Scientist Stories: Siniša Hrvatin, Mysteries of Animal Hibernation

Good afternoon, everyone. Welcome to today's webinar, Mysteries of Animal Hibernation. I'm Sebastian Loretto, a member here at Whitehead Institute where my lab studies the molecular principles of parasitism. I'm excited to be your host and moderator today. Today's webinar is part of a series of talks we've organized to share Whitehead Institute science and research with the wider community. In a few moments, we'll hear from our presenter, my fellow Whitehead Institute member, Suneesha Arvathin. First, I would like to let you know that this webinar is being recorded for future viewing. Also, we will be answering questions at the end of today's presentation, but you can submit a question at any time using the Q&A box at the bottom of your screen. I would recommend that you write your name next to your question because we've had some issues and you'll see a whole lot of Monicas in the audience, even though that's not your name. With that, let me introduce our speaker today. Sunita Arvatin earned an AB in biochemical sciences and a PhD in stem cell and regenerative medicine from Harvard University. He then went on to complete postdoctoral research at MIT and later served as a postdoctoral research fellow and an instructor in neurobiology at Harvard Medical School. Suneesh is the co-founder of ReadCube, a technology company that serves millions of researchers, publishers, academic and commercial organizations by providing access to scientific literature and reference management tools. In January 2022, he joined Whitehead Institute as a member and the MIT faculty as assistant professor of biology. At Whitehead Institute, he investigates states of stasis such as hibernation and the mysteries of how animals and their cells initiate, regulate, and survive these adaptations. Specifically, he is exploring 3 questions. How does the brain regulate torpor and hibernation? How do cells adapt to these states? And can inducing these states slow down tissue damage, disease progression, and even aging? Today, he will address these challenging questions and share his findings on the mysteries of animal hibernation. Siniša, welcome, and thank you for joining us.

Thank you so much, Sebastian. So let me just get set up with my screen. Could you confirm with me that you can see my screen?

Yes, we can.

