About the guest: Luis de Lecea, PhD is a neurobiologist whose lab at Stanford University studies the neural basis of sleep & wakefulness in animals.
Episode summary: Nick and Dr. de Lecea discuss: the neural basis of sleep; sleep architecture & sleep phases (NREM vs. REM sleep); orexin/hypocretin neurons & the lateral hypothalamus; cortisol & stress; circadian rhythms; neuromodulators (norepinephrine, dopamine, etc); sleep across animal species; sleep drugs; ultrasound technology; and more.
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Episode transcript below.
Full AI-generated transcript below. Beware of typos & mistranslations!
Nick Jikomes 3:44
Can you start off by just telling everyone a little bit about who you are and what your lab does?
Luis de Lecea 3:50
Yeah, I'm a professor in the Department of Psychiatry here at Stanford, and my lab has been studying the neural underpinnings of sleep and wakefulness for over 30 years.
Nick Jikomes 4:07
And sleep and wakefulness are really interesting, because in many ways, these are mysterious phenomena. But at the same time, we're all very familiar with these things at a personal level. You know, we all go to sleep and we're all awake. We all know what it feels like to be in a state of high vigilance, in a state of deep sleep and so forth. At a very high level, when neuroscientists talk about behavioral states and brain states, what do those terms mean, and how do you start to think about them?
Luis de Lecea 4:40
Yeah, yeah, that's a great question. So yeah, we talked on the basis that all animals sleep, so all animals have cycles of activity and rest, and we define sleep. Sleep is essentially behavioral state that has one particular a. Property, which is sleep rebound, or sleep homeostasis. That means, as you just mentioned, that when we are awake for, you know, longer period of time, then we feel this urge to go back to sleep, and this sort of sleep drive gets stronger, you know, the longer we've been awake. So that's different from just the circadian cycle where, you know, we just feel that we just want to go to sleep because it's, you know, nighttime. So the neural processes that govern this sleep drive are essentially, are essentially what we're studying when we're studying sleep. And the manifestation of this drive is in changes in the activity of in mammals cortical neurons, and we monitor those changes using an electroencephalogram, which can very broadly, measures cortical, cortical activity. So you know, sleep and wakefulness can be defined objectively with an electroencephalograph, by looking at the shape of the waves, the waveforms essentially in the electroencephalogram.
Nick Jikomes 6:23
So so there's behavioral changes that correlate with these different patterns of brain activity. So obviously, when animals are sleeping, they're not moving around as much. So there's macroscopic things you can see that that you can see in terms of an animal's external behavior, and those are mirrored by general patterns of global brain activity that you can measure with EEG and things like that. That's correct.
Luis de Lecea 6:46
And of course, you know the EEG can be measured only in a few species, in in lower vertebrates, and actually in insects and and other animals. It gets, obviously very difficult or impossible to get an EEG recording. So then we measure sleep strictly behaviorally and and we we look for quiescent periods of quiescence that, again have this property of sleep homeostasis. That means that when we interfere with the with with sleep, we extend the Wake periods, then we see this increased drive to fall asleep, and so
Nick Jikomes 7:36
with that kind of homeostatic regulation. So if you, if you deprive an animal of sleep, you prevent it from falling asleep for longer and longer periods of time, a sleep drive will build up, and then it will sleep more to make up for that. That would imply, right? That there's some, some essential function being served here by sleep.
Luis de Lecea 7:56
That's correct, and we're still trying to figure that out. There are several hypotheses out there as to what the main function of sleep is. Some researchers argue that it's Synaptic scaling. That means that you know that there is an increased synaptic, or communication between neurons. That's what the synapses are. So there's increased communication in the brain that needs to be reset every day so that we don't go just wild in terms of neural activity. So that's the synaptic downscaling hypothesis by Julia Tononi and Cara Chiari, essentially, and and then there's another one. They're actually several hypothesis. Another hypothesis is that sleep is necessary to clean up your brain so that during sleep, there's a there's this increased flow of in the in the brain that allow us to to clear metabolites and toxic substances that accumulate during wakefulness. But there are many others. There are other hypotheses that point to, for instance, you know, DNA repair as one of the main drivers for for sleep and also metabolic recovery. There, there are a bunch of hypotheses, none of which have been proven yet, or only have we only have partial pieces of evidence that that demonstrate that, yeah, there is a maybe, you know that sleep is essential for for some of these, for some of these functions,
Nick Jikomes 9:53
and so, you know, just being agnostic about the evolutionary purpose of sleep, when you look at brain. Activity with EEG in the mammalian brain, where you can measure, measure that activity. What are, what are the major phases of sleep, and what do they look like in terms of general levels of brain activity?
Luis de Lecea 10:17
I'm not sure if I understand that question. So,
Nick Jikomes 10:19
years, yes. So, if you compare sleep to wakefulness, and you compare, say, non REM to REM sleep, you know, these are different behavioral states, and they have very different, you know, there's very different patterns of brain activity you can observe through EEG. What do those actually look like for the different phases of sleep in comparison to wakefulness? Oh, I
Luis de Lecea 10:38
see. So, yeah. I mean, again, that's based on EEG and how we know or what we know about the EEG. So during wakefulness, there is this very complex pattern of activity throughout the cortex, which is what we are measuring in the EEG. And the EEG can be deconstructed into different waves, waveforms we call the alpha, beta and so forth and delta, so yeah, during wakefulness, there's a predominance of alpha and gamma and high frequency activity, okay? And that is in contrast to non REM sleep, which is characterized by a very high contribution of what we call the delta waves, which are slow, slow wave activity, and that is essentially reflects synchronized activity throughout the cortex. That means that many neurons are firing in synchrony during a non REM sleep. And in contrast to that, REM sleep is characterized by very high predominance of what we call the theta activity, which is a activity at the seven to 11 Hertz range, and that is also present, excuse me, during wakefulness. But that is, but again, REM sleep is very, very, is very prominent on that in that that frequency, and of course, REM sleep is also characterized by almost complete muscle atonia. That means that all muscles except for the eyes and and the breathing muscles involved in breathing are essentially lack activity. So, yeah, those are the, you know, the broad picture, the differences between the three and the three vigilant states.
Nick Jikomes 12:44
So there's these very different patterns of brain activity to characterize each of these states. What is like the neural basis for those brain state changes is it just that different neuromodulators are either, you know, being used a lot versus very little, what's actually accounting do we know what accounts for the very different brain states that you can, you can measure with EEG, yeah,
Luis de Lecea 13:08
well, that's we're still trying to figure this out. I mean, it's a very, very complex question, if you want to, if you want me to say that. So we don't know. We don't know, you know, what drives those complex patterns. We cannot really predict them very, very well, in general. And again, that's a very broad picture there. There are a bunch of excitatory transmitters and modulators that are more active during wakefulness than during sleep, but we see all sorts of patterns, both during wakefulness and during sleep. And in fact, there's this phenomenon that was characterized about 10 years ago by vladisovsky, which is called Local sleep. So there is a, you know, this sleep state within wakefulness and and also vice versa, like a wake state within sleep, that that that you can detect in different modules, quote, unquote, in the cortex. So there is not a simple answer to what is turned on during sleep, what is turned off during wakefulness. It's just, it's just very complex.
