Experiments on #sleep-deprived fruit flies suggest that reactive oxygen species build up in the gut & cut their lifespans in half.
Why do we need sleep? In their long search for answers, scientists have often uncovered only more thought-provoking mysteries about what sleep is, how it evolved and the benefits that it provides. In this episode, Steven Strogatz — the noted mathematician, author and host of The Joy of Why — speaks with Dragana Rogulja, an assistant professor of neurobiology at Harvard Medical School who recently discovered how sleep deprivation causes death in fruit flies. Then he continues the conversation with Alex Keene, a neurogeneticist at Texas A&M University who studies cave fish to understand more about the evolutionary history of sleep.
Steven Strogatz (00:03): I’m Steve Strogatz, and this is The Joy of Why, a podcast from Quanta Magazine that takes you into some of the biggest unanswered questions in science and mathematics today. Today, we’re going to be talking all about sleep.
Why do we sleep anyway? We spend about a third of our lives asleep, so it seems like it must be pretty important. But there’s still so much about it that we don’t understand. One thing that sleep researchers are pretty sure of is that every system in our body seems to be impacted by sleep. When we miss out on sleep, it impairs our circulation, our digestion, immune system, metabolism, and of course, brain function. And sleep deprivation doesn’t need to be long term to do damage. In fact, if you go without sleep long enough, you will die. But why, exactly?
Dragana Rogulja knows something about that. We’ll be talking with her in a minute. She’s an associate professor of neurobiology at Harvard Medical School. She studies why we need to sleep, and how the brain switches between being asleep and being awake. She also looks at the lethal effects of sleep deficiencies.
Later, we’ll be hearing from Alex Keene of Texas A&M University, who studies the neural regulation of sleep, and the role it plays in the bigger picture of how sleep has evolved. He does that by looking in an unexpected place — in fish that live in caves in Mexico. But first, Dragana Rogulja, thank you so much for joining us today.
Dragana Rogulja (01:35): Thank you so much for inviting me. This is incredible.
Strogatz (01:38): Yeah, I’m super excited to talk to you about your work. But first, I was hoping we could talk broadly about sleep in general. Like, we all understand that, you know, like, I remember asking my mother, “Why do I have to go to sleep?” when I was a little kid. And she said, well, because you’re tired, it’s going to help you get rest. But sleep seems like something really different than just mere rest. Because in sleep, we have this whole altered state of consciousness. How is sleep really different from rest?
Rogulja (02:07): Well, you’re probably sitting right now and kind of resting in some way. But you’re definitely not sleeping, right? So, yeah, what is it that’s so different? And I would say that, for me, what is the kind of most defining characteristic of sleep is that kind of loss of awareness of the external environment and of your internal state, in many ways.
Usually, when we study sleep in humans or other mammals, we do these recordings where we look at electrical activity of the brain, right, and you can see these waves change, and you can’t do that in simple animals that sleep. Yet we know that they really do enter these states where they disconnect. They stop moving, but you can stop moving anyway, right? So, you stop moving, but this is coupled with that loss of awareness, relaxation of the body. And I think it’s a tricky thing, asking why. I think we want to get at the why. But the way that we want to get to the why is by asking how. What are the most primitive things about sleep that we can understand?
Strogatz (03:09): I notice you mention other animals besides people. I mean, we have this very, naturally, human-centric view of what sleep is about, we think about our dreams. But as you say, maybe to get at the how questions we should be looking, possibly, at other animals? Who sleeps in the animal world?
Rogulja (03:24): What we think today is really that sleep is as old as animals themselves. So there are these animals that we refer to as the living fossils, because supposedly, they haven’t changed much throughout animal evolution. And as we look at the simple animals, like jellyfish, and Hydra now, so animals that have very, very primitive nervous systems. It is very clear that they engage in these forms of behavior that I would say, for all practical purposes are really like our sleep. They disconnect, they stop paying attention to what’s going on around them, they can’t respond to external stimulation, unless that stimulation is very strong. So we see that in basically the simplest animals.