Awesome. So yeah, thanks again on the introduction, and good afternoon, everyone, or I suppose morning or evening depending on where you are. I'm delighted for the opportunity to share with you some of the work that we and actually others have been doing towards solving some of the mysteries of mammalian hibernation and hibernation-like states. So personally, I love studying interesting biological problems that I believe could also have broad applications. And scientifically, I really enjoy thinking about how different species have developed remarkable behaviors and adaptations. So today I'll tell you about something you're all familiar with, hibernation. To me, one of the most amazing mammalian behaviors that we actually know surprisingly little about. And at the beginning of the talk, I'll introduce you to some of the reasons why I find these states so fascinating. And the bulk of the talk, I'll share with you a story about a discovery we recently made while dissecting how a hibernation-like state of torpor is regulated in mice. And then at the end, I'll touch on a handful of open additional questions that we and others are studying. So as you know, many birds and mammals, including some primates actually, have the ability to profoundly reduce their metabolic rate, breathing rate, heart rate, body temperature, and enter these states of torpor or hibernation. So here's an example of one animal, the Arctic ground squirrel. Work by Brian Barnes and others have shown that Arctic ground squirrels enter in a seasonal manner into hibernation, which is characterized by a decrease in core body from around 37 degrees Celsius, which is, let's say, normal mammalian temperature, to actually as low as -2 degrees Celsius. This drop in body temperature is called a bout of torpor, and hibernation consists of several consecutive periods of torpor interrupted by these arousals. Now, many scientists from fields spanning ecology, behavioral physiology, metabolism have studied torpor and hibernation, and there have been many, many excellent studies in hibernating organisms, but there's still a lot of unanswered questions. And here I just want to give you a sense of some of these questions that the field is trying to address and answer. They include: how do warm-blooded animals, which normally very closely control their body temperature, suddenly change their whole body metabolism and reset their body temperature to enter essentially torpor? Why do animals repeatedly come out and go back into torpor when this process is actually extremely energetically costly to them? How can the organs and the cells of hibernating organisms survive extreme cold when actually many non-hibernating organisms cannot? How did the ability to enter torpor and hibernation evolve across so many diverse species from bats to bears to rodents? And could we induce these states in non-hibernating animals? And finally, why do hibernators live longer than closely related non-hibernating species or Are the processes of aging and slow down or halted during hibernation? So these are just some of the many questions that remain unanswered, and of course, there are many more. So I want to give you a sense of these up front, and then I'll circle back to them at the end of the talk when I'll tell you a bit more about each one of them. And in addition to these being interesting fundamental questions of mammalian biology, I believe that studying states of torpor and hibernation could also have broad applications. Here are some examples. The neurobiological mechanisms by which animals adaptively regulate metabolism could provide new approaches to modulate human energy balance and treat metabolic diseases. The mechanisms that despite immobility maintain muscle tone during seasonal hibernation could be targeted to prevent muscle atrophy in bedridden cancer patients. The molecular mechanisms that slow down cell cycle and increase lifespan of hibernating organisms could be targeted to slow down cancer progression or even aging. And the protective that enable cells to survive these states might also be harnessed to facilitate neuronal survival during ischemia or brain injury. I don't think this is that far-fetched because in one example, traumatic therapeutic hypothermia, which is the process of exogenously cooling the body for a period of hours to days, is actually used in patients with cardiac arrest, ischemic stroke, traumatic brain injury. And it's actually a standard of care for hypoxic ischemic encephalopathy, which affects 1 in in 1,000 newborn babies where it essentially saves their life and limits long-term neurodevelopmental disability. So, despite its widespread use, there is relatively little known about the mechanisms by which cold protects cells and about generally physiology at lower temperatures. And perhaps if we understood these mechanisms better, we could develop alternative approaches to more safely, rapidly induce hypometabolic states and protect cells from ischemia. Finally, you know, in our wildest imagination, understanding these states could inform whether one day we can induce a hibernation-like state in humans, enabling everything from organ transplantation to space travel, lifespan extension. This is, of course, currently science fiction, but I hope that by studying these states, we may learn to harness their potential to advance medicine. And when I decided to begin to study this, I needed to first choose an animal model, as we all do. And most of you are probably familiar with hibernating bears, squirrels, and indeed, there are many mammalian species capable of these adaptations, including hamsters, lemurs, bats. But to begin to dissect the mechanisms that regulate these states, we needed at first really powerful molecular and genetic tools that are currently only available in mice. And I first became fascinated by this biology back in graduate school where I worked on a stem cell therapy for diabetes, and I often measured fasting blood glucose levels in mice. And I noticed one morning something peculiar. Several mice appeared very cold and lethargic, and initially I thought there was something wrong with them. And, um, but when they recovered about, I think, an hour or half an hour later, I went to look into the literature to try to explain this, and I found that fasted mice were conserving energy by entering a hibernation-like state called daily torpor. I was amazed. I had no idea at the time that laboratory mice can enter torpor when they're deprived of food. And as it turns out, this discovery actually dates back to the 1970s when Stephen Morton down in Australia[No speech] To me, this actually felt like a perfect initial model, and one of my goals during this presentation is to give you a sense of the type of experiments that we can do using powerful genetic tools to dissect the biology of torpor and hibernation. So this study was performed in mice, and I hope that in the near future we'll also be able to perform similar types of experiments in different hibernating organisms. So to establish a system to study mouse torpor in our own hands in the laboratory, we implanted mice