Nick Jikomes 14:43
So there's both external and internal variables that influence when animals sleep. So So for example, an obvious environmental variable would just be where you are in in the day. So for a diurnal. Animal like humans, we're generally awake when the sun is up, and then we're generally asleep when the sun is down. And you know, we all know that this has some influence on our sleep patterns. We tend to get more sleepy when the sun is down, and we tend to be more awake when the sun is up. But of course, there's internal factors as well. And if you sleep to private animal for long enough, you stay awake for long enough, eventually you'll get so tired that you'll even fall asleep in the middle of the day. And so what are some of these internal factors that build up that drive sleep? Are there, you know, are there? Is there something going on with metabolism, where certain molecules are building up and serving as sleep drivers? Is it sort of have to do with say, you know, the brain using up neurotransmitters or running out of fuel, in some sense, what are some of the major internal drivers of sleep? Yeah, no,
Luis de Lecea 15:47
that's a fantastic, fantastic question. So, yes, so we're again, we're still trying to figure out what those variables are, but, but we know that there are a bunch of them, and that some of them are in running an in conflict at many times, during the day and during the night. So So for instance, you actually mentioned already quite, quite a few of them. So the circadian rhythm, of course, is a main driver. Metabolism is a main driver. Of course, the brain uses 20% of the energy of the whole body. So it's important that the energy allocation to the brain is gets conserved. Then, of course, sleep helps with that. So what else? There's actually a bunch of variables, emotions, and I think we're going to, we can expand on today this. We all know that, you know, very strong positive emotions of excitement and in the wild that would be, you know, access to, for instance, high density of food or resources or the availability of mating partner, so that that stimulus obviously inhibits sleep, because the it's a Very good opportunity to engage in those you know, survival or you know or mating opportunities. So then you know that inhibits sleep, negative emotions. And in the wild, the most negative emotion is to have the risk of survival. So if you have a predator behind you, obviously you're not going to it's not a good idea to fall asleep. So a predator behind you engages the fight or flight response, the stress systems and that very, very powerful, powerfully inhibit sleep. So those variables are integrated in the in several circuits, in the in the central in the in the hypothalamus, and then those conflicts are computed, are sorted out, and then, then the decision to wake up or sleep is being made by that bunch of neurons. And then you know that that information is conveyed to several transmitter systems, including the monoamine the snrp, nefin, serotonin, dopamine and others, and that essentially would wake you up or inhibit, you know, activity to allow for a sleep bed
Nick Jikomes 18:47
and so with sleep deprivation, you know, so we've all had the experience where, you know, we stay up longer than we we should or that we would like to. And you know, gradually you have this build up of a sleep drive, we get more and more tired the longer we've gone without sleep. Is there anything that accumulates over or, you know, with that slow time course in the body that is at least partially responsible for that gradual buildup of sleep? Drive,
Luis de Lecea 19:16
yeah, again, a very interesting question. So if you ask that question 20 years ago, the vast majority of neuroscientists would have answered adenosine. Adenosine is the main molecule that accumulates during wakefulness and is released during sleep. And we all know, of course, that caffeine is an adenosine inhibitor, and that's why one of the mechanisms by which we can stay away for longer. But, you know, research into the past decade or so have has actually tempered that hypothesis and and it's less. There's, it's, the evidence is not so strong, it's, but it is. Way that adenosine is a main pleasure player. There's a lot of work in in metabolic metabolism field and and also in the sleep field, trying to figure out which substances, which metabolites could serve as markers of a sleep drive. And the truth is that it's unlikely that one, there's the single one, a single parameter will drive sleep needs. So it's probably a vector combination, multi dimensional construct that drives sleep. And there are, in addition to the neurometabolic parameters, there are also studies that indicate that there is a shift in the sensitivity of very, very small group of neurons that changes The sensitivity to neuromodulators, and that is this, and that is essentially what drives, you know, sleep pressure. But again, evidence supporting this is only strong in in invertebrates, in Drosophila, in the, you know, the in the flufly, and really not so much in in other organisms. So we still have to be cautious about, you know what? You know how to extrapolate those findings.
Nick Jikomes 21:45
Yeah, so, so there's many variables that that interact or compete with each other, in some sense that the term they get integrated together and determine whether an animal falls asleep or wakes up. And you know, this is a very complex phenomenon. You have. The brain has to keep track of whether or not you're in a rich social environment, whether or not you're safe or there might be predators around, what time of day it is, how long it's been since you last slept, all of these many factors. But you said a decision gets made in the hypothalamus, essentially, which is a deep structure in the brain, and I'm interested in that term, like a decision gets made. It's an interesting way of putting it. And one of the things that's kind of interesting is, you know, despite the fact that wakefulness can change on a slow time, of course, so we can sort of gradually get tired the longer and longer spin since we sleep, ultimately, the the decision or the switch in the brain happens quite abruptly. We fall asleep in a matter of seconds. How does that sort of integration happen within the hypothalamus? And why is it that? Why is it that this sort of very sudden transition ultimately happens where we fall asleep within a matter of just seconds?
Luis de Lecea 22:55
Well, it's really our perception that we fall asleep in a matter of seconds. The brain takes longer to fall asleep, actually. But we could also explain that, you know, that the speed of by which we fall asleep in terms of cortical dynamics, so that's there's essentially what we call, you know, they're emergent properties of very complex circuits, and at one point, when you synchronize, you know enough number of modules of the cortex, then you essentially have to fall asleep. That's, I mean, in very broad terms, that's, that's how we would explain this fast dynamic. But yeah, I mean, indeed, the decision is being made in the hypothalamus, because that's what, actually, that's what lots of neurons do, you know, they they integrate, they compute their inputs, and then, you know, based on those inputs, and of course, that's an online decision. Then the neuron fires or it doesn't fire, and that's a decision that is that has consequences downstream, so that activates or inhibits or doesn't or doesn't activate certain circuits. And that's, that's essentially how it works.
Nick Jikomes 24:13
And so is there a particular circuit or sub region of the hypothalamus where this decision is made? Ultimately?
Luis de Lecea 24:20
Yeah, we've learned that's actually work that we've been conducting for the last 20 something years, almost 30 years. So we identified this group of neurons in the lateral hypothalamus that we call the hypercretin neurons. Other people, other researchers call them orexins, and they're all localized in lateral hypothalamus. They're about, you know, 5000 neurons in the in in rodents, about 50,000 neurons in humans, that are responsible for making that decision. How do we know this? Essentially, because when we don't have those. Neurons. We have narcolepsy. And narcolepsy is a sleep disorder that is characterized by disrupted sleep wake patterns, essentially. Narcoleptic patients don't that lack hypercrete neurons. They cannot, you know, their brain cannot make that decision appropriately. So they don't consolidate sleep. They have serious issues and state boundary control. And
Nick Jikomes 25:31
so these hypocretin neurons, they're in a particular sub region of the hypothalamus. They're important for regulating sleep cycles. If you don't have those, you have narcolepsy, so you have very disordered sleep wake regulation. What is hypocretin or orexin? What type of molecule is it? What does it do at the cellular level?
Luis de Lecea 25:48
Yeah, well, the hypercretins are two orexins are two peptides. They're produced by the same precursor, and peptide transmitters are very widespread in the in the brain and also in evolution, there are molecules essentially that evolved to connect circuits at a slow pace, in a slower time scale than fast translators, and they work by binding to receptors in the postsynaptic what we call the postsynaptic neuron, the recipient neuron and and the you know that the binding of of these neuropeptide to the receptor triggers a whole bunch of intracellular events that result in either excitatory or inhibitory action on the potion optic root.
Nick Jikomes 26:46
So, so these neurons in the lateral hypothalamus, these hypocretin neurons, they are, in a sense, maybe this is simplifying things a bit, but they're the decision makers. They're really important for dictating whether or not the animal will fall asleep or wake up. But with certain exceptions, animals, generally, the entire animal falls asleep. And, you know, the entire brain more or less falls asleep, unless there's exceptions there that we might talk about, how does that decision in one region sort of get broadcast to the entire brain?
