us-east-1

Security risk here. Since there is no mention of checksum matching, might want to optionally call out a step to review/confirm the Terraform output script here before passing to eval

omit

and meet the following criteria:

are used for preventive strategies

runaways, homeless individuals, and...

For clarity, consider using “improve the quality of care” over “enhanced quality.”

add space and consider putting the 13 agencies in a bullet list format

If information about Inspection was provided, complete the chart with information about Palpation and the results of the test mentioned in the text.

Could also be removed and left to the cardiac section where it is formally presented.

Has been referred to in previous sections, should be removed.

working title

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I think I'll be able to adapt and handle it

the studying

I am going to be doing 6 classes a semester for my degree

2 semesters of dual credit at brazosport, then a 4 year degree at university of houston

I knew that these AND and OR search terms were options but I never thought about combining them before. This seems like it could be very useful if I were doing research and needed to filter out certain results but keep multiple keywords in the search. I had found searching with AND and OR is very helpful when I'm looking for a broad variety of information on specific topics.

Doesn't add much to the context of learning. Could be removed for an image of the actual form used or no image at all.

So ok.... this is saying that index number is n-1, and n is the final sequence of the character (or should I say number).

In what are the syntax for accessing the elements equivalent to accessing the individual characters of a string or all of them combined?

what is meant by special type?

interesting. from right to left: the negative numbers get more negative as you go more left.

So indexing is concerning string: its 1 less than what it is. This is saying that indexing operated is used on strings to enclose the index, and the characters are identified by their position or index value.

What the number?

So, ok.

This is something new to me that I didn't know before! There have been situations where I am trying to search something very specific where I need the words to be in order like someone's name or a specific product but when searching I would get nowhere because I couldn't figure out how to make the search order the first and second words how I was typing them out.

Yes, dust can be an issue over time, especially if your machine is over-oiled and even more if you're in high dust/pollution area of the world and leave windows open regularly or have pets with lots of hair and dander production.

However, if uber-collector Martin Howard can be trusted, perhaps when designing your dream typewriter display room, dust isn't as horrible a problem as you might imagine. See: https://lumicrest.com/showcase-lighting-martin-howards-antique-typewriter-collection/ One imagines that occasional dusting and a can of compressed air will do the trick.

You could also do a nicer dust cover/mat the way Tom Hanks does: https://boffosocko.com/2025/06/16/tom-hanks-ingenious-custom-typewriter-covers-mats/

For my refurbished machines displayed in the house (where we have had a monster German Shepherd who sheds all year long and lots of exterior dust from Southern California pollution), if they're not in regular rotation, I'll drop a sheet of tinfoil or plastic wrap under the hood to prevent dust coming in through the hood and that seems to be more than enough protection while not covering up the entire machine.

reply to u/harperjones at https://old.reddit.com/r/typewriters/comments/1whqodm/closed_cabinets_for_display/

I have noticed this in my person experience. When I search for products on my work computer it gives me advertisements and suggestions for pet-centered products and websites. I work in a veterinarian office so that makes sense from our previous searches. When I search on my personal computer I usually get results from sites that I have visited before like Reddit and YouTube. I also notice that some of the suggested results are for products that I have personally searched or looked at before. Even if I am searching for something specific I have noticed how the results can seem catered to me in some ways which is interesting. It can be beneficial since its personalized to me but also harmful since I may not be getting results that I am actually searching for and that could be helpful.

also known as 'bidmas' brackets, indices, division, addition, subtraction

This is so broad, his paper is helpful What is Knowledge Organization (KO)?

1. Após o 2º quesito (Autoria e Materialidade)

Se a defesa sustenta a desclassificação própria (ex.: "ele não queria matar, apenas ferir"):

Passo 1: Jurados confirmam a materialidade (quesito 1).

Passo 2: Jurados confirmam a autoria (quesito 2).

Passo 3: O juiz insere a pergunta da desclassificação: "O réu agiu com intenção de matar?" (ou "O réu assumiu o risco de produzir a morte?").

Resultado: Se a maioria dos jurados responder "NÃO", ocorre a desclassificação. Os jurados perdem a competência sobre o caso e o juiz presidente (juiz singular) assume o julgamento no mesmo momento para proferir a sentença pelo crime restante (ex.: lesão corporal).

1. Após o 3º quesito (Absolvição Genérica)

Se a defesa sustentar tanto uma tese principal de absolvição (ex.: legítima defesa) quanto uma tese subsidiária de desclassificação (ex.: se não for legítima defesa, que seja lesão corporal):

Passo 1: Materialidade (SIM).

Passo 2: Autoria (SIM).

Passo 3: "O jurado absolve o acusado?"

Resultado:

Se o júri responder "SIM", o réu está absolvido e a votação acaba.

Se o júri responder "NÃO" (não absolve), o juiz abre espaço para a tese subsidiária e formula o quesito da desclassificação.

Somente se os jurados rejeitarem a desclassificação é que se passa para a análise das qualificadoras, causas de aumento/diminuição e posterior fixação da pena.

I feel this section needs a bit of expansion in comparison to the previous sections. "The occipital lobe is primarily responsible for visual processing. It is associated with visuospatial processing, distance and depth perception, color determination, object and face recognition, and memory formation. The primary visual cortex transmits information through two pathways: the dorsal and ventral stream. The dorsal stream is associated with object location and carries visual information to the parietal lobe. The ventral stream has associations with object recognition and transmits visual information to the temporal lobe." Rehman A, Al Khalili Y. Neuroanatomy, Occipital Lobe. [Updated 2023 Jul 24]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2026 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK544320/

Amodei states that the public should have a say in what AI should be utilized for, but he does not dive into all of what that entails. Rather, he focuses on finances and national security. While they could be considered incredibly important, his interests are not entirely equivalent to that of average citizens. From A Common Good standpoint, the public should be the first to decide AI's limitations. What aspects of it do they want to be able to touch their life? Should Generative AI be accessible to the general population? Should AI be allowed to scrape the internet for information without compensating or letting those who own that information know? Should AI be used in science and medicine? What about data centers and the environmental threats they often pose? There are many questions, and many different uses for AI, some of which could benefit society more than others. These AI companies are making decisions that involve society as a whole without their consent. While Amodei's plan does benefit people by forming regulations on pacing and controlling the reach of AI, it still does not address a multitude of concerns.

From a Utilitarian lens, Amodei's arguement is solid. Bioterrorist attacks are real concerns that have major consequences. He is approaching the argument of pacing AI from a reasonable standpoint by measuring the greatest harms to humanity as a whole. It focuses on what could help the majority of people in terms of physical safety and economics. It doesn't delve into what exactly society wants or what they value, as I discussed while looking at it through a Common Goods lens, but rather what would benefit the most people.

hello

Madame Sosostris is based off of Huxley's character "Sesostris, the Sorceress of Ecbatana." In Crome Yellow, Sesostris is a fortune-teller whose theatrical predictions reveal people's eagerness to believe in prophecy. In The Waste Land, Sesostris represents the postwar culture of people searching for answers amid uncertainty. Madame Sosostris appears satirical to the reader as she uses her "wicked pack of cards" to tell the futures. Although Madame Sosostris seems to be a theatrical charlatan, her tarot readings introduce images that recur throughout The Waste Land, such as the Phoenician Sailor, the Hanged Man, and the crowd of the dead-like living. Thus, it is uncertain if her fortunes are of empty performance or genuine prophecy.

Issue: it inhibits his lifestyle

Who is they? Babies?

Oh wow. It's all about him, not her.

If I have an abortion, then she is back to being tolerated?

she feels she has to do it for things to stay how they are: change is more than scary

puts the responsibility on the girl

assurance

gets feedback from man, not herself

silence

abortion

asking for input

frequentist

¿Notas por proyecto o notas transversales a muchos proyectos y temas?

Pero ayuda... la parte importante siempre recae sobre la reflexión personal que se hace sobre la información. Las notas solo son el soporte sobre el que se debe hacer esto. Por eso no se puede automatizar el sistema ni delegar en la IA.

Tuples are similar to listd

given that in the results we were highlighting the local stability differed mainly due to the generative models, I am wondering if we are suggesting that we should use more than one generative model in the future e.g. predicting food web responses to environmental change? Or we just simply highlight that we need to understand the assumptions and limitations of each generative model?

I think we have to be careful here. Predictions of biomass equilibrum - I assume this means the network-level stability properties and local stability metrics. Because we currently don't have a PTA analysis with these stability metrics, we don't know whether they actually don't converge or how large the differece comparing to the PTA of realised network topologies.

I can add a PTA of these metrics later if you think this is needed.

we can discuss this at next meeting because the variation in reactivity is mainly because of the LTM model, other models seems to have similar reactivity.

functional??

Just realise that we don't need to use stability throughout the ms because I think it is weird to use wntwork-level stability metrics to describe persistence, biomass diversity, flux/interaction strngth distribution.

maybe we could use functional? which included the above metrics, resilience, and reactivity. Stability is one type of functional property. or ?