with telemetric temperature probes and built a system to monitor their activity and body temperature in real time without disturbing them. And here you can see an experiment monitoring the body temperature and activity of one fed mouse over a period of 24 hours. You can see, as you would expect in humans, that the body temperature is maintained around 37 degrees Celsius. This is the solid line right here, and it doesn't fall below 35 degrees Celsius, roughly this, this dashed line right here. The gray indicates the 12 hours of the dark cycle, and the light indicates the 12 hours of the light cycle. Now, when this same mouse— and on the bottom you can see actually its activity— when this same mouse is fasted, first thing that you observe is that the temperature actually increases. The mouse is moving around the cage searching for food, and then several hours into the fast, the mouse initiates the first bouts of torpor, which are characterized by this precipitous drop in core body temperature, a period of hypothermia, which can last up to several hours, and then finally spontaneous arousal from torpor. So, I think this is quite amazing. We think of mammals as maintaining their body temperature at 37 degrees Celsius. This is considered to be a defining feature of endothermy, but here's an example of a laboratory mouse that upon fasting lowers its body temperature to just a few degrees above room temperature. So I wanted to discover how mice enter and regulate this hypothermic state. Let me first show you what torpor looks like. So here is a fasted mouse. Its body temperature is around 37 degrees Celsius. It's moving around the cage, it's exploring, presumably trying to find food, as been described by others. Now, several hours later, we can see the same mouse now in torpor. Mouse is hunched over, its head is tucked in, the mouse is clearly still somewhat alert. So despite the fact that its body temperature is now around 30 degrees Celsius and it's still dropping. So this is what the behavior looks like in this case, and there are many aspects of this complex behavior that one could study, but I really wanted to figure out how the animal circumvents these highly conserved mechanisms that usually tightly regulate its body temperature to enter this hypothermic state. And decades of work by Helen Nakamura-Morris and many others studying mammalian thermoregulation have found that body temperature is centrally controlled by the brain. And they actually discovered several brain regions and neuronal populations that are critical for the process. But no one had to date applied modern genetic and neuroscience techniques to dissect how an animal initiates states such as torpor and hibernation. So using this model, we first wanted to explore whether there are specific regions in the brain that regulate and train to torpor. Because if that were the case, we'd be able to learn how animals initiate these states, and importantly, we'd be able to gain control over them. So the work that I'm describing here was done in collaboration with several exceptional colleagues in the lab, including Oren Wilcox, who was a technician at the time, now PhD student at UCSD, and Simiao Sun, who's a graduate student in the lab. So the first question we wanted to ask was a simple one: are there regions of the brain that are active during torpor, suggesting that those regions may regulate the process. So we collected brains of animals during torpor. We stained brain sections for CFOS, a protein that marks active neurons, to see if there are any regions active during torpor. And we indeed identified several brain regions containing a particularly high density of these active neurons. And here on the bottom, you can see regions of the hypothalamus. So this was actually a pretty good starting point, but How could we test whether these neurons were actually driving entry into torpor, not simply responding to torpor or being involved in a different process that's related to this fasted state? In order to do this, we needed to not only observe them, but we needed to gain control over these C-Fos-positive cells, over these active neurons, so that we can turn them on and see if that's sufficient to induce torpor. So we asked a bold question, which is, is the stimulation of the neurons that are active during torpor actually sufficient to induce a state of torpor? Or do the animals really need to be in this long-fasted metabolic state for several hours in order to enter torpor? And if the neuronal activity was sufficient, it would suggest the existence of a population of neurons that regulate entry into torpor. And importantly, it would give us a handle so that we can study and control the this process and can, for example, induce long-term torpor and study its effect on physiology later on. So to answer this question, we wanted to permanently label the neurons that are active during torpor in the brain and ask if we can induce torpor simply by reactivating these neurons. So we would first let animals naturally enter torpor, and active neurons express the cFos, as I've shown you in the previous slide. We then divide an approach that would allow us to genetically install a controllable receptor, which acts as a switch in these neurons, so that we can later, at will, reactivate these neurons using a chemical ligand that's specific to this receptor. That way, we can test whether by simply reactivating the neurons that were previously naturally active during torpor is sufficient to induce this state. Now, Now, to be honest, we didn't think that this would work because torpor is such a complex metabolic state and naturally requires hours of fasting. So the idea of recapitulating key aspects of torpor by simply reactivating a small set of neurons in non-fasted mice seemed far-fetched, but the results were actually rather astonishing. Now, you've seen before that there's many FOS-positive cells, many regions of the brain that were active. So what we needed to do is systematically look at quite a few brain regions to try to find if there is one that regulates this process. So we performed a screen across regions of the hypothalamus in which we re-stimulated neurons in different regions and analyzed around 270 different regions. And after that, we identified a region of the hypothalamus which we named the AV MLPA. Now, for most brain regions, when we re-stimulated the neurons that were previously active during torpor, we didn't see a change in body body temperature. So CNO here indicates the point of stimulation, and you can see that there isn't really a change in body temperature when we did that. But when we stimulated the neurons that were previously active in this relatively small region of the brain called the AVMLPA, what we observed is this immediate and beautiful drop in core body temperature as well as metabolic rate. So this was a remarkable finding. We discovered that stimulating neurons that were previously active during torpor is sufficient to induce torpor-like behavioral and physiological changes even in mice that were not fasted. Now, remember back again that 3D brain reconstruction and