Luis de Lecea 27:19
Well, that's because, again, the hypothalamus and that particular region of the hypothalamus where the hypertrophy neurons reside, this region connects to very broadly throughout the brain, and in particular to nodes that are really, really, really important to activate the whole cortex very quickly. And those are what I was referring to before, as the monoamines, norepinephrine, serotonin, dopamine, histamine, and in particular, you know, norepinephrine, that we also described a while ago as being really a very, very powerful and awake promoting transmitter is very, very tightly connected with with hypercreen. So a signal from hypercreen would activate norepinephrine very, very efficiently, and that would essentially light up the brain and wake you up again. In very simple terms,
Nick Jikomes 28:19
I see So, there's these neuromodulators in the brain. They the cell bodies where those neuromodulators are are made. They are in certain parts of the brain, but they send projections throughout large chunks of the cerebral cortex, and, you know, much of the brain. So these hypocretins can broadcast the signal widely by tapping into those neuromodulatory cells which are then going to, you know, sort of communicate broadly across the brain all at once.
Luis de Lecea 28:48
That's exactly what happens. So that's a very, very nice way of describing it. Yes.
Nick Jikomes 28:55
And so you mentioned norepinephrine, which is important here. So I think another name for norepinephrine is noradrenaline. So that might, people might naturally think that's a that's a wake proponent neuromodulator. Is that true? And if so, how exactly does an a neuromodulator like that one promote wakefulness? Is there something sort of special it does to the neurons, or something different than it does in comparison to other neuromodulators.
Luis de Lecea 29:22
Yeah. Well, norepinephrine is indeed a very special transmitter for several reasons, but one being that a lot of a lot of the ruins in the cortex do express these receptors for norepinephrine. So it's a signal that very efficiently reaches to very, very large areas of the cortex and very efficiently stimulates their activity. And what I'm saying efficiently, it's because, you know, at any given time there's a very. A tightly regulated balance of excitation and inhibition in the cortex and norepinephrine can, again, in very simple terms, override, you know, that balance so that it, you know, wakefulness predominates over pretty much anything else that is going on in the cortex at that time.
Nick Jikomes 30:26
Interesting. And so, what? Um, what are some so, so, I would imagine, so when you're awake, nor epinephrine levels are generally quite high, and when you're asleep, I would imagine they're quite low. Um, what is sort of the mix of the major neuromodulators when you're awake versus when you're asleep, say, a non REM versus REM sleep to those must have to change in different ways to define each of those phases.
Luis de Lecea 30:50
Yeah, well, I wish we knew, because we could only until now. Now we have the now we're starting to have the tools to answer that very question, and that is a very, very, very important question. Actually, you know, what's the mix of neuromodulators and transmitters at any given time? So norepinephrine, for instance, the activity of norepinephrine neurons varies actually relatively in very relatively little during wakefulness. So if we count, if we monitor the frequency of activity of norepinephrine neurons during wakefulness, it varies between three to five hertz, so not a whole lot of variation. And during sleep, then it falls to, you know, point 5.2, Hertz, to one. Hertz, okay. And during gram sleep is essentially 0.1, or zero, Hertz, okay. So that's, that's the variation. And on top of that, of course, we have a whole bunch of transmitters, and dopamine, for instance, dopamine has a tonic activity, you know, around again, five hertz also. But one of the characteristics, one of the features of the dopamine is that it has this phasic activity, so just bursting activity, essentially, that is responsive to stimuli or prediction of prediction errors, prediction of errors, as we call it, so and so, the active there's, you know, more complexity added onto that, you know, three to five hertz, a tonic activity in dopamine, serotonin is also has very variable, a very broad dynamic range during wakefulness. And again, we don't know exactly how it modulates transitions to sleep and what is necessary, what is permissive for sleep. But just to go back to your question, so we only know again, single channels so to speak, recording so recordings of a single transmitter at the time. And now we have sensors that have been developed recently by a few of our colleagues that will allow us to essentially have a better description with a multi dimensional vector that allows us to really understand which you know, what the contribution of each single transmitter to brain states.
Nick Jikomes 33:36
And so I want to talk a little bit about sleep architecture so we fall asleep. But sleep itself is not just one state. There's different phases of sleep that have very different characteristics. So there's non REM sleep, there's different phases of non REM sleep, including deep sleep. There's REM sleep, and we've talked a little bit about those already, but what actually, where does this architecture arise from, and what's the significance of transitioning into non REM, and then REM, and then non rem and REM sleep throughout a period of sleeping?
Luis de Lecea 34:12
That is a very difficult question, and I don't think we know the answer. So if so, for people interested in memory consolidation as one of the functions of sleep. They could argue, well, we need to cycle through those phases because that's essentially what you know, that's essentially the way that memory circuits work. Okay? I would argue that that's actually very, not a very solid argument, because their animals have very different sleep architectures, with very different brain configurations. And we can talk about, you know, also very special cases of sleep and but, uh, anyway, so. So we really don't know why there are these phases of sleep. And if we compare, for instance, rodents and humans, we essentially go through very similar phases. But of course, humans have consolidated sleep, so we sleep over eight hours, and we have several cycles during these eight hours, rodents don't have consolidated sleep, so they awake. They wake up a lot during their during their day, which, you know, they're not going to animals, so to sleep during the day. And the other cycles are much shorter and their transitions are much faster. So why do we need those cycles? Why do we need those the sequence of those of the of the phases of the sub states between sleep? We don't really know very well.
Nick Jikomes 35:54
And so you mentioned that there's, there's obviously a lot of variety in nature in terms of what sleep architecture looks like. Some animals sleep much more than others. Some of them, their sleep is more fragmented. You know, they wake up more times, and their sleep is broken up more Are there any? Are there any animals that have sort of bizarre or deviant sleep patterns that you think tell us something important about some of these different phases of sleep and what it might be doing, you know, animals that either, you know, spend a lot of time in one phase or another phase and and just have very different sleep architecture.
Luis de Lecea 36:32
Yeah, I think, well, that is also a fascinating topic in sleep, of course. Yeah, the the ratio of REM sleep versus non compared to non REM sleep varies dramatically across species. We have the I think the extreme is the bats, which are only awake for a few hours, essentially at the dusk, which is when their insects are available, their food is available, and and then they fall asleep for the rest of the day, for 20 hours, and most of this time is REM sleep. Again, we don't know why. I mean, that doesn't it really doesn't fit with memory consolidation, that you need REM sleep for memory. I mean, we don't know why these animals have so much REM there is also some correlation between metabolism and REM sleep, and that was brought up but by Jerry Siegel, so ruminants that have slower metabolism have more REM sleep. Marine mammals don't have much of a much of REM sleep at all. And also, one very peculiar aspect of sleep in these marine mammals is that they sleep with half of the brain, so while the other half is active. So what we call the uni hemispheric sleep. So, yeah. And also, very recently, this example of the penguins. So there was a group that recorded penguins in in the wild, and they showed that up while they're incubating eggs, which is a period of their of their life, or when they're very exposed to predators, so they have to be very vigilant. And they sleep at in two to three second intervals. So it's essentially they are, but they have these sleep episodes like 10,000 times a day. So over overall, they sleep 12 hours. But they sleep only in very, very short sleep bouts. Wow. So
Nick Jikomes 38:50
when they're incubating the eggs, they have to stay on the eggs to keep them warm, especially in the cold environment. Obviously, if they can't move, they're really vulnerable to predators and things. So you're saying they essentially just engage in many, many small little bouts of micro sleep throughout the day. That's
Luis de Lecea 39:04
correct. That's right. Well, these the same animals when they are at sea. They have, they have much more relaxed sleep, they can sleep, and they have much, you know, completely different sleep architecture. And also, you know, there are other examples of extreme sleep, like the flag of birds, for instance, that when they migrate, they are essentially sleepless for a very, very long, you know, two week migration period, right? And also the big changes in in their metabolism as they migrate. And, you know, there are some really incredible adaptive strategies for, you know, under for those those conditions.