Yes there is no rewiring in the species interaction links. The feasible network is just a subset of initial interaction network.

I think we may demonstrate more like dynamic redistribution of realised interaction strengths (but not sure whether this is a good term). The initial network sets the initial interaction structure. the initial biomasses and biological rates set the initial energy fluxes and interaction strength distribution. During the dynamic simulation, changes in species biomasses alter energy fluxes and therefore realised interaction strengths. The system finally moves to a feasible steady state where the realised interaction strength distribution is redistributed into a configuration that supports species coexistence.

Does the mapping of migration actually lead to better population outcomes, or is the protection of mapped migration corridors more important?

Local/indigenous knowledge is an interesting component to further understand lost migration patterns. I love the connection of "living history" here.

I think this would be interesting to overlap with research on seed dispersal, and if the migration area vs non-migratory areas differ in plant diversity, composition, or functional traits.

eLife Assessment

This fundamental study, supported by convincing evidence, identifies the specific neuronal components that enable HIF-1 signaling in specific serotonergic neurons to regulate effectors in peripheral tissues. Specifically, stabilizing HIF-1 in ADF and NSM serotonergic neurons is sufficient to extend lifespan and healthspan in C. elegans, and these physiological benefits require several oxygen-sensing neurons, as well as neurotransmitters (GABA and tyramine), and a neuropeptide (NLP-17). The strengths of the study include rigorous experimental design, high quality data, the use of multiple genetic tools, and functional dissection using the well-validated C. elegans model.

Reviewer #1 (Public review):

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

Summary:

In this study by Kitto et al., the authors set out to identify specific signaling components regulating the hypoxic response from the neurons to the periphery and which components are required for lifespan extension. Their previous work had shown that expression of a stabilized HIF-1 mutant in the nervous system extends lifespan through the serotonin receptor SER-7 and leads to the induction of fmo-2 in the intestine. In the current study, they mapped the precise neural circuits required for this response, as well as the signaling mediators. Their work reveals that neurotransmitters GABA and tyramine, and the neuropeptide NLP-17, act downstream of neuronal HIF-1 to convey a "hypoxic signal" to peripheral tissues. Through cell-type-specific expression studies, targeted knockouts, and comprehensive lifespan analysis, the authors provide robust evidence to support their conclusions. The insights gained from the study are both moving the field forward as they advance our understanding of neuro-peripheral hypoxic signaling, but they also lay the groundwork for potential therapeutic strategies aimed at the modulation of such signaling pathways.

Strengths:

(1) This study provides new evidence further delineating signaling components required for hypoxic signaling-mediated longevity, from the nervous system to the periphery. Using a rigorous approach where they express stabilized HIF-1 mutant selectively in ADF, NSM, and HSN serotonergic neurons, followed by cell-type-specific tph-1 knockouts to pinpoint ADF-dependent serotonin signaling as essential for both lifespan extension and intestinal fmo-2 induction.

This was followed by generating 11 transgenic lines that drive SER-7 expression under distinct neuron-specific promoters, to systematically tease out in which of 27 candidate neurons SER-7 functions to mediate hypoxia-induced longevity. This ultimately highlighted the RIS interneuron as the required signaling hub.

(2) As the intestine lacks direct neuronal innervation, the authors employ neuron-specific RNAi (TU3311 strain) and dense core vesicle analyses to identify that the neuropeptide NLP-17 is required to transmit the hypoxic signal from RIS to induce fmo-2 in the intestine.

(3) Overall, the paper is very well written. The experiments were carried out carefully and thoroughly, and the conclusions drawn are also well supported by the results they are showing.

Reviewer #2 (Public review):

Summary:

The authors aimed to identify the specific neurons, neurotransmitters, and neuropeptides that mediate the longevity effects of the hypoxic response in C. elegans. By genetically dissecting the pathway downstream of HIF-1, they define a neural circuit involving ADF serotonergic neurons, the SER-7 receptor in the RIS interneuron, tyraminergic signaling from RIM, and neuropeptide NLP-17, ultimately linking neuronal hypoxic sensing to pro-longevity signaling in the intestine.

Strengths:

The study employs a diverse genetic toolkit, including neuron-specific transgenes, tissue-specific knockouts and rescues, RNAi knockdowns, allowing the authors to pinpoint causality, sufficiency, and necessity with high resolution. The comprehensive mapping of cell-nonautonomous signaling adds depth to our understanding of how HIF and serotonin signaling interface with aging pathways. The conclusions are supported by consistent survival assays and fmo-2 gene expression analyses.

Reviewer #3 (Public review):

Summary:

This study found that ADF serotonergic neurons have a significant role in extending lifespan mediated by HIF-1, as well as serotonin receptor SER-7 in the GABAergic RIS interneurons. The author focuses on the sufficiency and necessity of components from the central nervous system and how they contribute to aging upon hypoxia.

Previous work from the lab has identified that the stabilization of HIF-1 in neurons is sufficient to extend lifespan through the serotonin receptor, SER-7, which subsequently activates fmo-2 in the intestine and leads to lifespan extension. Building on this, the author sought to determine which serotonergic neurons are involved and found that serotonin signaling in ADF neurons is required for lifespan extension mediated by HIF-1.

The author next tested which subset of neurons requires Ser-7 expression to rescue hypoxic response. They found that ser-7 expression in multiple neurons is sufficient to induce fmo-2, with the top candidate being the RIS neuron. Ablation of the RIS neuron did not extend lifespan, suggesting that ser-7 expression in the RIS neuron is required for lifespan extension, positioning it as a key component in the longevity signaling pathway.

The author also investigated neurotransmitters and found that GABA and tyramine are important components in this circuit. They showed that the tyramine receptor called tyra-3 is required for vhl-1-mediated longevity. Given that tyra-3 is expressed in oxygen- and carbon dioxide-sensing neurons, the author demonstrated that these sensing neurons work downstream of serotonin signaling. Lastly, the author screened neuropeptide/receptor binding pairs and identified NLP-17 as playing a role in hypoxia-mediated longevity.

Originality and Significance:

This research is significant in that it uncovers components that are sufficient and necessary for lifespan extension via the hypoxic response. It provides comprehensive data supporting longevity induced by HIF-1-mediated hypoxic response, in conjunction with fmo-2, a longevity gene, as demonstrated in previous work from the lab. Moreover, it provides a number of new transgenic worm tools for C. elegans and aging communities.

Conclusions:

This study provides insights into how hypoxic response regulates aging in a cell non-autonomous manner, outlining a potential circuit involving neurons, neurotransmitters, and neuropeptides.

Author response:

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

We thank the reviewers and editors for their time and valuable input into improving our manuscript. The reviewers recognized the value of this work while identifying places where further explanation and/or additional experiments could strengthen the manuscript. We greatly appreciate this feedback and have addressed reviewer comments through additional experiments and necessary textual edits.

In response to the reviewer comments, our principal data-driven changes to revise the manuscript include: 1) testing how stabilizing HIF-1 in the ADF and NSM neurons affects healthspan; 2) measuring for interactions between HIF-1 stabilization in serotonergic neurons and the mt-UPR; and 3) measuring nlp-17 expression downstream of HIF-1 stabilization in serotonergic neurons. We also made textual changes including: 1) clarifying that our study focuses specifically on the vhl-1-mediated genetic hypoxic response; 2) summarizing which parts of the working model are experimentally validated vs. speculative; and 3) expanding our discussion of future directions needed to understand the epistasis of the many signals acting in this circuit. Our responses to each reviewer comment are below.

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this study by Kitto et al., the authors set out to identify specific signaling components regulating the hypoxic response from the neurons to the periphery and which components are required for lifespan extension. Their previous work had shown that expression of a stabilized HIF-1 mutant in the nervous system extends lifespan through the serotonin receptor SER-7 and leads to the induction of fmo-2 in the intestine. In the current study, they mapped the precise neural circuits required for this response, as well as the signaling mediators. Their work reveals that neurotransmitters GABA and tyramine, and the neuropeptide NLP-17, act downstream of neuronal HIF-1 to convey a "hypoxic signal" to peripheral tissues. Through cell-type-specific expression studies, targeted knockouts, and comprehensive lifespan analysis, the authors provide robust evidence to support their conclusions. The insights gained from the study are both moving the field forward as they advance our understanding of neuro-peripheral hypoxic signaling, but they also lay the groundwork for potential therapeutic strategies aimed at the modulation of such signaling pathways.

We appreciate the reviewer’s positive assessment of the topic and general interest in this work.

Strengths:

(1) This study provides new evidence further delineating signaling components required for hypoxic signaling-mediated longevity, from the nervous system to the periphery. Using a rigorous approach where they express stabilized HIF-1 mutant selectively in ADF, NSM, and HSN serotonergic neurons, followed by cell-type-specific tph-1 knockouts to pinpoint ADF-dependent serotonin signaling as essential for both lifespan extension and intestinal fmo-2 induction.

This was followed by generating 11 transgenic lines that drive SER-7 expression under distinct neuron-specific promoters, to systematically tease out in which of 27 candidate neurons SER-7 functions to mediate hypoxia-induced longevity. This ultimately highlighted the RIS interneuron as the required signaling hub.