many different active regions. It's remarkable in many ways that among the many different brain regions that showed neuronal activity during fasting-induced torpor, we're able to identify one region in which the reactivation of this torpor-associated circuit is sufficient to induce torpor-like changes, suggesting that these neurons represent a critical node in the circuit that regulates natural entry into torpor. It was also comforting that in this region, that this— the specific region that we identified here is a part of, of the preoptic area, which is a key thermoregulatory hub in the brain and contains neurons that can sense local body temperature as well as receive information from receive information about ambient temperature. So these torpor-regulating neurons are pretty well positioned to integrate this relevant information to regulate torpor. So we started looking across many brain regions. We narrowed down on one region, and now we wanted to look inside this region. And as you can imagine, every brain area contains many different neuronal cell types, and the preoptic area of the hypothalamus here is no exception. Decades of work have actually shown that the preoptic area contains neurons that regulate temperature, but also thirst, sleep, mating, parental behaviors. And recent work by Jeff Moffett, Catherine Dulac, and Xiaowei Zhuang identified actually over 50 molecularly distinct neuronal cell types in this preoptic area. So we next wanted to catalog the diversity of neurons that exist in this in this area, and then identify which among them were the ones that are marked here in dark, which are active during torpor. So to do this, we microdissected this region, dissociated it into thousands and thousands of neurons, and then we sequenced using high-throughput microfluidics each one of these cells. And here I'm showing you the result of this experiment. This is a UMAP plot. It is a representation of the diversity of neurons in this region of the brain. Each dot here actually represents one neuron. There is about 28,000 of them plotted here, and neurons cluster on the basis of how similar they are into these different color-coded neuronal subtypes. So we identified in total about 36 different cell types in this fairly small brain region. Now, as I mentioned, not all of these neurons were active during torpor. Only a small number of them were, and the rest probably play a role in a different circuit or different behavior. And to identify which among all of these neurons were the ones that were active during torpor, we mapped the expression of that receptor that we installed in the torpor-active neurons. And among the 28,000, we identified only 3,042 neurons that were active in the torpor, and they're plotted here on this graph in red. We observed that there was actually a particularly high density of these neurons in this little cluster up here on the top, which represented excitatory PACAP-expressing neurons. So we decided to further investigate this, this specific population. So we, we at this point had, had certain genetic markers of these cells, and we could avail ourselves of the full spectrum of neuroscience tools that were available to us. And the first thing we asked is, if we monitored the activity of these neurons in real time, could we confirm that they were active during torpor and right as the animals were entering and maintaining torpor? So to answer this, what we did is we installed a fluorescent sensor of neuronal activity deep in the the brain into these neurons. And we also installed an optical fiber in the brains of these animals so that we can monitor these neurons in real time. And here you can see on the bottom the body temperature of one mouse over a period of about 8 hours. And you can see that this mouse is entering several bouts of torpor during this period of time. And here on top, you can see the activity of these neurons, these calcium transients. And hopefully you can appreciate from the plot that there is a distinct pattern of activity when the animals are entering the state of torpor. And in fact, this signal was so tightly linked to when the animals were entering torpor that we could fairly accurately predict if the mouse was in torpor just by looking at the activity of these neurons on the computer screen and not even looking at the animals. So this is one of the things that we did. Another one is we asked what would happen if we inhibited these neurons? What we observed is that when we inhibit these neurons using a neurotoxin, animals fail to naturally enter torpor. So the orange line here are the control animals which are entering torpor, and the purple line here are the animals in which excitatory neurons in this brain region were inhibited. And you can hopefully see that after inhibiting these neurons, animals no longer display the natural patterns of torpor suggesting that these neurons that were identified are also necessary for normal torpor. And then finally, we were able to show that if we simply stimulated this molecularly and anatomically defined population of neurons, we can induce the large drop in core body temperature shown here in blue, while stimulating other neurons in the area shown here in orange doesn't change the body temperature. Suggesting that the activity of this specific defined population of neurons is sufficient to phenocopy this decrease in body temperature and metabolic rate that we observe during natural torpor. So taken together, our work discovered a population of neurons in the brain that regulates torpor. We show that these neurons are active during torpor, that their activity is necessary for natural torpor, and that by simply stimulating them, we could induce a torpor-like state in mice. And I think this is an important discovery and milestone in the field because it enables us one point of genetic access to now monitor, initiate, manipulate, and study this behavior. So what did I tell you about so far? It was a story about trying to understand how mice enter hibernation-like state called torpor. And we now have a key component of this pathway and have the ability to start asking several additional important questions. For example, how do these neurons sense the body's fasted metabolic state to know when to initiate torpor? Also, when they're activated, these neurons rapidly signal to the body to change its metabolism and body temperature and enter torpor. How do they do that? Is it through specific secreted factors, peripheral neurons? So in addition to these and many others being unanswered basic questions about entry into torpor, I think that broadly understanding how the brain senses and regulates the whole body metabolism could uncover new mechanisms through which we could modulate human human metabolism and treat metabolic diseases. So, this was a first part of the talk. And now we have 5 more parts remaining. I'm kidding there. So, this is the main story that I wanted to tell you about. And I wanted to contextualize our work in terms of some of these mysteries of mammalian torpor and hibernation that I very briefly introduced at the beginning of the talk. And