Nick Jikomes 39:51
So there's a lot of diversity in nature of sleep architecture, you know. And it's really complicated. So it's going to relate to a lot of things, obviously. The, you know, the lifestyle of the animal when it's foods available. You mentioned, you know, bats, their food is only available one time of day, so they're sort of set up to be awake at that time. They don't need to be awake at other times. They can also hang upside down from the cave, so they're pretty safe, and they can afford to do that, in contrast to the penguins, who have to be prepared to make a run for it or whatever, when they're staying awake to incubate their eggs. So obviously, all these ecological variables that constrain and dictate what sleep architecture actually looks like in a given species, but within a single species, so an experimental species like a mouse, say, Have people been able to manipulate sleep architecture in ways that allow us to understand the contribution of individual parts of sleep to whatever it is that sleep is doing. So, for example, can you selectively inhibit REM sleep but not non REM sleep or vice versa, and see what kind of effects that has on memory consolidation or something like this? Yeah, that has been
Luis de Lecea 40:55
done. And beautiful, beautiful experiments in that in that direction, have shown that, yeah, by inhibiting REM sleep, and not only REM sleep, but actually just the theta oscillation that I was referring to, so that is driven by one brain area in particular during REM sleep is essential for memory consolidation. So that is, you know, those are beautiful experiments. The problem is that it's very hard or impossible to disentangle one part of sleep without affecting the other. So if you affect REM sleep, you're going to necessarily affect non REM sleep. So I would say that the evidence again, supporting that there is something going on during REM sleep for memory consolidation is quite solid. But we were also quite excited a few years ago when this story about memory replay during sleep came about. So that essentially started to, you know, to introduce this concept. Essentially people researchers were recording the activity of neurons in the hippocampus as an animal was exploring a maze and and then there, there are these neurons that are called place cells that fire whenever these animal encounters you know particular space in that maze. So you can actually predict the position of the animal in the maze based on the activity of those neurons. It's actually, it's quite amazing. So there's this group, but MIT Mike Wilson, who showed that during sleep, there was a the sequence of activity that had occurred during wakefulness. The animal was exploring the Maze was replayed during sleep. So, so that provided a very attractive hypothesis that, yeah, so, you know, the animal is replaying so that it remembers where it has been, you know, the day before. And, you know, it potentiates the synapses, blah, blah, blah, blah. So, you know, their whole, whole bunch of theories came from, from the from that observation, but then later studies show that, you know, there's replay also during wakefulness. So it's not sleep is not necessary for that replay. So what is sleep for? Then, you know, that's uh. Again, that's a question we're trying to address. And, you know, going back again to your question. So is REM sleep more important than than non REM? It's still not clear. If you manipulate one, one aspect of sleep, you're I mean, because everything is connected, it's very likely that you're gonna affect others, and then you're gonna, you know, the conclusions are gonna be, you know, much harder, too.
Nick Jikomes 44:09
So, so if you put a mice, a mouse through a maze, it will explore the maze. It will take some trajectory through the maze, and you can essentially see the trajectory the animal is taking by just looking and recording from specific neurons in the brain, and the pattern with which they fire tells you the trajectory the animal took through physical space. And you're saying that during sleep, but also when they're awake and they're just in a state of quiet waking, not really doing anything, the same patterns that define that that fired when they were actually going through the maze will replay in certain circuits of the brain while the animal is disengaged from navigating the maze, either while it's awake or while it's asleep. That's, that's exactly right, yeah. And so, so, you know, I would imagine the natural interpretation of this, when you see this replay during sleep is, you know. Naturally makes you think about dreaming like, Okay, I often dream about things that happened to me recently. Maybe I dream about important things or emotionally salient things more and so maybe, you know, the mouse, if we speculate, is dreaming about what happened to it, and that essentially would correspond, in this interpretation, to the brain replaying certain patterns of activity for the purpose of memory consolidation, is that the basic idea, and if so, it sounds like maybe people actually demonstrated that's really what's going on.
Luis de Lecea 45:32
Well, in regards to dreaming, no, they're actually separate. You know, dreaming is, is a it's a different rearrangement of of activity that occurs during, usually during REM sleep, but also during a deep non REM sleep. So that the replay that that this experiment was referring to, essentially, it's a, it's a, it was a snapshot of the activity of neurons during sleep. And it was surprising that indeed, was almost identical to what the animal had experienced the previous day, so and so. You know, I also became very excited about this, I think, honestly. But again, one has to put in the context of, well, if we only record one channel of activity, and only those cells, those, you know, the place cells we're missing, really the big picture of what is going on during sleep, and that is, yeah, again, that is what we're trying to get, you know, with the current with the current technologies, yes, when
Nick Jikomes 46:38
that replay activity is observed during sleep. What phase of sleep is it in? Is it a non REM sleep or REM? It's
Luis de Lecea 46:44
non REM. Yeah, it's non REM,
Nick Jikomes 46:45
I see. So that would be if REM sleep is most closely associated with dreaming, then, I mean, so, so if presumably, presumably, the rodents were not dreaming in non REM sleep, that's correct. That's correct. Interesting. And so one of the things I want to talk about as well is just like overall sleep depth and sleep quality. So taking those in two pieces, when we talk about sleep death or excuse me, sleep depth, I would imagine that the coarse grained way you measure that is just you. You try and poke the animal or wake it up, and the harder it is to wake it up, the deeper its sleep is. And obviously, you know, again, we've all had experiences like this, where we try to wake sometimes you try to wake someone up, and they wake wake up very easily. Sometimes it takes much more effort to wake someone up. What are the deepest phases of sleep, and do we know anything about what's causing it to be deep?
Luis de Lecea 47:46
Well, the sleep step. Sleep depth is defined by the amplitude of the slow wave. The slow wave activity essentially the delta waves were I was referring to at the beginning. So, and that correlates, as you mentioned, with the the arousal threshold. So you know, the the higher the amplitude of those delta waves, the more difficult it is to wake up an animal or an individual. So that is, again, in general terms, but it's not always the case. And also, there is a very interesting, I would say, disconnect between what we perceive as a good night's sleep, or a sleep where we have slept very deeply, and again, our perception of sleep and the actual, you know, EEG recordings, they sometimes, I mean, most often, they do connect well, but they do overlap well, but in some instances they don't. And so it so happens that there's actually a bunch of people who complain about insomnia. They so they go to their primary doctor. The primary doctor refers them to the sleep clinic or speech sleep specialist. So they do sleep study. And in terms of that, many of these people don't have, they have perfectly normal sleep, but they do complain about not sleeping well. So, and it's not, it's just not an anecdote, it's really many, a lot of people you know, have these disconnect. So, what is it that so are? So what happens then is that the case that we're missing something in the EEG, in. In the electrical recordings that do not really reflect what is going on really in the brain. Is it that we are missing some features of the EEG in the normal protocols of analysis? So we still don't know. But, you know, it looks like, yeah, you know, sleep in these patients seems not to be, almost seems not to be, actually, perfectly fine. So if you there are some parameters that are that are different from in these patients, from the from the healthy controls, yeah. So,
Nick Jikomes 50:42
obviously, so, so if you measure sleep wake cycles with EEG, EEG is really good at defining when you're asleep, when you're awake. Non numbers is REM sleep, but it's a pretty coarse grained measurement, and it sounds like you're basically saying there's many examples of people who their EEG looks perfectly normal. They're going through their sleep wake cycles like people who are well rested do, and yet they say that they're not well rested. So the natural, the natural thing to think there, is that the EGS just not able to tell us the full story. There's something going on that we can't
Luis de Lecea 51:14
see. That's, that's exactly right, yeah, that's so, yeah, with the experimental animals, of course, we have the ability to go deeper at the EEG analysis. We can add more electrodes. We can have, we can really have more, a better idea of what is going on in the brain. We can actually, you know, be invasive and then insert electrodes in the brain the animals, and really see if there's, you know, what else the EEG is not telling us and so. But of course, we cannot, we cannot ask the animal whether you know they feel tired or not, and if they had had a good night or not. So, so that's essentially we're trying to come up with additional measures in the EEG that will be able to tell us in humans, whether this person has is has a healthy, healthy sleep or not.