(2) As the intestine lacks direct neuronal innervation, the authors employ neuron-specific RNAi (TU3311 strain) and dense core vesicle analyses to identify that the neuropeptide NLP-17 is required to transmit the hypoxic signal from RIS to induce fmo-2 in the intestine.

(3) Overall, the paper is very well written. The experiments were carried out carefully and thoroughly, and the conclusions drawn are also well supported by the results they are showing.

Weaknesses:

Overall, I don't see many weaknesses. One point relates to their read-outs, which rely heavily on lifespan measurements and fmo-2 induction without evaluating other physiological processes that serotonin or NLP-17 might affect. For translational relevance, it would be valuable to assess or mention potential adverse effects, such as changes in reproduction, pharyngeal pumping, or proteostasis capacity (proteostasis capacity specifically in the tissue showing fmo-2 upregulation).

We thank the reviewer for the positive review and fully agree and acknowledge that the primary readouts used in this work, fmo-2 induction and lifespan, may not reflect other important elements of health and physiology. To address this weakness, we have performed three measurements of healthspan (pumping, thrashing, and maximum velocity), in the ADF and NSM HIF-1 stabilized strains at young adulthood and at middle age. We focused on examining these HIF-1 stabilized strains rather than our nlp-17 knockout animals because stabilizing HIF-1 in the NSM or ADF neurons is sufficient to extend lifespan. nlp-17 is necessary, but it remains unclear whether nlp-17 signaling is sufficient to extend lifespan. Our new data show that both ADF- and NSM-specific HIF-1 stabilization had no effect on pumping rate in young (day 1 of adulthood) worms. In aged animals (day 12 of adulthood), however, NSM-, but not ADF-, specific HIF-1 stabilization rescued the pumping rate decline in hif-1 knockout compared to WT worms (new Fig. S1C). Similarly in the new thrashing data, NSM-, but not ADF-, specific HIF-1 stabilization rescued the thrashing rate decline in the hif-1 knockout young and aged worms (new Fig. S1D). Lastly, our new data show that ADF and NSM HIF-1 stabilization had no effect on average or maximum movement speed at days 1 and 5 of adulthood (new Fig. S1E-F). Together, these results indicate that genetic activation of the hypoxic response in NSM neurons but not in the ADF neurons could improve healthspan.

While we did not examine reproductive capacity in these strains, we have expanded our discussion section to mention the importance of fully characterizing other elements of physiology, health, and behavior in future work.

“Another limitation of this work is that it uses lifespan as the main readout for organismal health. While genetic manipulations that extend lifespan often improve stress resistance and healthspan [8,80], longevity manipulations can also have adverse effects on reproduction [81,82] and behavior [83,84]. In this study, we find that HIF-1 stabilization in the ADF neurons does not prevent the deleterious effects of hif-1 knockout on mobility, but that HIF-1 stabilization in the NSM neurons may attenuate age-related decline in pumping and thrashing (Fig. S1). However, future work should examine whether other modifications to this pathway, such as manipulations to RIM, RIS, or NLP-17 signaling, influence healthspan in addition to lifespan. It will be important for future studies to determine whether various components of this pathway affect both longevity and the response to different types of stressors like oxidative stress, proteotoxic stress, and infection, as HIF-1 activity also interacts with multiple stress responses [41,42,39,40,43].”

While lifespan assays and fmo-2 expression do provide strong evidence, incorporating additional markers of stress resistance could strengthen the link between hypoxic signaling and organismal health as well.

We also measured hsp-6 expression via qPCR in the serotonergic neuron-specific HIF-1 stabilized strains to determine whether these conditions that lead to upregulated fmo-2 may also affect the mt-UPR. Interestingly, we find that stabilizing HIF-1 in either the ADF or NSM serotonergic neurons decreases hsp-6 expression relative to WT worms (new Fig. S1G). This could suggest either that the mt-UPR response is impaired in these worms, or that HIF-1 stabilization decreases proteotoxic stress leading to a lower basal level of hsp-6. Although this method of measurement did not allow us to interrogate whether these changes occur in the specific tissues where fmo-2 is upregulated, we have expanded our discussion to emphasize that further investigation of cell- and tissue-specificity within the hypoxic response should be a focus of future work.

Finally, we agree that it is important to examine whether activating the hypoxic response promotes stress resistance in addition to longevity. We did not focus on this element of the hypoxic response in this work because HIF activity is known to promote adaptive stress-responses to some stressors like infection [7,8] and oxidative stress [9,10], while simultaneously impairing the proteosasis stress response [11]. We have added this important information to our introduction section, and have expanded our discussion section to emphasize that a key future direction will be to test which specific components of this pathway also facilitate stress resistance.

Introduction Section Modification:

“However, the physiological changes induced by the hypoxic response are broad and involve adaptations such as increased vascularization, metabolic rewiring, and changes in cell survival pathways. In mammals, some of these same adaptations can be detrimental, as mutations in components of the hypoxic response have been linked to conditions like cancer and cardiovascular disease [12-15]. Additionally, HIF activity is essential to promote some forms of stress resistance to infection [9,10] and oxidative stressors [7,8] but can have detrimental effects on proteostasis [11].”

Discussion Section Modification:

“Another limitation of this work is that it uses lifespan as the main readout for organismal health. While genetic manipulations that extend lifespan often improve stress resistance and healthspan [1,2], longevity manipulations can also have adverse effects on reproduction [3,4] and behavior [5,6]. In this study, we find that HIF-1 stabilization in the ADF or NSM neurons has little effect on mobility in young and aged animals. However, future work should examine whether other modifications to this pathway, such as manipulations to RIM, RIS, or NLP-17 signaling, influence healthspan in addition to lifespan. It will be important for future studies to determine whether various components of this pathway affect both longevity and the response to different types of stressors like oxidative stress, proteotoxic stress, and infection, as HIF-1 activity also interacts with multiple stress responses [7,8,9-11].”

Reviewer #2 (Public review):

Summary:

The authors aimed to identify the specific neurons, neurotransmitters, and neuropeptides that mediate the longevity effects of the hypoxic response in C. elegans. By genetically dissecting the pathway downstream of HIF-1, they define a neural circuit involving ADF serotonergic neurons, the SER-7 receptor in the RIS interneuron, tyraminergic signaling from RIM, and neuropeptide NLP-17, ultimately linking neuronal hypoxic sensing to pro-longevity signaling in the intestine.

Strengths:

The study employs a diverse genetic toolkit, including neuron-specific transgenes, tissue-specific knockouts and rescues, RNAi knockdowns, allowing the authors to pinpoint causality, sufficiency, and necessity with high resolution. The comprehensive mapping of cell-nonautonomous signaling adds depth to our understanding of how HIF and serotonin signaling interface with aging pathways. The conclusions are supported by consistent survival assays and fmo-2 gene expression analyses.

Weaknesses:

A key limitation is the lack of clear evidence showing epistasis of so many identified molecular/neuronal components downstream of HIF-1 and serotonin. Thus, the mechanisms of how a diverse set of molecules/neurons coordinate and mediate neuronal HIF-1 effects on intestinal fmo-2 and longevity remain murky.

We thank the reviewer for these important points. We agree that the epistatic relationships between ADF serotonin, RIM tyramine, RIS GABA, and neuropeptide NLP-17 signaling remain unclear within this pathway. Determining the epistatic relationships of each signal within this complex pathway will require: 1) generating genetic manipulations to each identified signaling component that may mimic vhl-1 knockout to promote longevity; 2) crossing these new strains into multiple genetic knockouts we identified as required for vhl-1 mediated longevity; and 3) measuring the lifespans of each double and triple mutant. We are very interested in testing the epistatic relationships between all of these molecules and neurons, and believe this extensive follow-up exploration will generate significant future results.

To better address these limitations of the current work, we have added a summary table to Fig. 7 as well as a paragraph to our discussion section. This table and paragraph better explain which components of our working model have been tested for necessity, sufficiency, and epistasis, and which components of this model remain unclear (Fig. 7). See updated Fig. 7 with added table.

Updated discussion section detailing this limitation:

“While many individual neurosignaling components are essential for genetic activation of the hypoxic response to extend lifespan, their epistasis is unclear (Fig. 7B). Most components of the pathway identified in this work act downstream of vhl-1 depletion, and upstream of fmo-2 induction (summarized in Fig. 7A-B). However, the order of each signal between these two endpoints is only predicted based on C. elegans neural wiring and the overlap between various identified signals and cells. For example, we hypothesize in our working model that GABA may be produced by the RIS neuron in this circuit because RIS is the primary GABAergic neuron required for vhl-1-mediated longevity. Alternatively, it is possible that GABA is produced by a different cell that either acts in series or in parallel with RIS signaling. In order to determine the order of each signaling component, future studies should generate genetic manipulations to each signaling component that may mimic vhl-1 knockout to promote longevity, cross these new strains into knockouts of other signals required for vhl-1 mediated longevity; and measure the lifespans of each double and triple mutant. This approach would also narrow down which signals are downstream of the genetic activation of the hypoxic response, and which are sufficient to extend lifespan upstream of the hypoxic response in a normoxic environment. One notable target for further exploration is the SER-7 expressing RIS neuron, which plays a role in sleep [16] and stress resistance [17], and can extend lifespan when optogenetically activated under normoxic conditions [18].”