now I want to spend a little bit of time, but really just one slide on each one of these other topics to give you a broader sense of some of the many questions that the field, as well as our own lab, is interested in. So one interesting question in the field is, to what extent is the regulation of torpor and hibernation similar? So here is an example of hibernating hamsters. These animals enter deeper bouts of torpor. They stay in torpor longer than mice. And what's really interesting is that to prepare for hibernation, hamsters need to be exposed to winter-like conditions of a shorter light cycle and cold temperatures for several weeks. So how animals are sensing these seasonal changes and recording this information in the brain to regulate when to enter hibernation, when to come out of hibernation, remains largely unknown. And we're hoping to build a lot of tools to be able to answer these questions and study hibernating organisms in similar ways that we studied mice in the past. This is, of course, a long-term goal. Another long-standing mystery in the field is why do animals come out of hibernation so frequently? Here you can see the body temperature of a 13-lined ground squirrel who has in this example aroused from torpor about 20 times during the course of the hibernation season. And each of these arousals is very energetically demanding. Animals spend as much as 90%, it's estimated, of their energy during hibernation on these arousals. So why, why are they doing this? And I can tell you that in this case, it's not that these animals are coming out of torpor to eat or drink or urinate or defecate, because ground squirrels don't appear to to perform these bodily functions during arousal. And instead, they're actually found to be in a sleep-like state during this time. So again, why is this the case? And there's some interesting hypotheses and data in the field, but I still don't think we completely understand this. Now, one of my favorite mysteries concerns cold tolerance. Mammalian cellular processes are optimized for function at 37 degrees Celsius, and relatively little is known about basic cell biological processes in decreased temperature, in torpor, in hibernation. And what's remarkable is that if you place many human cells in the cold, essentially take a plate and put them in the fridge, they begin to lose key structural— key cellular structures, in this case, I think marked here, microtubules. And they may also not survive for more than a couple of days in this environment. Environment. In contrast, cells isolating from hibernating organisms retain these structural fibers, and they can also survive cold temperatures for many days to weeks. So, this is a striking difference at a cellular level. And it may be really interesting to ask— and again, several people in the field have started to ask these questions— what are the genes and pathways that are responsible for for extreme cold tolerance, and actually, could we transfer some of these pathways into human cells so that we can improve cold storage and transplantation of human organs, as an example? There have been many scientific debates also on the topic of the evolution of hibernation, given that so many mammalian species are capable of entering hibernation. You can see many of these marked in blue here. The question is, is it more likely that these behaviors independently evolved in each of these species? Or is the ability to enter these states of torpor and hibernation actually a trait that's left behind from our cold-blooded ancestors quite a long time ago? And simply, it's just been retained in some species and lost in others. So if it is the latter, are these ancestral circuits also somewhat present in non-hibernating animals, in all of these other animals in between? And would it be possible to actually reactivate them in non-hibernating organisms to induce a state of torpor or hibernation? An interesting experiment performed recently by Takeshi Sakurai and colleagues that I think shed some light on this question was to stimulate the same brain region that we identified previously as regulating stimulating mouse torpor in an animal that doesn't naturally enter torpor or hibernation. In this case, it was a laboratory rat. And what was remarkable is that by stimulating these neurons shown here with this arrow at the point in which CNO was injected, they observed a pretty profound decrease in body temperature of about 5 degrees Celsius, which suggests that these animals were or entered a torpor-like state. So of course, a lot more work is needed to better understand whether similar neurons and similar circuits might exist across different hibernating and non-hibernating organisms, including humans, and what are the effects of stimulating these cells. But, but this finding is rather encouraging, and it supports this hypothesis that we could perhaps induce aspects of torpor and hibernation in other non-hibernating animals. And then finally, an interesting study that caught my eye a few years back is a study done in the 1980s by Charles Lyman and colleagues who took a large number of hamsters and placed them in a laboratory environment where they would naturally hibernate. Hibernate. And although the animals were all genetically fairly similar, there are considerable differences in how long they hibernated. So they grouped them into animals that were poor, moderate, and good hibernators. And actually, for each animal, the scientists recorded the length of hibernation as well as their eventual lifespan. And what they observed is that the longer the animals hibernated, the longer their lifespan. And importantly, if you look closely into the math, turns out that for every day the animals hibernated lived about 2 to 2.5 days longer. So to me, this was a pretty striking result that raises the question of whether lower body temperature, the states of torpor and hibernation, slow down processes such as aging or even induce some type of regenerative state. Because again, for every day of hibernation, these animals appear to have gained more than a day of lifespan. So our ability, actually, now that we have identified a population of neurons in in the mouse brain that we can stimulate and induce torpor, and we can do so for much longer than animals naturally enter torpor. Our ability to synthetically induce torpor offers a pretty nice opportunity to investigate the extent to which processes such as aging and disease progression are affected in these long-term states of torpor. So life on Earth evolved amazing adaptations to survive extreme environments. I believe that by studying how organisms adapted to low temperatures, we'll be able to advance our understanding of this biology and eventually harness these mechanisms to advance medicine, perhaps even one day open the door to possibilities of space travel and lifespan extension. I am extremely grateful to everyone who's been instrumental in this work to date, particularly all my collaborators in the Greenberg our growing lab here at the Whitehead Institute, our funding, and you for your attention. Happy to take questions at this point.