Strogatz (03:25): That’s amazing. I hadn’t really heard about this
Rogulja (03:26): The biggest problem to me is that there’s too much focus on the relationship between the brain and sleep because we tend to think of it from our human-centric perspective, like you said, because we dream, et cetera. But if we accept the fact that these simple animals sleep, then we really have to think beyond the brain because we did not appear with these big, beautiful brains, you know, just out of nowhere.
Strogatz (04:33): Yeah, it’s a really interesting point. So, okay, so let’s try to think a little less about brains. Allan Hobson had this remark that sleep is “of the brain, for the brain and by the brain.”
Rogulja (04:44): Yeah, I don’t think that’s right. I don’t think that’s right. I don’t think that’s right. I mean, it is for the brain for sure. But it is also for many other — for all the other, you know, pieces of our body, I think, really. So I don’t think that that’s right. Even saying it’s by the brain or of the brain, I mean animals that don’t have a brain, that have very simple nervous systems, very simple nervous systems, really rudimentary nervous system, they do sleep. So I guess that could be a sort of semantics issue, you know, is it a brain or not a brain, but I don’t even think it’s just for the nervous system.
And actually, we have some evidence now that, you know, it’s not even just of the brain, other places in the body can regulate sleep. I mean, we and others have evidence for that, that, you know, signals to regulate sleep can come from other places in the body. And it’s not just for the brain, we also have evidence for that.
Strogatz (05:35): So much evidence has accumulated now, thanks to work by you and your colleagues. You have made use of a wonderful model organism, we call it from our, again, from our human-centric perspective. I mean, I don’t know if they think of themselves as model organisms: the fruit fly, we know so much about them genetically, developmentally. And now you’re using them to teach us about what sleep might be for.
Rogulja (05:59): Yeah. So it was shown several decades ago that sleep in flies checks off all those boxes. You know, there’s certain criteria that you have to pass to be considered sleeping. And it was shown that this is really the case in flies. They do enter these states where they stay immobile for many hours, and of course, being immobile does not mean you’re sleeping. But again, they do enter this state where they disconnect from their external environment to a large degree. Same thing as happens to us, right? When you’re sleeping, you just — you don’t respond to stimulation.
They’re amenable to all kinds of genetic manipulations. And it became clear that they need sleep, in a sense that if you prevent them from sleeping, bad things can happen and they can die. We’re using flies, primarily, in the lab. And then when we find something that we think is kind of a, you know, an important discovery, then we test those findings in mice, and that does give us more confidence. And so far, things that we have been cross-checking, it’s pretty much the same, you know, and I’ve really come to kind of think that flies are just like us. Plus, they can fly. So it’s very, very cool.
Strogatz (07:08): That’s amazing. I mean, because it’s — not many — other than the movie The Fly, I mean — not many people would think of themselves as connected to flies, but it’s, it’s fantastic the unity of life on Earth, how we can learn so much about ourselves from flies and mice. I mean, we are kind of relatives in a deep way.
Rogulja (07:26): Oh, my god, absolutely. I’ve come to think that we’re all — essentially we’re all the same. The more biology you know, the less can you think, I think, of ourselves as separate from everything else.
Strogatz (07:39): Suppose you do the kinds of experiments that you have done and that people before you have been doing for decades, where you deprive an animal, in this case a fly or maybe a mouse, of sleep, and then ask, if you do that enough, if you make them go without sleep long enough, and they die from it, what exactly killed them? And you have a clue, a very important clue
Rogulja (08:04): Yeah, that’s exactly where we started. So, when I started my lab, this is a question I’ve been interested in for a long time. Why, right? Why do you need to sleep? And “why” is an interpretation, right? We can say, like, okay, this is why it happens. But what we can experimentally, really, show is like, what happens, right? Like how things go.
When I started the lab, a postdoc came to my lab, Alex Vaccaro, who was just ideal for this. And we talked about, kind of, how to approach this question. And we decided to take a new approach where we would be agnostic about the reason for why animals would die without sleep. Exactly trying to stay away from that thinking of sleep is of, for, by the brain. So we just thought, okay, let’s see, if we really deprive flies of sleep, and we try to do it in different ways. So, to have different methodologies, non-overlapping methodologies, and then try to look at their lifespan and see if there’s a certain time that they die. And then, can we find what happens preceding that?