Some rescue strategies may inadvertently cause non-physiological expression.

This is a great point. We bring attention to this limitation in the discussion section. Our cell-specific knockouts (ADF tph-1 KO, Fig. 1E) or ablations (RIS ablation, Fig. 2C) data showed that the neurons identified using rescue strains (i.e., tph-1 in the ADF and ser-7 in RIS) are likely not false positives. However, we did not generate a RIM-specific knockout or ablation strain to confirm our tdc-1 results and have added this limitation to the discussion of these results.

Discussion of rescue strategy limitations and the need for a RIM-specific knockout:

“The circuit-mapping approaches employed in this work are also impacted by limitations in cell-specific genetic modifications and in the use of RNAi knockdown. For example, cell-specific rescue constructs can sometimes lead to unintended rescues in other cell types due to cell-nonautonomous signaling. Because all serotonin-producing neurons also express the serotonin reuptake transporter mod-5, serotonin produced by one cell in our rescue strains could be taken up by other serotonin-producing neurons, leading to unintended signaling effects. This may also be true of the uv1 and RIM tyraminergic rescue strains, although little is known about tyramine reuptake in C. elegans. Two cells identified via tissue-specific rescue experiments (ADF and RIS, Fig. 1G and Fig. 2D) were also found to be necessary via cell-specific knockout (ADF tph-1 KO, Fig. 1E; RIS ablation, Fig. 2C), decreasing the likelihood of a false positive from the rescue strain technique. However, the role of the RIM neuron was identified via a tdc-1 rescue strain and was not validated using a RIM-specific knockout (Fig. 7B). Therefore, the contribution of RIM signaling to this circuit is less well-validated, and a RIM-specific tdc-1 knockout strain should be examined in future work.”

Additionally, environmental hypoxia was not tested in parallel, so the claim on "hypoxia response" throughout the manuscript is not justified by genetic manipulation alone, and the translational relevance of the genetic manipulations remains somewhat uncertain.

We thank the reviewer for identifying the need for additional specificity in our language. We agree that it is critical to make it clear that this paper only examines genetic mimics of hypoxia, rather than environmental hypoxia, and have replaced every occurrence of “the hypoxic response” with either 1) “genetic activation of the hypoxic response”, or 2) describing the specific manipulation used in that experiment (e.g., “vhl-1 mediated longevity”). We also agree that examining the similarities and differences between genetic and environmental activation is a key next step towards evaluating the translational potential of this pathway. We discuss this in Discussion and are excited to interrogate these differences in future work.

“While this study identifies many neural signals required for vhl-1 knockdown or knockout to extend lifespan, one key limitation of this work is the potential differences between genetic and environmental methods of inducing the hypoxic response. While vhl-1 knockdown or knockout leads to HIF-1 stabilization by blocking its proteasomal degradation, it also results in hydroxylated HIF-1. This contrasts with environmental hypoxia, in which HIF-1 remains stable because it cannot be hydroxylated. While HIF-1 is stabilized and localized to the nucleus in both cases, there are differences in transcriptional outcomes between stable hydroxylated and unhydroxylated states [19,20]. Additionally, we did not explore the effects of alternative genetic activators of the hypoxic response such as HIF-1 hydroxylase PHD/EGL mutants, which may provide further insight into how different manipulations of the hypoxic response impact longevity. Future work should interrogate the similarities and differences between the circuits driving longevity in response to environmental hypoxia, vhl-1 knockdown, HIF-1 stabilization, and PHD/EGL knockdown.”

Reviewer #3 (Public review):

Summary:

This study found that ADF serotonergic neurons have a significant role in extending lifespan mediated by HIF-1, as well as serotonin receptor SER-7 in the GABAergic RIS interneurons. The author focuses on the sufficiency and necessity of components from the central nervous system and how they contribute to aging upon hypoxia.

Previous work from the lab has identified that the stabilization of HIF-1 in neurons is sufficient to extend lifespan through the serotonin receptor, SER-7, which subsequently activates fmo-2 in the intestine and leads to lifespan extension. Building on this, the author sought to determine which serotonergic neurons are involved and found that serotonin signaling in ADF neurons is required for lifespan extension mediated by HIF-1.

The author next tested which subset of neurons requires Ser-7 expression to rescue hypoxic response. They found that ser-7 expression in multiple neurons is sufficient to induce fmo-2, with the top candidate being the RIS neuron. Ablation of the RIS neuron did not extend lifespan, suggesting that ser-7 expression in the RIS neuron is required for lifespan extension, positioning it as a key component in the longevity signaling pathway.

The author also investigated neurotransmitters and found that GABA and tyramine are important components in this circuit. They showed that the tyramine receptor called tyra-3 is required for vhl-1-mediated longevity. Given that tyra-3 is expressed in oxygen- and carbon dioxide-sensing neurons, the author demonstrated that these sensing neurons work downstream of serotonin signaling. Lastly, the author screened neuropeptide/receptor binding pairs and identified NLP-17 as playing a role in hypoxia-mediated longevity.

Originality and Significance:

This research is significant in that it uncovers components that are sufficient and necessary for lifespan extension via the hypoxic response. It provides comprehensive data supporting longevity induced by HIF-1-mediated hypoxic response, in conjunction with fmo-2, a longevity gene, as demonstrated in previous work from the lab. Moreover, it provides a number of new transgenic worm tools for C. elegans and aging communities.

We thank the reviewer for the positive assessment of the manuscript. We appreciate all suggestions for further improving the work and have made changes based on these suggestions (see details below).

Data and Methodology:

(1) The experiments were thoroughly conducted, especially the generations of strains using different neuron-type promoters and crossing into mutant strains to demonstrate sufficiency and necessity.

(2) Some figure legends from the text do not match what the data show. (Figure 6E, F, G).

We have made changes to the legends accordingly to make sure figures and legends are consistent.

(3) The lifespan graph legends are confusing and could use some revamping for better clarification.

We have updated the lifespan graph legends to provide the statistics in the same section as the labels, indicating which line is which condition (see updated Figure 1E). We hope this change better clarifies the lifespan graph legends.

Conclusions:

This study provides insights into how hypoxic response regulates aging in a cell non-autonomous manner, outlining a potential circuit involving neurons, neurotransmitters, and neuropeptides.

Recommendations for the authors:

Reviewing Editor Comments:

As suggested by the Reviewers 2 & 3, including environmental hypoxia will broaden the impact of the study. If not, the authors should consider clarifying their response as "genetic activation of the hypoxic response" (see Reviewer 2 below).

We thank the editor and reviewers 2 and 3 for this clarification. We agree that it is critical to make it clear that this paper only examines genetic mimics of hypoxia, rather than environmental hypoxia, and have replaced every occurrence of “the hypoxic response” with either 1) “genetic activation of the hypoxic response”, or 2) describing the specific manipulation used in that experiment (e.g., “vhl-1 mediated longevity”). We also agree that examining the similarities and differences between genetic and environmental activation is a key next step towards evaluating the translational potential of this pathway. We have also expanded our discussion of this important future direction.

“While this study identifies many neural signals required for vhl-1 knockdown or knockout to extend lifespan, one key limitation of this work is the potential differences between genetic and environmental methods of inducing the hypoxic response. While vhl-1 knockdown or knockout leads to HIF-1 stabilization by blocking its proteasomal degradation, it also results in hydroxylated HIF-1. This contrasts with environmental hypoxia, in which HIF-1 remains stable because it cannot be hydroxylated. While HIF-1 is stabilized and localized to the nucleus in both cases, there are differences in transcriptional outcomes between stable hydroxylated and unhydroxylated states [19,20]. Additionally, we did not explore the effects of alternative genetic activators of the hypoxic response such as HIF-1 hydroxylase PHD/EGL mutants, which may provide further insight into how different manipulations of the hypoxic response impact longevity. Future work should interrogate the similarities and differences between the circuits driving longevity in response to environmental hypoxia, vhl-1 knockdown, HIF-1 stabilization, and PHD/EGL knockdown.”

Suggested minor changes by Reviewer 3 should also be made to improve the readability of the paper.

We have made changes suggested by Reviewer 3 to improve the readability of the paper.

Reviewer #2 (Recommendations for the authors):

(1) Suggestions for additional experiments or analyses:

(a) To clarify the hierarchical relationships among the components of the identified circuit, epistasis experiments between serotonin, tyramine, NLP-17, and oxygen-sensing neurons (e.g., double mutants or sequential rescues) would strengthen the proposed model and help determine whether these signals act in parallel or downstream of each other.