Great, thank you so much, Sinesha. I would like to invite everybody to start typing in your questions and remember to add your name if you're one of the Monicas in the audience. I'll start by maybe asking whether this preoptic area has been associated in any cases of human diseases or human injuries with elements that are similar to hibernation or torpor.

Yeah, it's a good question, and I am not aware of studies of lesions of the preoptic area in humans. It is in part possible that there aren't many because it's so deep in the brain, and by the time you get to it, you lesion a lot of other areas. But there have been experiments in the past in cats and other animals back at the beginning of the 20th century where specifically one had lesioned different areas of the brain to see what happens when that is the case. In those studies, it appears that lesion in the preoptic area had some effects on thermoregulation, and that were some of the earliest studies that identified the preoptic area as a hub for thermoregulation. And I, I'm not as familiar with whether those studies actually monitored several of those other behaviors, but since then, other studies that more specifically looked at neurons in that area have identified neurons which, if you lesion them or change their activity, you can affect thirst and parental behaviors and so on, though primarily in rodents.

Really fascinating. There are a couple of questions around the sort of cellular changes either in the cytosol or the cytoskeleton that are associated with torpor and the, the association physiologies. And so I'm wondering if beyond the microtubule effects that you were describing, there were perhaps other characterized changes to those cells.

Yeah, so I think we— at least the way it's helpful for me to sometimes think about it is there is, of course, a big gradient of temperatures from 37 degrees Celsius and as you go lower. And at each one of those stages, there could be different changes that happen to the cells. So there are certain genes that are known to be induced when the temperature is lowered just a little bit, so there's cold-induced genes. A lot of those have to be RNA-binding proteins and so on. There's also big changes in translation that happen, with the translation of the majority of the genes, I think, decreasing at a point in which you cool, but also some intriguing possible observations about certain genes that are not that way. I think we've started thinking more and more as scientists about phase separation and different phase compartments inside the cell, whether it's in the nucleus or the cytosol. And a lot of those are really temperature dependent, and it'll be interesting to see what happens at different temperatures to the biophysics of the cells. Of course, we know that the membrane itself and the fluidity of the lipids is So there's a good number of observations that I think have been made. And they weren't as systematic as I think we can do them today, especially looking at a variety of different temperatures. And then there's, of course, the question of these are temperatures, how similar is this to natural torpor and hibernation? And I'm sure there's similarities, but there's also differences because the whole body torpor phenomenon I think might involve something in addition to just the cell autonomous effect.