Nick Jikomes 52:15
And something that's very obvious from experience is that stress affects sleep quality and sleep duration. And you know, when people are really stressed out, they generally report having trouble sleeping, or they wake up frequently things like this. Cortisol is the major stress hormone that you usually hear about as a marker for overall stress levels. How does cortisol change throughout a normal sleep wake cycle for a human. And what is the role of stress and cortisol in sleep?
Luis de Lecea 52:48
Yeah. Well, cortisol has a circadian cycle, right? And so it takes during the during the early hours in morning. And so what does it tell us? So that is, those are again similar to what I told you with the dopamine tone. So cortisol has its day, night oscillation, regular oscillation. But when we have a stressor, then you know that oscillation gets disrupted. So you know, the presence of a stressor
increases dramatically the the release of of cortisol. And that affects, you know, the whole body. Puts the the individual in a state of either ready to fight or ready to flee. And that, of course, affects the immune system, affects just arousal levels, affects the, you know, cardiovascular it affects the whole physiology of the organism,
Nick Jikomes 54:06
interesting. So the other thing that we've mentioned too is there's, there's a lot of external cues that regulate circadian rhythms, besides just, besides just the light dark cycle. So the presence of food can constrain when animals want to be awake versus asleep, and obviously the food is the source of all the energy of the animal. And what's one thing that's interesting to think about, for me is, obviously animals can change their diet. Sometimes their diets change seasonally. Experimentally, you can manipulate an animal's diet. We all have experiences, firsthand experiences, where you know, eating a big meal or eating certain types of food compared to certain other types of food can cause you to get sleepy more than eating other types of food. Do we know about the relationship between food and nutrition and sleep beyond just food availability, obviously, if you're if you're hungry, it's going to be harder to fall asleep. You've all. That experience. Obviously, if you gorge on a huge meal, you could have a sugar spike and then a sugar crash, and that can affect sleep. But, you know, other than those things, can eating certain types of food affect metabolism in ways that that affect sleep architecture. Yeah.
Luis de Lecea 55:14
I mean, other than the what, what you just described, there have been in a number of studies trying to dissect, you know, whether certain lipids, or you know protein, you know protein rich foods, or you know, would affect sleep depth or sleep architecture. And the evidence is not overwhelming. You know, it's, it's a again, you know how we extract nutrients out of the food is, you know, it's a slow process, and it's very regulated. And one would be, I mean, I would be very surprised if, if you know standard, acute changes, or even not you know even, even long term changes in in diet would affect sleep architecture significantly, sleep architecture being really also extremely tightly regulated. Having said that, there's evidence that, for instance, changes in the microbiome do affect, do significantly affect the amount of sleep and that, of course, you know microbiome. Can you know changes with with different diets and so forth, and how you know and how our body extracts nutrients from from those sources. So there, there is, there is a relationship between diet and and sleep architecture, but, but it's not easy to dissect out which components of the diet really are critical to affect sleep, because there there are a bunch of layers in between, and they're difficult to dissect out.
Nick Jikomes 56:59
And how about so? So there's a lot of correlations between sleep deprivation and lots of stuff. I mean, when you sleep derived animals, lots of things tend to go wrong, but my understanding is there's, there's quite a bit of metabolic dysregulation that correlates, and potentially that's even caused by sleep deprivation. So for example, obesity rates correlate with sleep quality. The more sleep deprived you are, the more metabolically unhealthy people tend to be. What do we know about sleep deprivation and metabolic dysregulation? Is there much known about the specific ways that sleep deprivation can influence metabolism?
Luis de Lecea 57:32
Yeah, the general at the general level, yes. And you know sleep deprivation induces insulin resistance. And a whole bunch of you know, especially glucose metabolism is affected by by sleep deprivation, quite significantly, actually, in a way that is similar to, and we can talk more about this. You know, sleep deprivation and aging go are two sides of the same coin in many ways. So the body reacts to sleep deprivation in very much the same way as it reacts to aging. So, you know, as we age, as we get older, our ability to respond to, you know, glycemic challenges is, you know, is impaired. And same thing as with sleep deprivation our, you know, the error rate, so to speak, or the accumulation of errors in our metabolic state also accumulates more rapidly as we age and also with the sleep generation. So there's a really a very good parallel between sleep restriction or not sleeping well, or, you know, insomnia, or, you know, these conditions and aging. So there's no, it's not. It doesn't come as a surprise that there are many indicators of aging that are accelerated in people who don't sleep well.
Nick Jikomes 59:19
Yeah. Well, speaking of aging, I wanted to ask you about sleep across the lifespan. So again, from experience, we know that sleep obviously changes across the lifespan. Newborn babies sleep all the time, more or less, most of the day, and as we get older, we sleep less. Many of us will know that. You know, our grandparents tend to sleep less in a given day than we do. Sleep quality also often changes as we age. In general, people report that their sleep quality seems to decrease as they get older. Can you talk a little bit about how sleep quality and sleep architecture change throughout the lifespan? And I want to maybe differentiate between developmental. Changes that are sort of supposed to happen, or pre programmed versus dysregulation that happens due to aging or accumulated damage or something like that?
Luis de Lecea 1:00:08
Yeah, yeah. No, that's, that's a great question. So yeah, the it's certain that the sleep architecture changes throughout lifespan, and we all know, of course, that infants sleep most of the day. They are essentially growing. They're growing their brain, myelating their synapses. You know, there's a whole really, as you mentioned, the developmental aspect of it, that is for which sleep is essential. And as we reach adolescence, then it becomes more and more important to either just brain development or and or memory consolidation as as as it goes. But of course, at a certain age, yes, there's accumulation of as mentioned, metabolic and also genetic errors that result in in what I would say, you know, disruption of sleep architecture, mostly due to sleep fragmentation. So it's not that older people sleep more. And they do tend to sleep more, but also it's the there's sleep becomes disrupted. And therefore, you know, the sense of, you know, a recovery from sleep is, is decreased. And so we, we published the unimportant paper a couple of years ago, indicating that one of the reasons why, you know, sleep is fragmented in aged individuals is because there are circuits in the hypothalamus affecting the hypercreen neurons that we're referring to before. So these that hypercrete neurons become more excitable as we age, and that means that the brain, quote, unquote, is easier to wake up, and therefore any either spontaneously or due to external stimuli, the sleep becomes more disrupted and therefore less efficient
Nick Jikomes 1:02:21
I see. So the Eureka neurons, those were the neurons in the lateral hypothalamus we mentioned before. If you're missing those, you have narcolepsy, which is one way to have fragmented sleep. And these neurons promote wakefulness. It sounds like so if they become more sensitive across a lifespan, essentially the arousal threshold decreases. So it's easier to wake someone up, the more sensitive the erection neurons
Luis de Lecea 1:02:44
are. That's exactly right. That's, that's something that we're, we're able to to prove in that paper. And
Nick Jikomes 1:02:52
do we know what drives that? What's actually making them more sensitive?