We thank the reviewer for identifying this important caveat. As described in the public review response, we completely agree that understanding the epistasis of serotonin, tyramine, NLP-17, and oxygen-sensing neuron signaling within this pathway is important to fully test our working model. We hope to address these questions about epistasis and interactions between different signals in upcoming projects.

To clarify that the order of many of these signals remains speculative in our working model, we have added a table to Fig. 7, that summarizes what is known and unknown about the epistasis of these pathway components. We have also expanded our discussion of this limitation. See updated Fig. 7 with added table.

Updated discussion section detailing this limitation:

“While many individual neurosignaling components are essential for genetic activation of the hypoxic response to extend lifespan, their epistasis is unclear (Fig. 7B). Most components of the pathway identified in this work act downstream of vhl-1 depletion, and upstream of fmo-2 induction (summarized in Fig. 7A-B). However, the order of each signal between these two endpoints is only predicted based on C. elegans neural wiring and the overlap between various identified signals and cells. For example, we hypothesize in our working model that GABA may be produced by the RIS neuron in this circuit because RIS is the primary GABAergic neuron we found to be required for vhl-1-mediated longevity. Alternatively, it is possible that GABA is produced by a different cell that either acts in series or in parallel with RIS signaling. In order to determine the order of each signaling component, future studies should generate genetic manipulations to each signaling component that may mimic vhl-1 knockout to promote longevity, cross these new strains into knockouts of other signals required for vhl-1 mediated longevity; and measure the lifespans of each double and triple mutant.”

(b) Testing whether environmental hypoxia (e.g., 0.5-1% O₂ exposure) elicits similar neuronal requirements and fmo-2 induction as the genetic HIF-1 stabilization would validate that the described pathway is relevant to the actual hypoxic response and improve translational relevance.

We thank the reviewer for identifying the need for clarification. As also discussed in the public review section, we agree that it is important to emphasize that this paper exclusively examines genetic mimetics of hypoxia, rather than actual exposure to a hypoxic environment. To clarify this point, we have replaced every occurrence of “the hypoxic response” with either 1) “genetic activation of the hypoxic response”, or 2) describing the specific manipulation used in that experiment (ie “vhl-1 mediated longevity”). We also agree that examining the similarities and differences between genetic and environmental activation is a key next step towards evaluating the translational potential of this pathway. We discuss this in discussion and are excited to interrogate these differences in future work.

“While this study identifies many neural signals required for vhl-1 knockdown or knockout to extend lifespan, one key limitation of this work is the potential differences between genetic and environmental methods of inducing the hypoxic response. While vhl-1 knockdown or knockout leads to HIF-1 stabilization by blocking its proteasomal degradation, it also results in hydroxylated HIF-1. This contrasts with environmental hypoxia, in which HIF-1 remains stable because it cannot be hydroxylated. While HIF-1 is stabilized and localized to the nucleus in both cases, there are differences in transcriptional outcomes between stable hydroxylated and unhydroxylated states [19,20]. Additionally, we did not explore the effects of alternative genetic activators of the hypoxic response such as HIF-1 hydroxylase PHD/EGL mutants, which may provide further insight into how different manipulations of the hypoxic response impact longevity. Future work should interrogate the similarities and differences between the circuits driving longevity in response to environmental hypoxia, vhl-1 knockdown, HIF-1 stabilization, and PHD/EGL knockdown.”

(c) Functional readouts beyond lifespan and fmo-2 induction (e.g., neuronal activity monitoring or optogenetic modulation of key neurons) could help clarify how information flows through the circuit.

We thank the reviewer for this valuable suggestion. We are hoping to be able to implement these experimental techniques in future work. We agree that in combination with genetic epistasis analyses, direct measurements of neuronal signaling will greatly improve our understanding of the directionality and interactions between signals in this circuit. Our discussion section recommends employing these techniques in future studies.

“Finally, while the use of RNAi knockdown and genetic knockouts establishes the necessity of many signals within the vhl-1-mediated longevity circuit, the exact directionality of these signals remains unclear. It is possible that increased, decreased, or pulsatile changes in signaling through these bioamines and neuropeptides are required for genetic activation of the hypoxic response to extend lifespan. Work on C. elegans reversal behavior has also revealed an antagonistic relationship between RIM and RIS activity facilitated by both chemical (neuropeptide and tyramine) and electrical (gap junction) signaling [16,21]. This known interaction should also be interrogated in the context of how these cells may communicate following genetic induction of the hypoxic response. Future work in this area could use tools to measure or modify neuronal activity, such as calcium imaging or optogenetics, to begin answering these questions.”

(2) Recommendations for improving the writing and presentation:

(a) The manuscript is well written overall, but clarity would be improved by explicitly stating in the abstract and introduction that the study is based on genetic activation of the hypoxic response, rather than environmental hypoxia.

We appreciate this valuable suggestion and have updated the abstract and introduction to clarify that this work examines genetic activation of the hypoxic response.

Updated sentences from the abstract:

“Here, we interrogate the cell-nonautonomous signaling pathway downstream of genetic activation of the hypoxic response.” 

“Together, these insights develop a circuit for how genetic induction of the hypoxic response cell-nonautonomously modulates ageing and suggests valuable targets for modulating ageing in mammals.”

Updated sentences from the introduction:

“In this study, we uncover key neural components of the longevity circuit initiated by genetic induction of the hypoxic response. Within this circuit, we identify individual cells, signals, and receptors necessary and/or sufficient to extend lifespan downstream of genetic activation of the hypoxic response. More specifically, we find serotonin signaling in the ADF serotonergic neurons is both necessary and sufficient to extend lifespan through genetic activation of the hypoxic response. This pathway signals through the serotonin receptor SER-7 in the RIS interneuron. We further demonstrate additional neurotransmitters (GABA and tyramine), and a neuropeptide (NLP-17) are critical for mediating these longevity effects. Finally, we identify that oxygen sensing neurons (URX, AQR, PQR and BAG) act downstream of neuronal HIF-1 in this circuit. Our insights into this longevity pathway provide a mechanistic understanding of how genetic activation of the hypoxic response delays aging and improves health.”

(b) In the discussion, clearly delineating which parts of the proposed pathway are firmly established versus inferred would aid interpretation.

We thank the reviewer for this idea, and have added the following text to the discussion:

“Evidence for the necessity, sufficiency, and epistatic relationships between each signal are summarized in new Fig. 7B. In brief, all signaling molecules presented in this work are necessary for vhl-1 to extend lifespan. Rescuing ADF serotonin production, RIS ser-7 expression, and RIM tyramine production is sufficient for vhl-1 to extend lifespan. The sufficiency of oxygen sensing neurons BAG and UPA/PQR/AQR, and the neuronal signals of GABA, NLP-17, and TYRA-3 to restore vhl-1 mediated longevity remains unclear. All signals act downstream of vhl-1. ADF HIF-1 stabilization and SER-7 signaling act upstream of fmo-2 induction, and the oxygen sensing neurons (BAG, UPA/PQR/AQR) act upstream of or in parallel to ADF HIF-1 stabilization.”

(c) Adding a summary table or schematic that visually distinguishes necessity vs. sufficiency for each component (e.g., ADF, RIS, RIM, NLP-17) would make the overall model more accessible.

We thank the reviewer for this great suggestion and have added a table to new Fig. 7B that summarizes what is known about necessity for vhl-1, sufficiency for vhl-1, and sufficiency to extend lifespan independently of vhl-1 for each signal (table included in response to Reviewer 2, comment 1a).

(3) Minor corrections and clarifications:

(a) Define or replace "hypoxic response" with "genetically induced hypoxic response" where appropriate to avoid conflating genetic manipulations with actual environmental hypoxia.

We appreciate this valuable suggestion and have replaced “the hypoxic response” and with “genetic activation of the hypoxic response” or “genetically induced hypoxic response” throughout the manuscript to clarify this point.

(b) All genes and alleles should be italicized per worm nomenclature.

We thank the reviewer for this comment and have reviewed the manuscript to italicize all gene names and alleles. In some locations, the protein is referred to instead of the gene using the conventional uppercase non-italicized format.

Reviewer #3 (Recommendations for the authors):

(1) Using vhl-1 RNAi as the sole approach to demonstrate hypoxic response appears somewhat limited, as vhl-1 is also involved in HIF-1 independent processes that can influence lifespan in C. elegans. Including additional downstream effectors of HIF-1, such as egl-9, or having HIF-1 nondegradable strain as validation could strengthen the findings.

We thank the reviewer for this valuable comment and agree that an important next step is to test whether these signals are also required for other genetic (HIF-1 stabilized, egl-9) and environmental activators of the hypoxic response to extend lifespan. We have worked to clarify that this paper focuses primarily on vhl-1 mediated longevity throughout the text and have also included this limitation in our discussion section (for details, please see response to Reviewer 2 public review).

(2) Investigating how the healthspan is affected by serotonergic neuron-specific hypoxic responses would be interesting and could enhance understanding of the physiological mechanisms underlying lifespan extension. 