Relatedly, Yukiko asks whether the observed cold tolerance of microtubules in the squirrel model might imply that torpor might not be possible for humans, right, in the event in which our own microtubules are much more cold sensitive.

No, I think that's quite possible. I mean, in the study whose figures I drew on for that slide, Wei Li's work, they actually have gone on to think more about what gave rise to that and observed changes in mitochondria and lysosomes that preceded the changes in the microtubules. And they have been able to, using certain targeted small molecules, inhibit some of these stress pathways in human cells that give rise to, at the end, microtubule depolarization. So, not to a complete extent, but to a good extent. So I think I've been somewhat hopeful that if we understand more of these components of what's contributing to the deleterious effects, that we may actually be able to slow a lot of them down or pause them. But to what extent we'll be able to do that is still very much unknown.

So Nancy Devine had kind of a related question about this microtubule observation. So you're suspecting that those characteristics render these microtubules then kind of more resistant to cold and the neurons that depend on them then more resistant as well, right?

Yeah, so I think the expectation is that hibernating species have slightly different, I guess, metabolism and slightly different genes expressed at the right compartments, to speak very broadly about it, that hopefully will give rise to resistance to the polarization of microtubules. And that's just one of the features that I think I highlighted, but we don't completely understand why the cells from non-hibernating organisms die during this cold exposure yet.