(09:04) We jumped into this with some faith, but I had very little hope, honestly, that we would find something like what we ended up finding, because it just seemed so — I had a feeling, you know, like, all kinds of things could be falling apart, all over the body, right? Even if it’s not the brain, there could be many, many different things happening in the body, and it might be really difficult to pinpoint the exact cause of death.
The first thing that really surprised me was that when you deprive animals of sleep, they crash, they die prematurely. This is in flies, and it was very reproducible when they die. So it really depended on how much sleep they lost. So the more sleep you lose, the faster you die. But if you have different methodologies, which all produce the same loss of sleep, you ended up dying with the same kind of kinetics. So that really surprised me. That happens at a specific time.
The reason why that was important is because it suggested to us that there really might be some specific events, something that we could dig up somewhere in the body. That was the first thing that we were like, okay, if we can find a real correlation between survival and loss of sleep, then maybe we’ll be able to find what’s going on.
Strogatz (10:17): Can I ask you to pause right there? Because you use the word kinetics, and I have a guess what you mean, tell me if this is the right picture. Like, suppose I had 100 flies, and then I start depriving them of sleep, that maybe a certain amount survive one day, and then a certain amount survived to the second day, and so on. And you’re making a graph like that.
Rogulja (10:35): Exactly. That’s right. And then, so what happens is that in the beginning, they all look — everybody’s 100% alive. And then the controls keep on living. And at some point pretty early on, depending on how much sleep you lose. The more sleep you lose, the sooner you crash, these survival curves start going down. So it’s like 80% of sleep-deprived animals are alive, 60%. And so, if we remove all sleep, lifespan can be caught in you know, you live a quarter, your lifespan is a quarter of the control, you know, so it’s a really, yes, it’s a very strong effect.
Strogatz (11:11): That’s brutal. So when you make a graph of this thing that you’re calling the survival curve, the number surviving as a function of the amount of time that you’ve sleep-deprived them, what does the curve look like?
Rogulja (11:22): Yeah, that’s a great question. And something that was really critical in this whole journey. So it seems like that there is a certain point, and that point depends, seemingly, solely on how much sleep you lose, where all of a sudden, these sleep-deprived animals massively start dying. So, under this condition that we looked at first, for example, where the controls live up to 40 days, around day 10, it’s about 90% sleep loss. So around day 10, they start crashing, and by day 20, they’re all dead. So the last of the surviving of these sleep-deprived animals is dead by day 20. And then the controls live to 40 days. And so at day 10 is that inflection point where they start crashing, and that really gave us a window, when to look for bad things that were happening in the body. Yeah, so that was a really critical point.
Strogatz (12:16): And so did you start, like, looking at different organs?
Rogulja (12:18): Yeah, so it was actually very simple. I mean, the idea was very, very simple. What we started from Alex, the postdoc, who started this project, and then Yosef Kaplan Dor, who joined her, another postdoc. The idea was to, now, take all organs that we could take out from the fly, just dissect the whole animal, do pathology on it, so to speak, and look at anything that we could think of, what are some markers of bad things happening? Markers of cell death, markers of DNA being damaged. And so we just looked at everything all over the place. Okay, all over the body. And that was really the critical thing is that we did not limit ourselves to the brain, we just thought, okay, bad things could be happening anywhere, let’s just take all the organs.
And then when we did that, it was actually really, really quick that we got to this surprising answer, which was the bad things were happening in the gut, specifically.
Strogatz (13:12): The gut. That is not obvious. I mean, right? Losing sleep somehow messes something up in the fly’s gut. So, whoa, that’s, that’s really, really interesting.
Rogulja (13:23): Yeah, it was shocking. You know, it’s one of those things that, now, I’ve gotten used to it. It’s been a few years. And it’s just like, yeah, of course, it’s that, you know? But when we first got these results, yeah, it was really weird, you know?