We appreciate this suggestion, and have performed three measurements of healthspan (pumping, thrashing, and maximum velocity), in the ADF and NSM HIF-1 stabilized strains at young adulthood and at middle age. We found that both ADF- and NSM-specific HIF-1 stabilization had no effect on pumping rate in young (day 1 of adulthood) worms. In aged animals (day 12 of adulthood), however, NSM-, but not ADF-, specific HIF-1 stabilization rescued the pumping rate decline in hif-1 knockout compared to WT worms (new Fig. S1C). Similarly, NSM-, but not ADF-, specific HIF-1 stabilization rescued the thrashing rate decline in the hif-1 knockout young and aged worms (new Fig. S1D). ADF and NSM HIF-1 stabilization also had no effect on average or maximum movement speed at days 1 and 5 of adulthood (new Fig. S1E-F). Together, these results indicate that genetic activation of the hypoxic response in NSM neurons but not in the ADF neurons could improve healthspan.

(3) While the experiments were thoroughly performed, the connections between components such as NLP-17, GABA, and tyramine in regulating aging appear critical for establishing a "cell non-autonomous circuit." Additionally, how the potentially antagonistic roles of RIM and RIS neurons influence this axis could be interesting to further explore.

We thank the reviewer for identifying this important caveat. As described in the public review response to Reviewer 2, we completely agree that understanding the epistasis of serotonin, tyramine, NLP-17, and oxygen-sensing neuron signaling within this pathway is important to fully test our working model. Our current data showed that intestinal fmo-2 is required for neuronal HIF-1 stabilization to extend lifespan, indicating information must be communicated between the nervous system and the intestine through serotonin, tyramine, NLP-17 and responsible neurons using a “cell non-autonomous circuit” [22]. However, we will continue to address questions about epistasis and interactions between different signals in upcoming projects to fully establish the circuit.

With respect to RIM and RIS, we value this suggestion and agree that there could be interesting signaling occurring between RIM and RIS in this circuit, as is observed in initiation of reversal behaviors [16,21]. We have updated the discussion section to mention this interesting antagonistic relationship between RIM and RIS signaling in the context of reversal behaviors:

“Finally, while the use of RNAi knockdown and genetic knockouts establishes the necessity of many signals within the vhl-1-mediated longevity circuit, the exact directionality of these signals remains unclear. It is possible that increased, decreased, or pulsatile changes in signaling through these bioamines and neuropeptides are required for genetic activation of the hypoxic response to extend lifespan. Work on C. elegans reversal behavior has also revealed an antagonistic relationship between RIM and RIS activity facilitated by both chemical (neuropeptide and tyramine) and electrical (gap junction) signaling [16,21]. This known interaction should also be interrogated in the context of how these cells may communicate following genetic induction of the hypoxic response. Future work in this area could use tools to measure or modify neuronal activity, such as calcium imaging or optogenetics, to begin answering these questions.”

(4) Does serotonergic neuron-specific rescue impact the mitochondrial unfolded protein response (mtUPR), given that serotonin signaling has been shown to modulate mtUPR?

We appreciate this question and suggestion. To determine whether activating the hypoxic response in serotonergic neurons modifies the mt-UPR, we measured hsp-6 expression via qPCR in the ADF and NSM-specific HIF-1 stabilized strains. Interestingly, we find that stabilizing HIF-1 in either the ADF or NSM serotonergic neurons decreases hsp-6 expression relative to WT worms (new Fig. S1G. This could suggest either that the mt-UPR response is impaired in these worms, or that HIF-1 stabilization decreases proteotoxic stress leading to a lower basal level of hsp-6. Although this method of measurement did not allow us to interrogate whether these changes occur in the specific tissues where fmo-2 is upregulated, we have expanded our discussion of these results to emphasize that further investigation of this response should be a focus of future work.

(5) To remain consistent with the flow of Figure 1, the authors should include fmo-2 expression in NSM:HIF-1S in addition to the ADF:HIF-1S (Figure 1I).

We appreciate this suggestion and have added NSM::HIF-1S data to Fig. 1I. We found that stabilizing HIF-1 in either the ADF or the NSM has a similar effect on fmo-2 induction.

(6) It would greatly strengthen the RIS observation if the authors demonstrated that ablation of another neural subtype from their screen does not abolish lifespan extension by vhl-1 RNAi. This would be a good supplemental figure, but it is not necessary for the overall story.

We thank the reviewer for this suggestion and agree that ablating a ser-7 expressing neuron that was not a hit from our screen would be an excellent additional control. We did find that ablating a non-ser-7-expressing neuron (the RIC, Fig. 3E) did not affect vhl-1 mediated longevity, suggesting that impairing the signaling of any interneuron is not sufficient to disrupt the phenotype. In addition, ablating a ser-7 expressing neuron other than RIS would provide much stronger support for this finding. We have suggested this approach to further validate this working model in the future directions section of our discussion.

“Finally, additional genetic controls could better support the role of RIS-specific ser-7 expression in genetic activation of the hypoxic response. For example, a ser-7 expressing neuron that was not a hit in our screen could also be ablated and tested for necessity in vhl-1 mediated longevity. This experiment would test whether the ability of RIS ablation to attenuate vhl-1-mediated longevity is not a false positive driven by any disruption to ser-7 expression.”

(7) For consistency with the rest of the manuscript, it would strengthen the hypothesis if modulating the expression of nlp-17 or its receptors impacted the intestinal activation of fmo-2 transcription.

This is a great point. We attempted this experiment, but were unable to achieve consistent results (see Author response image 1). This result could be due to indirect effects of the overexpression of nlp-17 signaling modulating fmo-2 induction in a complicated circuit, variability in expression of its receptor, or other complexities within the circuit.

Author response image 1.

(8) It would strengthen the manuscript to determine whether serotonergic, GABA, and/or tyramine signaling activate the expression or secretion of this nlp-17 neuropeptide.

We thank the reviewer for this great idea of experiment. To address this suggestion, we performed qPCR to measure nlp-17 mRNA in WT, hif-1 KO, ADF HIF-1 stabilized, and NSM HIF-1 stabilized strains. Compared to WT and hif-1 KO controls, we observed no change in nlp-17 expression when HIF-1 was stabilized in the ADF or NSM serotonergic neurons (new Fig. S5F). This could suggest either that nlp-17 signaling acts in parallel to serotonergic signaling following genetic activation of the hypoxic response. Alternatively, neuronal HIF-1 stabilization may modify nlp-17 splicing or translation without resulting in detectable differences in mRNA levels. Together, these data indicate NLP-17 signaling is required for longevity following genetic activation of the hypoxic response, although whether this peptide is synthesized or released in response to hypoxic response remains unclear.

We agree that it is also important to connect nlp-17 expression and/or secretion to other components of this pathway. However, we believe the most effective experiment to confirm a connection between GABA and tyramine signaling and nlp-17 in the context of hypoxia would be to measure nlp-17 expression in strains that manipulate GABA and/or tyramine signaling in a manner that mimics vhl-1 knockdown and extends lifespan. Because we have not yet validated hypoxic-response mimetics for these specific signals, we hope to first generate these strains and then measure their effect on nlp-17 expression in future work. The importance of identifying manipulations to GABA and tyramine signaling that promote longevity has been added to our discussion section. 

“While many individual neurosignaling components are essential for genetic activation of the hypoxic response to extend lifespan, their epistasis is unclear (Fig. 7B). Most components of the pathway identified in this work act downstream of vhl-1, and upstream of fmo-2 induction (summarized in Fig. 7A-B). However, the order of each signal between these two endpoints is only predicted based on C. elegans neural wiring and the overlap between various identified signals and cells. For example, we hypothesize in our working model that GABA may be produced by the RIS neuron in this circuit because RIS is the primary GABAergic neuron required for vhl-1-mediated longevity. Alternatively, it is possible that GABA is produced by a different cell that either acts in series or in parallel with RIS signaling. In order to determine the order of each signaling component, future studies should generate genetic manipulations to each signaling component that may mimic vhl-1 knockout to promote longevity, cross these new strains into knockouts of other signals required for vhl-1 mediated longevity; and measure the lifespans of each double and triple mutant. This approach would also narrow down which signals are downstream of the genetic activation of the hypoxic response, and which are sufficient to extend lifespan upstream of the hypoxic response in a normoxic environment. One notable target for further exploration is the SER-7 expressing RIS neuron, which plays a role in sleep [16] and stress resistance [17], and can extend lifespan when optogenetically activated under normoxic conditions [18].”