Luis de Lecea 1:02:57
Yeah. Well, in that, in that study, we showed that there's a specific channel. A channel is a protein that allows the flow of ions into the cell and is essential for the neuronal excitability. So one of these channels becomes less efficient, and that makes the cell more hyperexcitable, essentially, intrinsically more hyperexcitable. What makes that protein less efficient? We don't know. But there are studies indicating that there's oxidation in the milieu, in the external milieu, that causes this child to be a little bit dysfunctional, and therefore affecting, you know, the physiology of the hazarding neurons and other neurons as well. And so
Nick Jikomes 1:03:49
it sounds like there could be the extra excitability that you see emerge in these neurons, the dysregulation of sleep could be related, at least in part, to the accumulation of oxidative damage over the lifespan. That's correct. And so with that in mind, what are you know? What are the some, some of the things that we know about how to preserve high quality sleep in into late life. You know, are there certain lifestyle factors that might help someone preserve a high quality to their sleep architecture as they age, such as not being constantly sleep deprived, or, you know, engaging in other aspects, engaging or not engaging other and other types of behaviors that that lead to this regulation of sleep?
Luis de Lecea 1:04:35
Yeah, other than the common sense our unfortunately, the the basic science has not been able to come up with with a protocol that would, you know, result in less oxidative stress. It's just that's an intrinsic part of our of our brain activity, and I again, other than the common sense, you know. To lead a healthy lifestyle, I don't think we can recommend anything in addition to that.
Nick Jikomes 1:05:05
And so obviously, as we age, you know, people often have these sleep problems. One way that people deal with those is through pharmaceuticals. So Ambien and other medications that promote sleep. When you think about, when we think about sleep medications, medications that are meant to help people fall asleep and stay asleep, to what extent do those drugs actually give you genuine sleep? Are they putting you into a natural sleep state? Are they putting your brain to some other type of quiescent state that doesn't resemble natural sleep? And what are the consequences of that?
Luis de Lecea 1:05:43
Yeah, that's also a fantastic question. So, so an acute administration of one of these drugs is essentially reduces the latency to sleep. So it what these drugs do, most of the sleep aids. What most of the sleep aids do is to increase the overall inhibition in the brain. So those are ligands for inhibitory transmitter receptor transmitters, so they reduce the sleep latency. So then the person who takes those drugs has the perception that they slept better because they fell asleep faster, okay, but the so one acute administration, in principle and as prescribed by the physician, of course, has not much of a consequence. The problem is when this becomes a chronic administration of the drug, so then the brain tries to adapt not to that, to this new state of inhibit, inhibited, inhibitory transmission. So and then the sleep architecture becomes affected, indeed, and the sleep becomes less natural. And there's also sleep inertia, as many of the patients have, you know, experience sleep inertia, meaning that, you know, they, there's, they feel, they feel sleepy during the morning and after they wake up, and they, you know, again. And that's part because of the drug itself, and also because of the adaptation to the drug, right? So, yeah, there's definitely an over prescription of sleep aids in our society, for obvious reasons. It's, you know, relatively easy to they're very cheap, you know, drugs and most physicians don't have, you know, primary care physicians, they don't have the training, you know, to really got deeper at what caused insomnia. So they, you know, the prescribed sleep aids. And that's, you know, it's easy, relatively safe as well. So one would say, Well, it's a it's a shorter remedy for for a problem that may be deeper. And obviously, the sleep dates are not the best way to address the problem.
Nick Jikomes 1:08:26
I want to ask you a little bit more about REM sleep. So, so we mentioned it briefly earlier. Obviously, REM sleep is different from waking. It's also different from other phases of sleep, like non REM sleep, and you mentioned earlier the EEG signature of REM compared to non REM. So as opposed to non REM, where you get these large, slow waves of synchronized activity in the cerebral cortex, REM sleep has a very different EEG pattern. Superficially, at least, it almost looks more wake like than it does like non REM sleep. And yet, obviously you're not awake, and so it's not going to be identical to the waking patterns that you see in the EEG, what are some of the neuromodulatory factors that influence the REM EEG? Which neuromodulators are, say, increase or decrease relative to waking in REM sleep?
Luis de Lecea 1:09:15
Yeah, most. It's an interesting question. So many neurons in the or sleep regulating neurons have are active both during wakefulness and REM sleep. So there are very few that are exclusively REM sleep active and so still calling seems to be one of the transmitter that is, the transmitters that is critical for the non REM to wait, non REM to REM transition. And so that is one of the, you know, basic it's. Are the basic components of the RAM on circuitry, as we as we call so, but there are others. And, you know, dopamine being also one, one transmitter that is wake on Remon, and serotonin, also, it's part of, at least part of it is Ramon. So it's, again, there's, as I mentioned previously, there's not a single, you know, it's not, it's not a simple, there's not a simple answer to that. It's just a, you know, a complex regulation that leads to, leads to REM sleep.
Nick Jikomes 1:10:33
I see So, so it's, it's complex, but there are some differences between rem and non rem and waking. So it sounds like acetylcholine is one of the neuromodulators that's quite high, high activity during REM sleep, as opposed to some of the other ones you mentioned, like neuroadrenaline, which is very low, little to no activity during REM sleep.
Luis de Lecea 1:10:52
Yeah, that's correct.
Nick Jikomes 1:10:53
And you mentioned earlier too that you know the the theta rhythm that characterizes REM sleep, um, also seems to be important for memory consolidation. So there's some evidence that what, whatever exactly is going on in terms of brain dynamics during REM that seems to have some some role in memory consolidation. Perhaps one of the things that's weird about REM sleep and dreaming. So dreaming is usually observed during REM sleep is, again, from experience. We all have. We all have many examples in our lives where we wake up and we know that we've dreamed, we remember the dream, but then we very quickly don't remember it anymore. It seems to slip right through your fingers. So there's this amnesic quality to dreaming where the experience is very ephemeral, ephemeral and difficult to remember, but simultaneously, it sounds like there's evidence that REM sleep might be important for me. Sleep might be important for memory consolidation. So do we understand much about that, why there's this amnesic quality to Vivid dreaming that we experience, and yet, there's probably something going on during REM sleep that's important for memory consolidation.
Luis de Lecea 1:11:56
Yeah, I think I would distinguish very clearly what, what is the dreaming aspect of sleep and the basic function of sleep during REM, during during REM. So, and, you know, we could get into a deeper at what, what maybe, and this is not really part of my research that I've been I've been very interested in this topic for a long time. You know what is, what is dreaming? What is, what is actual? What is going on during dream? Is it an epiphenomenon? It's something that you know all I do, all animals, dream or not, and so forth. So you know that feeling of, you know, not, not remembering, you know, the dream that you just had. It's usually happens when you're, when you, when you wake up during a REM sleep, which is actually occurs quite frequently, but it's even more so when it's even more more obvious, when you're, you're, you wake up during deep, non REM sleep. So there is dreaming during non REM sleep, but the there is during non REM sleep, there's no concatenation of of experiences, of of events. And therefore you either don't remember that you were what you were doing at all, or you just have a snapshot of the dream, and then you forget about it immediately. So yeah, what characterizes dreaming during REM sleep is this again, that flow of experiences, ideas that were reactivated or came up again during the dream. So it's interesting. You know, the dreams are essentially, you know, are an accumulation of re activation of experiences, right? We know, for instance, that people who are born blind, they they don't have the they don't have they don't dream images. And, you know, most of the dreams are images, right? So they do dream, they do have dreams, but they don't have a visual component to dream. Okay? So, again, it's a reactivation of sensory experiences. Whatever you can sense, you can dream essentially, right? But we all know that, you know, the connection of those dreams to reality is obviously not. It is just not there and and it's likely, again, there are a few studies we have, you know, brain imaging during during dreams. But again, not so solid, I would say, again, the conclusion of those studies is that, essentially, you dream what the cortex has the. Of lets you dream, or what is available, what kind of, the kind of experiences that are available during REM sleep, as you as you dream, and those get the you know, filled and connected, as if it was a a conscious episode, but, but the fact, of course, it's not, it's not connected to reality. It's not it's just an internal process that that gets bound so and that process of streaming, again, is very, very different from the physiological events that underneath that are really an essential part of sleep.