So what Alex did was, one of the things that she looked at was levels of reactive oxygen species. We can talk about that a little bit, these are molecular derivatives of oxygen that are extremely chemically active, they’re very labile. And she saw that exactly around the time when these animals start massively dying, sleep-deprived animals, so there’s a huge, huge, huge increase in reactive oxygen species, specifically in the gut. That was immediately preceding that.
Strogatz (14:04): I’ve heard people talk about free radicals. Is that a different thing?
Rogulja (14:07): So, reactive oxygen species, like the name says, it’s derived from oxygen, very reactive. Free radicals are the most reactive forms of these reactive oxygen species, okay. They’re very, very damaging, but free radicals can be derived, also, not from oxygen, you know, from some other thing. But the critical thing there is that you have an unpaired electron in their outer orbital, in their valence orbital. So that’s the orbital that engages in chemical reactions. And you need electrons to be paired for stability. So these molecules are kind of wobbly. And they attack cellular molecules. They steal electrons, so to speak, from DNA from protein from, from fats, they oxidize them. So this is very similar to rusting, right? Or, like, when you cut an apple, you expose it to air, it gets oxidized. That’s the brown stuff, okay? So you turn these cellular molecules also into dangerous molecules, free radicals, which then attack other things.
Strogatz (15:03): Oh boy.
Rogulja (15:04): Yeah, it was crazy, and so, what happens, what we saw is that you have these reactive oxygen species accumulating, and then you track oxidation of the gut, or what is called oxidative stress. And that’s the thing that I refer to, the fact that you steal electrons from cellular molecules, and you destroy them, and so you — eventually, you see cells dying, massively dying in the gut. And this all happens, and then they die.
Strogatz (15:29): Just to make sure, since it’s been a while since I did chemistry or biochemistry, maybe some of our listeners the same thing. The basic point is, if you, a fly, go without sleep, or are forced to go without sleep for too long, you’re going to build up very abnormally high levels of these reactive oxygen species in your gut. I see them abbreviated as ROS; do you pronounce it as “ross”?
Rogulja (15:49): “Ross”, or reactive oxygen species. ROS, yeah.
Strogatz (15:52): So these reactive oxygen species are kind of like an internal rust or some kind of poison. That’s the point, right?
Rogulja (15:58): Yeah, that’s exactly the point. I think of it as, like, rusting, you know, rusting of a pipe, expose apple to air, it turns brown, that’s oxidation. Oxidation is essentially, you oxidize other molecules means you steal electrons from them. Okay.
So, that’s what happens. That’s what oxidative stress is. And what’s really dangerous about it is that it can propagate, because one molecule attacks another, turns it bad, and that one attacks another. And so this is what we saw. And we saw it in flies, and we saw it with every method of sleep loss that we could think of. And then we checked in mice, and you see the same thing. But the most interesting thing came when we tried to show causation between that and the death that follows. Just because something precedes death, it doesn’t mean that it’s causal, right? It could be correlation.
And so what Alex and Yossi did, and others that, others on the team, was to try to neutralize these molecules. So to get rid of reactive oxygen species in the gut specifically, and then see if this could allow survival, normal life without sleep. And we tried this thinking, okay, it’s a logical thing to do. But did I have any kind of faith that that would work? I would say no, I mean, it really seemed like a fantasy, you know.
(17:17) It was shocking, it was absolutely shocking. We would all gather around and look at these flies every day. I mean, they were just simply fed certain antioxidants, you could neutralize their reactive oxygen species, and they could survive. But when we did this by feeding antioxidants, we were thinking, okay, you — you know, when you eat something you don’t know where that could go and act anywhere in the body, right, just because we’re not seeing ROS in other places, it doesn’t mean that they’re not there. So, then what we wanted to do was to, basically, through genetic manipulations, only do this in the gut.
So you can imagine, if you express an antioxidant, and if you put an antioxidant enzyme — we have these tricks, right? So, antioxidant is something that neutralizes oxidants, like ROS, you only put it in the gut. And then you ask, can this rescue survival? And it does, as long as we get rid of these things in the gut, animals can survive.