References:

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(3) Yuan, R., Hascup, E., Hascup, K. & Bartke, A. Relationships among Development, Growth, Body Size, Reproduction, Aging, and Longevity - Trade-Offs and Pace-Of-Life. Biochemistry (Mosc) 88, 1692-1703 (2023). https://doi.org/10.1134/S0006297923110020

(4) Mautz, B. S., Lind, M. I. & Maklakov, A. A. Dietary Restriction Improves Fitness of Aging Parents But Reduces Fitness of Their Offspring in Nematodes. J Gerontol A Biol Sci Med Sci 75, 843-848 (2020). https://doi.org/10.1093/gerona/glz276

(5) Duric, V., Clayton, S., Leong, L. M. & Yuan, L.-L. Comorbidity Factors and Brain Mechanisms Linking Chronic Stress and Systemic Illness. Neural Plasticity 2016, 1-16 (2016). https://doi.org/https://doi.org/10.1155/2016/5460732

(6) Mariotti, A. The Effects of Chronic Stress On Health: New Insights Into the Molecular Mechanisms of Brain–Body Communication. Future Science OA 1 (2015). https://doi.org/https://doi.org/10.4155/fso.15.21

(7) Bellier, A., Chen, C.-S., Kao, C.-Y., Cinar, H. N. & Aroian, R. V. Hypoxia and the Hypoxic Response Pathway Protect against Pore-Forming Toxins in C. elegans. PLoS Pathog 5, e1000689 (2009).

(8) Palazon, A., Goldrath, W. A., Nizet, V. & Johnson, S. R. HIF Transcription Factors, Inflammation, and Immunity. Immunity 41, 518-528 (2014). https://doi.org/https://doi.org/10.1016/j.immuni.2014.09.008

(9) Vora, M. et al. The hypoxia response pathway promotes PEP carboxykinase and gluconeogenesis in C. elegans. Nature Communications 13 (2022). https://doi.org/https://doi.org/10.1038/s41467-022-33849-x

(10) Nakazawa, S. M., Keith, B. & Simon, C. M. Oxygen availability and metabolic adaptations. Nature Reviews Cancer 16, 663-673 (2016). https://doi.org/https://doi.org/10.1038/nrc.2016.84

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(16) Costa, S. W. et al. A GABAergic and peptidergic sleep neuron as a locomotion stop neuron with compartmentalized Ca2+ dynamics. Nature Communications 10 (2019). https://doi.org/10.1038/s41467-019-12098-5

(17) Wu, Y., Masurat, F., Preis, J. & Bringmann, H. Sleep Counteracts Aging Phenotypes to Survive Starvation-Induced Developmental Arrest in C. elegans. Curr Biol 28, 3610-3624.e3618 (2018). https://doi.org/10.1016/j.cub.2018.10.009

(18) Busack, I. & Bringmann, H. A sleep-active neuron can promote survival while sleep behavior is disturbed. PLOS Genetics 19, e1010665 (2023). https://doi.org/10.1371/journal.pgen.1010665

(19) Powell-Coffman, J. A. & Coffman, C. R. Apoptosis: Lack of oxygen aids cell survival. Nature 465, 554-555 (2010). https://doi.org/10.1038/465554a

(20) Kruempel, J. C. P. et al. Hypoxic response regulators RHY-1 and EGL-9/PHD promote longevity through a VHL-1-independent transcriptional response. Geroscience 42, 1621-1633 (2020). https://doi.org/10.1007/s11357-020-00194-0

(21) Bach, M., Bergs, A., Mulcahy, B., Zhen, M. & Gottschalk, A. (2023).

(22) Leiser, S. F. et al. Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span. Science (2015). https://doi.org/10.1126/science.aac9257

I dont see this in the Relations button as an option Listed as Predecessor and Successor

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eLife Assessment

This important study combines anatomical tracing, tissue clearing, and functional manipulations to demonstrate lateralized brainstem control of hepatic glucose metabolism and identify a site of sympathetic nerve crossover supplying the liver. The evidence supporting the anatomical organization of hepatic sympathetic innervation is compelling, and the functional studies provide solid support for a role of asymmetric sympathetic outflow in regulating glucose homeostasis. While some uncertainty remains regarding the contribution of sensory innervation and the extent to which these findings generalize beyond mice, the work provides an invaluable advance in understanding neural regulation of liver metabolism.

Reviewer #2 (Public review):

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

Summary:

The manuscript by Wang and colleagues aims to determine whether hepatic glucose metabolism is differentially regulated by the left and right sides of the LPGi and to reveal decussation of hepatic sympathetic nerves.

The authors used tissue clearing to identify sympathetic fibers in the liver lobes, then injected PRV into the hepatic lobes. Five days post-injection, PRV-labeled neurons in the LPGi were identified. The results indicated contralateral dominance of premotor neurons and partial innervation of more than one lobe. The authors then activated each side of the LPGi, resulting in a greater increase in blood glucose levels after right-sided activation than after left-sided activation, and in changes in protein expression in the liver lobes. These data suggested lobe-specific modulation of HGP. Chemical denervation of a particular lobe did not affect glucose levels due to compensation by the other lobes. In addition, nerve bundles decussate in the hepatic portal region.

Strengths:

The manuscript is timely and relevant. It is important to understand the sympathetic regulation of the liver and the contribution of each lobe to hepatic glucose production. The authors use state-of-the-art methodology.

Weaknesses:

(1) Image clarity was improved in some cases, but not in others. For example, Figure 3I, showing c-Fos expression, is not convincing due to the image quality and lack of orientation.

(2) The methods section states that 8-week-old male mice were used in the experiments without specifying the experiments (e.g., brain injection with AAVs or PRV organ inoculation). The authors should include these details.

(3) The authors should use the exact location of pre- and postganglionic neurons, as they often refer to neurons in the sympathetic chain. Their findings should be compared with the existing literature on the location of preganglionic cells.

(4) Figure legends should be revised and matched with the text.

Reviewer #4 (Public review):

Summary of General Strengths & Weaknesses:

The studies here are highly informative for anatomical tracing and sympathetic nerve function in the liver in relation to glucose levels, but because they are conducted in a single species, it is challenging to translate them to humans or determine whether these neural circuits are evolutionarily conserved. Dual-labeling anatomical studies are elegant, and the addition of chemogenetic and optogenetic studies provides mechanistically informative. Denervation studies lack proper controls, and sensory innervation in the liver is overlooked.

Specific Weaknesses - Major:

(1) The species name should be included in the title.

(2) Tyrosine hydroxylase was used to mark sympathetic fibers in the liver, but this marker also labels a portion of sensory fibers that need to be ruled out in whole-mount imaging data.

(3) Chemogenetic and optogenetic data demonstrating hyperglycemia should be described in the context of prior work demonstrating liver nerve involvement in these processes. The Discussion currently mentions this only briefly, but comparing methods and observations would be helpful.

(4) Sympathetic denervation with 6-OHDA can drive compensatory increases in tissue sensory innervation, and this should be measured in the liver denervation studies to implicate potential crosstalk, especially given the increase in LPGi cFOS that may be due to afferent nerve activity. Compensatory sympathetic drive may not be the only culprit, though that is clearly assumed. The sensory or parasympathetic/vagal innervation of the liver is altogether ignored in this paper and could be better described in general.

Comments on the revised version.

Across all reviewer comments, the revised resubmission has adequately addressed all concerns.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #2 (Public review):

Summary:

The manuscript by Wang and colleagues aims to determine whether hepatic glucose metabolism is differentially regulated by the left and right sides of the LPGi and to reveal decussation of hepatic sympathetic nerves.

The authors used tissue clearing to identify sympathetic fibers in the liver lobes, then injected PRV into the hepatic lobes. Five days post-injection, PRV-labeled neurons in the LPGi, which were identified. The results indicated contralateral dominance of premotor neurons and partial innervation of more than one lobe. Then the authors activated each side of the LPGi, resulting in a greater increase in blood glucose levels after right-sided activation than after left-sided activation, and in changes in protein expression in the liver lobes. These data suggested lobe-specific modulation of HGP. Chemical denervation of a particular lobe did not affect glucose levels due to compensation by the other lobes. In addition, nerve bundles decussate in the hepatic portal region.

Strengths:

The manuscript is timely and relevant. It is important to understand the sympathetic regulation of the liver and the contribution of each lobe to hepatic glucose production. The authors use state-of-the-art methodology.

Weaknesses:

(1) Image clarity was improved in some cases, but not in others. For example, Figure 3I, showing c-Fos expression, is not convincing due to the image quality and lack of orientation.

We sincerely apologize for the insufficient image clarity and anatomical orientation in the original Figure 3I. To resolve this issue, we have performed the following revisions in the revised Figure 3I:

(1) Replaced the original panels with the high-resolution confocal images showing clear c-FOS immunofluorescence in the LPGi.

(2) Included explicit anatomical orientation indicators (Bregma −6.75 mm) to clearly demarcate the boundaries of the LPGi.

(3) Added ROI outlines surrounding the LPGi region.

(2) The methods section states that 8-weeks-old male mice were used in the experiments without specifying the experiments (e.g., brain injection with AAVs or PRV organ inoculation). The authors should include these details.

We thank the reviewer pointing out this oversight. We have updated the Methods section under "Animals" and specific procedure subsections to clearly state the exact age of animals.

(1) For retrograde trans-synaptic PRV tracing, 8-week-old mice received intrahepatic viral injections and were sacrificed 5 days post-injection.

(2) For chemogenetic and optogenetic manipulations, stereotaxic AAV injections were performed at 8 weeks of age. Mice were allowed 4 weeks for viral expression and recovery before undergoing metabolic tests or light stimulation at 12 weeks of age.