Nick Jikomes 1:15:44
So we often wake up during REM sleep, but we don't always do that, and I'm wondering what the connection there might be to this concept of sleep inertia that you mentioned before. So very often in my life, for example, I've had the experience where maybe I have a full night's sleep and I wake up naturally, 30 minutes before my alarm was supposed to go off, and I wake up. I was just dreaming. I feel perfectly awake and rested, but, you know, I've got an extra 30 minutes, so I just sort of lie in bed and and think to myself, well, maybe I'll just relax. I don't need to get my day started quite yet. And then sometimes I fall back asleep. And even though I felt perfectly rested before, and now I'm sleeping more. I wake up and then I feel much more tired than I did just a few moments ago. Does that have something to do with which phase of sleep I'm waking up in in terms of what dictates the level of wakefulness you experience when you actually come out of sleep?
Luis de Lecea 1:16:36
Yeah, I'm not I know there are a bunch of researchers that study that very aspect of sleep. I'm not very familiar with that literature, to be honest. I don't I don't know exactly what, what is going on and what are the consequences of waking up at different phases during in the human human sleep.
Nick Jikomes 1:17:00
One of the things you have been doing more recently, it looks like, is using ultrasound technology to actually manipulate what's going on in the brain. And I want to give people an idea for what's going on here and how this technology is developing. Obviously, when people hear ultrasound, the thing they're probably most likely to think about are the, you know, the images of the baby in a mother's stomach, which we get from ultrasound. So what is ultrasound, and how is it actually being used by your lab and other labs now to manipulate what's going on in the brain?
Luis de Lecea 1:17:31
Yeah, so indeed, ultrasound is sounds. There are sound waves emitted at frequencies that are beyond our perception, our sensory perception.
So there's a very, very broad range of that's, you know, sound is a mechanical wave that transmits through the air, and the ultrasound comprises, sounds that you know from from 50 kiloHertz, 70 kilohertz to, you know, megahertz. So it's a very, very broad range of frequencies.
And the imaging, you know, technology uses very high frequencies for, you know, for imaging, for that to to record, essentially, those sound waves back and forth the tissue, in the soft tissue. So, you know, the way we're using ultrasound is slightly different, because we're using ultrasound in the brain and most of the so. So in ultrasound, as in many other waveforms, you know, the higher the frequency, the higher the energy, okay? And but most of the for the high frequency ultrasound, most of the most of that energy is absorbed by the skull. So for transcranial ultrasound, for neuromodulation, we need to, we needed to choose a frequency that that gets through the skull and then gets distributed throughout the brain, essentially. So that limits our frequencies, or frequency range to, you know, up to 500 to 700 kilohertz, and in turn, you know, the lower the frequency, the lower the energy, and also the broader the focus. So that you know the focus ultrasound, the transferring of focus ultrasound has this limitation, that the size of the focus is relatively large compared to high frequency ultrasound. Hmm, but, but we can. We can design, you know, tools or tricks to make that focus smaller. But anyway, the reason why we got started into this is because all of the other methods for neuromodulation to essentially target the brain tissue that used magnetic stimulation or electrical stimulation, they don't penetrate deep in the brain. They only affect the surface of the cortex. And I mentioned earlier that the critical players in sleep, weight regulation and in many other behaviors in the brain are subcortical. Are structures deep in
Nick Jikomes 1:20:53
the brain I see, so you needed a way to get down to that depth and manipulate neurons. And most existing ways of manipulating neurons weren't able to get down to the hypothalamus, that's
Luis de Lecea 1:21:06
great. So hypothalamus, or even the thalamus, which is also another structure that you know that is very important in in in the sleep wake regulation. So yeah, that was our motivation, our initial motivation, we started more than five years ago, trying to figure out ways to use ultrasound along other methods to to reach, to target deep brain structures and ultrasound, you know, after consultation with some of our colleagues here at Stanford, we figured, well, ultrasound is probably the the method that has not really been fully explored yet, and that, you know, there's a lot to be learned from, from what you know, how ultrasound, how neurons respond to ultrasound, and how we can actually manipulate brain activity using ultrasound
Nick Jikomes 1:21:54
and so. So ultrasound, very high frequency sound waves, so high frequency, we can't hear them, but they can penetrate tissue. The frequency that you use with your ultrasound technique is going to dictate how deep the manipulation can penetrate into the brain and also how focused that energy is. So you know, you've probably played around a lot with the frequency, and what that means for how deep in the brain you can stimulate neurons versus how focused the era area of stimulation is, but it sounds like you've actually been able to stimulate neurons and influence neurons using ultrasound all the way down in structures in the hypothalamus. Presumably, you guys looked at ones that were going to affect arousal and sleep and wakefulness. Can you talk a little bit about what you found. How specific are these manipulations? Can you get down to very small regions of brain tissue, and you know, are you exciting neurons? Are you inhibiting neurons? Does that change based on the frequency? What does the manipulation look like?
Luis de Lecea 1:22:53
Yeah, no, that's that's a great question. So, you know, in short, yeah, we can manipulate neurons. We can excite neurons, we can inhibit neurons, we can inhibit neurons depending on the frequency of the stimulation. But so our hypothesis, and still hypothesis, because we haven't proved it yet, is that different neurons respond to ultrasound based on the expression of mechanosensitive channels.
Nick Jikomes 1:23:14
So the physical sound waves are there's mechanosensors that are evolved to respond to physical stimulation, and that might be what's being tapped into here. That's correct.
Luis de Lecea 1:23:27
So there's people who still argue that this may not be the case, but the fact of the matter is that, indeed, neurons do express mechanisms. We know exactly why and what for, but ultrasound leverages that, you know, the expression of the of those channels which is is not uniform across across the brain, so there are certain neurons express more channels than others, and therefore are more responsive to ultrasound than others. But again, ultrasound is a very the response to ultrasound is non linear. That means that it's actually very complex. It depends on many factors and and we're with our study. We're trying to essentially proof of concept. We're not really trying to get very, you know, very, get very lots of details from these but essentially public concept, do different neurons respond differentially to ultrasound? That was the question we're trying to address. And so in order to do that, we Keith Murphy was a postdoc in the lab. He used a measure of calcium activity using an optical fiber that we use all the time, you know, in Systems Neuroscience, as an objective measure of what was going on in neurons in response to ultrasound. And he showed that different different brain. Patients respond very differently to ultrasound, and if you vary the frequency, you're going to have differences in the in the in the response. And also, if you vary the repetition of the pulse, so that will also affect how neurons respond to ultrasound. And indeed, they can respond in a positive way so they can increase their activity, or they can respond in a negative way. That means the ultrasound inhibits their activity. But that's a since we had an objective measure of what was going on with ultrasound at individual cell types, now we can build sort of an atlas of how the brain responds to ultrasound in in both, you know, deep structures, and also in the cortex.