Strogatz (18:15): It’s so, it’s so brilliant. I mean, these experiments are so ingenious and sort of so simple that it must make all your colleagues feel like, ah, why didn’t I do that? This field has been — you know, right? But of course, you have the power of genetic engineering now, that’s really been helpful.
Rogulja (18:30): Yeah. I don’t know if they feel like that. But I will say this, yeah, it was very simple. That’s the thing, though, I will tell you. I’ve never been a part of something that went smoother. Like, I’ve never worked on a tougher question, I think, and never been a part of something that went smoother.
Strogatz (18:46): So let me get it. It’s sort of like saying, again, it’s oversimplifying, but it’s sort of like if I did rust remover — not rust remover — but these antioxidants. Just in the gut, that’s enough to save the flies that would have otherwise been dead.
Rogulja (18:58): That’s absolutely right. That’s exactly what happened. And another question that we wanted to understand in the lab is exactly how do you do this sensory disconnect? Like how do you enter this state of sensory disconnect? Why is it that you do not process information the same way when you’re asleep as when you are awake?
And so Iris Titos, another postdoc in the lab, she did this screen, and screen is something where you just try to get rid of a bunch of genes, like, one by one, right? So, we’re looking for manipulations where it would — it would make animals extremely responsive, now, during sleep, or extremely unresponsive, you know, someone who can sleep through an earthquake. And what she found is a signal that originates in the gut. And so this was a completely separate study that also led us to the gut. She found this molecule that’s secreted from the gut, in response to high-protein diet, and that signals to the brain to put you in deeper sleep, to put you in the state of greater sensory disconnect. So that’s an example of where the gut is really dictating the quality of sleep.
Strogatz (20:05): This is wild. This, this reminds you of people saying, after a turkey dinner on Thanksgiving, you’ll have too much tryptophan and you’re going to want to go to sleep.
Rogulja (20:13): In our case, we showed it’s not tryptophan. At least, you know, this molecule is made not in response to specific amino acids, but it’s just sensing how proteinaceous food is. And so for me, the way that I interpret it is, you know, I used to say this thing, which we all say, working in sleep, it’s like, “It’s dangerous. Why would you be in that state, you can’t do anything?” But I actually think you’re better off if you had a good meal, you’re better off hiding somewhere, and not moving, and sleeping, right? So you can essentially afford to disconnect and sleep deeply. And you don’t have to run across the field and look for food and expose yourself to danger.
Strogatz (20:52): Huh, very interesting. So sleep is really tied to digestion, or something.
Rogulja (20:58): Yeah, I think so. I think so.
Strogatz (21:00): Well, that would explain why every animal up and down the, whatever, the tree of life, is going to need to do this, we all have to eat. Everyone knows eating is important. Metabolism is important.
Rogulja (21:11): Well, there’s also something else, you know, like the gut is one of the first organs that, that appeared in animal evolution. And I used to think of it as — before this study, I would not have been fascinated by the gut because you just think, oh, you eat to survive. But think about, like, everything that you’re made of essentially, it has to come through your gut. You have to eat it, extract some molecules, turn it into yourself.
This is like a place close to the middle of your body where wild things happen. You have to break down tissues of other animals, of plants, of different things without harming yourself. It’s nothing else that you can think of in your body is exposed to anything approaching what your gut is exposed to.
Strogatz (21:52): That is a fascinating note to end on. Thank you so much for this really enlightening and delightful conversation, Dragana.
Rogulja (21:59): Thank you, Steve.
Announcer (22:05): The Joy of Why is a podcast from Quanta Magazine, an editorially independent publication supported by the Simons Foundation. Funding decisions by the Simons Foundation have no influence on the selection of topics, guests or other editorial decisions in this podcast, or in Quanta.
Strogatz (22:26): My next guest is Alex Keene. He’s a neurogeneticist who studies sleep. He also heads up the biology department at Texas A&M University. Alex has studied the molecular basis for sleep, and memory formation, in fruit flies. And he’s looked at Mexican cave fish to gain a better understanding of the neural mechanisms of sleep, and how they’ve evolved. Alex, thank you so much for joining us today.
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