(3) For chemical denervation (6-OHDA), 8-week-old mice were injected into targeted lobes and examined 7 days post-denervation.

(4) For postnatal innervation mapping, neonatal mice at postnatal week 0 (P0), week 1 (P7), and week 2 (P14) were harvested for tissue clearing.

(3) The authors should use the exact location of pre- and postganglionic neurons as they often refer to neurons in the sympathetic chain. Their findings should be compared with the existing literature on the location of preganglionic cells.

We appreciate the reviewer for this feedback. We agree that our original description lacked precise anatomical localization regarding the pre- and postganglionic neurons, and it was inaccurate to state that descending fibers pass through the sympathetic chain (SyC).

Based on our whole-mount tissue clearing data, we observed that the preganglionic neurons of the brain-liver sympathetic circuit are primarily located in the T6–T12 segments of the thoracic spinal cord. Accordingly, we have revised the text in Results 4 to specify these exact locations.

Manuscript Revision (Results 4):

"Using whole-mount clearing, we visualized the brain–liver sympathetic circuit and found that preganglionic neurons in the thoracic spinal cord (T6–T12) send descending fibers via the splanchnic nerves to innervate postganglionic neurons in the CG-SMG (Figure 4A)."

Furthermore, following your valuable suggestion to compare our findings with existing literature, we reviewed a recent study published in Nature Communications (Harima, Yukiko et al. Parallel labeled-line organization of sympathetic outflow for selective organ regulation in mice. Nat Commun. 2024;15(1):10478). In that study, researchers injected retrogradely transducible AAVs directly into the CG-SMG and traced the preganglionic neurons predominantly to the T8–T13 segments. Their results are largely consistent with our findings. Interestingly, the broader anatomical range observed in our trans-synaptic liver-to-brain mapping (T6–T12) compared to their CG-SMG-specific tracing (T8–T13) reveals a slight discrepancy. This observation suggests an intriguing anatomical hypothesis: a subset of sympathetic preganglionic nerves may bypass the CG-SMG relay entirely and project directly to the liver.

(4) Figure legends should be revised and matched with the text.

We apologize for the oversight. We have conducted a comprehensive audit of all figure and legends to ensure precise matching between the main text and the figures.

Specifically, we have corrected a typographical error in the Figure 1 Legend where panel (C) was mistakenly labeled as a second panel (B), and we fixed a spelling error ("LPG" corrected to "LPGi"). Additionally, we corrected a miscitation in Results (Section 3) regarding Figure 3. In the original text, Figure 3C was incorrectly grouped with blood glucose data, whereas it actually displays the Western blot validation of sympathetic denervation.

We have revised the corresponding sections in the manuscript as follows:

Manuscript Revision (Figure 1 Legend):

“(C) Quantification of PRV-labeled neurons in left and right LPGi across different hepatic lobes: left lateral, median, right posterior, right anterior, caudate, and porta hepatis (n = 3).

(D) Sankey diagram showing projection patterns from left and right LPGi to individual hepatic lobes. (E and F) Representative slices of EGFP+ and mRFP+ neurons in left (top) and right (bottom) LPGi following PRV-EGFP (right anterior lobe) and PRV-mRFP (median lobe) injections. Proportions of EGFP+, mRFP+, and co-labeled neurons in left and right LPGi (F, n = 3). Scale bars, 100 μm.”

Manuscript Revision (Results 3):

“Despite the absence of directly sympathetic input to denervated lobes, systemic blood glucose levels were unchanged compared with controls (Figures 3A-3B, Figure S5A), indicating functional compensation through the remaining intact liver.”

Reviewer #4 (Public review):

Summary of General Strengths & Weaknesses:

The studies here are highly informative for anatomical tracing and sympathetic nerve function in the liver in relation to glucose levels, but because they are conducted in a single species, it is challenging to translate them to humans or determine whether these neural circuits are evolutionarily conserved. Dual-labeling anatomical studies are elegant, and the addition of chemogenetic and optogenetic studies provides mechanistically informative. Denervation studies lack proper controls, and sensory innervation in the liver is overlooked.

We sincerely thank the reviewer for their time and evaluation. We respectfully note that these comments mirror those raised during the previous round of review. We would like to kindly direct the reviewer to the extensive revisions we implemented in our previous resubmission, which directly and comprehensively addressed these exact concerns. These revisions remain intact in the current version of the manuscript. Below, we briefly summarize how each point was previously addressed for your convenience.

Specific Weaknesses - Major:

(1) The species name should be included in the title.

As addressed in our previous revision, we fully agree with this suggestion. We updated the title of the manuscript to explicitly include the species: "Symmetric brain-liver circuits mediate lateralized regulation of hepatic glucose output in mice." We also clarified the species used throughout the main text to ensure accuracy.

(2) Tyrosine hydroxylase was used to mark sympathetic fibers in the liver, but this marker also labels a portion of sensory fibers that need to be ruled out in whole-mount imaging data.

As detailed in our previous response, we acknowledge this important limitation. In our prior revision, we addressed this concern through both additional data analysis and text revisions:

(1) We provided SyGlass 3D reconstruction data demonstrating that the TH-positive nerve fibers originate from the celiac-superior mesenteric ganglia (CG-SMG), a well-established sympathetic ganglion (Figure S5F).

(2) In parallel, we collected dorsal root ganglia (DRG) from spinal segments T1-6 and T7-12 five days after intrahepatic PRV injection. While the T7-12 DRG segments are historically known to contain the sensory neurons that innervate the liver (Anat Rec A Discov Mol Cell Evol Biol. 2004; Auton Neurosci. 2024), we detected only a remarkably sparse number of PRV-positive neurons in these segments. This effectively functionally distinguishes this efferent pathway from primary sensory afferents (Supplementary figure B).

(3) We explicitly added this methodological limitation to the Discussion section (paragraph 6) of the current manuscript, noting that more selective approaches, such as genetic targeting of sympathetic lineages, will be important for future validation."

(3) Chemogenetic and optogenetic data demonstrating hyperglycemia should be described in the context of prior work demonstrating liver nerve involvement in these processes. There is only a brief mention in the Discussion currently, but comparing methods and observations would be helpful.

As outlined in our previous response, we incorporated this crucial context into our revised manuscript. Specifically, we expanded the Discussion section (paragraph 3) to contrast our precise cell-type-specific chemogenetic and optogenetic approaches with historical studies that relied on coarse electrical stimulation. This addition highlights how our current methodology reveals the contralateral and lobe-specific architecture of brain-liver sympathetic control that was previously obscured.

(4) Sympathetic denervation with 6-OHDA can drive compensatory increases in tissue sensory innervation, and this should be measured in the liver denervation studies to implicate potential crosstalk, especially given the increase in LPGi cFOS that may be due to afferent nerve activity. Compensatory sympathetic drive may not be the only culprit, though that is clearly assumed. The sensory or parasympathetic/vagal innervation of the liver is altogether ignored in this paper and could be better described in general.

We appreciate this insightful physiological perspective, which we addressed comprehensively in our previous revision. As we previously agreed, the central nervous system integrates a broad range of afferent signals, and compensatory sensory or parasympathetic mechanisms likely contribute to the observed LPGi activation following hepatic sympathetic denervation.

To address this, we significantly expanded our Discussion section (paragraph 4) in the prior revision. We explicitly proposed a model wherein hepatic glucose production is regulated by an integrated afferent-central-efferent loop, acknowledging that our current study primarily resolves the efferent component. We clearly noted the lack of direct assessment of sensory or parasympathetic innervation as a limitation and highlighted this dynamic crosstalk as a critical avenue for future investigation.

Comments on the revised version.

Across all reviewer comments, the revised resubmission has adequately addressed all concerns.

Recommendations for the authors:

Reviewer #4 (Recommendations for the authors):

No further recommendations aside from tempering the CGRP language, as marking all sensory fibers.

We appreciate the reviewer for pointing out this important anatomical distinction. We entirely agree that CGRP specifically labels peptidergic sensory afferents and does not represent the entirety of the sensory nervous system.

We have carefully reviewed the entire manuscript and tempered our language accordingly. Wherever CGRP is mentioned, we have clarified that it serves as a marker for peptidergic sensory fibers, rather than functioning as a pan-sensory marker.

Manuscript Revision (Results 1):

"Unlike the NTS, a well-established hepatic sensory center served here as a positive control, the LPGi contained few CGRP-positive cell bodies (Figure S1G), indicating a lack of peptidergic sensory projections."

This quote sits with me because this is something I've personally been guilty of when I don't agree with an article or it makes me uncomfortable, so I just view it as biased and don't bother reading it. This quotation I've highlighted confirms that we tend to trust sources that agree with our point of view and if it doesn't, then we disregard it. I think the outcome this article is looking for is to get students to evaluate sources more thoroughly rather than getting students to believe less. Healthy skepticism should not mean believing that no sources are trustworthy or that all sources are equal. In my classroom I want to have my students turn their minds on and ask "is this source well supported?" instead of does this support my point of view. This is the kind of unbiased and skepticism I want to bring to my classroom.