Nick Jikomes 1:25:46
And so obviously, having a way to non invasively modulate the activity of neurons even very deeply in the brain, very useful from an experimental standpoint, because now you can, you can excite or inhibit neurons in different regions, non invasively and study circuits in the brain. And what the brain is doing when we think about the applications of this technology to humans. Is there any are people using it in humans? And what are some of the limitations here? Can you actually stimulate down to the full depth of the human brain? Are people doing that? And could this be useful clinically, to, say, treat people with certain psychiatric conditions? Yeah, of course. That's,
Luis de Lecea 1:26:27
that's, you know, a very clear application that from from ultrasound. So the depth of the of ultrasound is essentially, it's, it's unlimited. So, so ultrasound is actually being used already in the clinic for for tumor ablation at very high frequency, though, you know, again, I mentioned the high frequency, small focus, but so high frequency generates a lot of heat, and that's why it is restricted in the clinic to very, you know those applications, and you know it needs the the patient needs to be cooled. You know, the needs to be cooled in order to, you know, to avoid other consequences. So,
Nick Jikomes 1:27:14
so they're using super, super high frequency ultrasound that's focused on a tumor in the brain. And so someone's got a really bad cancer tumor, you could essentially cook the tumor in their head Exactly,
Luis de Lecea 1:27:25
exactly, yeah, and that is being used, you know, has been used in the clinic for a while already. So again, it's, it's non invasive, so you don't have to drill, you know, through, through this goal, but, but it has its risks, because, you know, the heat generate, and, of course, also the the precision of the ultrasound. But anyway, so that's one application. But what we're not talking about that, you know, that type of ultrasound, you know, high, high frequency, high energy. We're talking about low, low intensity focused ultrasound and but the role it still applies that you know, you can reach pretty much any region of the brain, because the sound waves propagate through the brain tissue at the essentially the same speed, because, for some purposes, the brain is quite uniform, so it's a and, and you can get the waves pretty much anywhere, at any depth. So going back to your question, is it possible to in the future? Will it be possible in the future to treat conditions where neural circuits are not functioning deep, well, neural circuits deep in the brain are not functioning. I would anticipate that, yes, that's possible. We don't, I don't know of anyone who is doing it right now. You know, deep in the brain, but, but it's, it's definitely suddenly possible. So
Nick Jikomes 1:29:04
it sounds like we probably don't understand all the stimulation patterns that might be useful for human applications, but in principle, you can stimulate with ultrasound all the way through the depth of the human brain, and it's not invasive. So once, once the technology is developed, a bit more and we understand, like the consequences of different stimulation protocols and frequencies and things. I would imagine this would naturally be an alternative to something like deep brain stimulation, where perhaps you can stimulate regions deep in the brain without having to cut the person's skull open and implant an electrode deep in the brain. That's
Luis de Lecea 1:29:37
exactly right, of course. The main caveat now is that, again, the focus is not super precise yet, but there are ways that one could envisage to narrow the focus, by using interference, by using a based arrays, by using a whole bunch of physical and. A tricks, essentially, to partners the power of ultrasound and make it more, you know, more confined to a small volume.
Nick Jikomes 1:30:09
And so what are some of the ways that you are in your lab using this technology to study sleep weights, wake circuits in the brain? What are some of the questions you guys are asking?
Luis de Lecea 1:30:19
Yeah, well, why is essentially, can we reach areas of the brain that are complex, like the hypothalamus, where, you know, you have a weight promoting cell right next to a sleep promoting cell, and are these cells really differentially responsive to ultra cells? So that's one, you know, question that we're trying to address the other is, you know, for sleep and wakefulness and essentially cortical activity. So we know that, you know, there are very complex patterns of activity in the cortex during wakefulness and during sleep at the 100 millisecond timescale. So essentially, there's a lot going on in many different brain modules in the cortex and with very complex dynamics. So our question with ultrasound would be, if we use ultrasound for a, you know, for one millimeter. So essentially, we stimulate a column, a cortical column, one millimeter wide. How do, how does that affect, you know, that complex dynamic of brain activity during wakefulness and during sleep, and during and during and during REM sleep. So can we how does the brain adapt to those, you know, disturbances, and you know questions like that, yes,
Nick Jikomes 1:31:51
and from what I can gather, it sounds like so when we think about human applications of non invasive, non invasive neuromodulation like this is so you have something called transcranial magnetic stimulation, which which has been used on people. It's also a pretty coarse grained way of manipulating brain activity. It's not very focused. Would ultrasound be a more spatially focused or specific way of non invasively manipulating the brain, yeah,
Luis de Lecea 1:32:22
and especially the most salient feature of ultrasound is, again, that it can reach deep brain structures, which transfinium magnetic stimulation cannot, despite the efforts over, you know, the last Few years. You know these, you know, deep TMS coils, they do not function well. So, yeah, so that it would be that would overcome the limitation of of TMS. Interesting.
Nick Jikomes 1:32:51
So in terms of what your lab is doing, what are some of the experiments you're working on right now, some of the questions you're investigating in terms of sleep and wakefulness and the underlying circuitry that you're excited about and that you think will have maybe some interesting answers to in the next year or two.
Luis de Lecea 1:33:08
Yeah, well, I mentioned, you know, the very complex patterns of activity in the cortex. Those are in you know, I think it's really fascinating. You know, what the new tools are bringing us new tools in the form of, you know, sensor for neuromodulators. And we discussed that, you know, that we only have an idea of, you know, single channel recordings of, let's say, you know, norepinephrine. But now with the new sensors, we can have simultaneous recordings of multiple transmitters and multiple neuromodulators and that that, I think it's, it is super, really, super exciting. Also, you know, the calcium sensors for genetically encoded voltage calcium and voltage sensors, they're bringing incredible, incredible new images of cortical activity. And, you know, cortical dynamics that have not been we haven't we haven't seen before. So it's, you know, trying to decipher what those dynamics mean and what is going on. You know, what are the drivers of those dynamics? Is, is also fascinating. And we're really trying very, very hard to to get those questions. What else so, of course, we're very interested in, in the nature of the sleep home, is that we, you know, that's, I think, a fundamental question in sleep research. And we discussed about this in a little bit, what, what is? What is that, you know, increases during wakefulness, that is released during sleep? So well, adenosine was a good candidate, but it's certainly not the only one. And so there we have a few candidates that we're trying to you. You know, evaluate as to their potency for driving sleep. Also, another hypothesis that we're very actively pursuing is whether, as I mentioned, also, DNA repair, is it the driver for sleep. So we're monitoring DNA repair in real time in cortical neurons as the animals sleep and see whether we can, you know, we can actually prove that that's
Nick Jikomes 1:35:30
true. Oh, wow. So I would imagine the hypothesis is that some significant amount of DNA repair is happening during sleep.
Luis de Lecea 1:35:38
That's correct. And the reason why is because, as we discussed with the synaptic homeostasis, essentially, sleep provides a very interesting window of opportunity to repair DNA that has been damaged during wakefulness. And why do we need sleep? Is because, of course, DNA repair also happens during wakefulness, but the rate of damage is much less because there's less activity in general, and that allows you to, essentially to catch up with the with the DNA repair rate. So, and also, we are seeing that not only that, but also during sleep there, there's a higher rate of DNA repairs. So that would also rebalance, you know, that, and needless to say, that DNA integrity is a fundamental property of neurosystems, and that would explain why, even with very, very different brain architectures we have, we see sleep across, you know, fire, you know, you know, in all animal phyla. So again, we believe that the sleep has really to subserve a really, really fundamental function. And one of these could be, it could be
Nick Jikomes 1:36:58
DNA repair. Interesting. We covered a lot already. This has been fascinating. Is there anything that you want to reiterate for people that we went over, or any final thoughts that you want to leave people with regarding sleep?
Luis de Lecea 1:37:10
Well, I just want to restate that. You know, sleep is universal, and it is universal for a reason. I think we need to take care of of our sleep very you know, very well, I think the the consequences of of not sleeping are really very, you know, they're very detrimental to our health. So we cannot ignore the fact that we're not sleeping. Well, I would really, you know, for those who have trouble sleeping, I would really, really encourage them to get the help of a physician and and hopefully get preferred to a sleep clinic, because it's, you know, it's something that can be fixed in the hands of professionals now, but, but it cannot be ignored. So that's that'll be my final point. Yeah.
Nick Jikomes 1:38:02
All right. Well, Professor Lewis delacea, thank you for your time. This was fascinating.
Luis de Lecea 1:38:08
Thank you so much. I really enjoyed this. This episode
Nick Jikomes 1:38:20
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Sleep: Neural Circuits, Orexin/Hypocretin, Hypothalamus, Neuromodulators, Stress & Cortisol, Sleep Drugs & Ultrasound Technology | Luis de Lecea | #168