Steve Engler asks whether you tested any other areas for similar torpor-inducing effects, other areas in the brain, and relatedly, whether the areas that were targeting in the rat study were analogous to the areas that you had identified.

Yeah, it's a great question. So we did in our study at the beginning, and in the interest of time and in part how complex it was, I didn't discuss it we did actually look into quite a few different regions, but we focused on the hypothalamus. So we took about 50 different animals, and in each animal we injected different regions, and we stimulated all of those, and then we looked in which animals did we see this effect of a decrease in body temperature, which was really a key signature effect of entry into torpor. And we were not we were able with the screen that we did to find other regions in addition to the region that we described, this AVMLPA. Now, it is possible that we missed a region and that there is— because of course the brain is a lot more vast than what we were able to explore deeply. With regards to which region it was, yeah, it was actually pretty much the same region. It was so close enough that I think we'd consider it the same region that was stimulated in the rats that observed that effect.

Someone asks whether there are any dangers that you predict with inducing torpor.

So, you know, I think, I think it's fascinating that animals do come out of torpor every few days or a few weeks, and there's several hypotheses, and one of them is that deep torpor is incompatible with normal sleep patterns, which may be required for normal cellular physiology or circuit physiology. So if one is inducing torpor for long periods of time, we would need to understand what are the natural limitations that are causing animals to naturally come out, and then could we overcome those in order to bypass them and induce torpor? Torpor longer. Yeah, so this is in the context of hibernation. In the context of mice, they come out of torpor sometimes every half an hour, other times every 8 hours, and to be honest, we've been able to keep them in torpor for several days. So I don't think that there is a limit within that period of time, but probably much longer than that, we might run into something.

It's fascinating. See, you only says great talk, very interesting, as I'm sure many of us are thinking. And asks whether you could elaborate on how inducing torpor might be affected by thermogenesis in mice, sort of that relationship between thermogenesis and the actual neuronal program that induces it.

Yeah, so basically, essentially, is, yeah, mice need to continuously generate heat, as in activate thermogenesis, in order to keep their body temperature at 37 degrees Celsius in a room temperature of 22 degrees Celsius. That's a pretty big difference for a small mouse. So we think that one of the reasons, or one of the ways, that the animals are entering torpor is to inhibit this thermogenesis, and that may be happening by stimulating these neurons. And we don't think that that's the entire story, but I think it is likely that the animals are inhibiting brown adipose tissue thermogenesis, possibly also thermogenesis from muscle, possibly also from other tissues. But that's a big component of how they enter into torpor. Now, it's interesting to ask, why are they— why are all of the thermoregulatory pathways not responding back and saying, okay, we're too cold, we have we have to, we have to heat back up. So there is some evidence that we are lowering the set point. So normal temperature set point of animals is around, or of mammals, is around 36, 37. There is some evidence that by stimulating these neurons, we're lowering that set point. So that could in part be contributing to the neurons not— the, the neurons that usually gate the cold temperature are not being turned on. But there's still a lot to be understood there.

Yeah, I think there's a lot of curiosity also around the kind of applications to human physiology, of course. And so a couple of our attendees ask whether it might be possible that inducing torpor during normal sleep cycle would extend the lifespan, or does torpor need to last for a specific length of time and then relatedly, can humans enter torpor through meditation, breath control, or some other methods?

So I would say on the sleep and aging aspect, you know, we just— this is so early days that we just simply don't know. We don't yet know whether inducing torpor in mice extends lifespan. I think there's some reasons to think that it might. Parallels from hibernating organisms, but also the fact that fasted mice have a longer lifespan, and fasted mice routinely enter torpor. And if you fast mice and block torpor, the lifespan extension is not quite as dramatic. So I think there's reasons to think that, but I would say it's still early days to know whether inducing, you know, torpor in addition to sleep short-term could have some beneficial effect. And the second question, can you actually remind me what—

whether meditation might kind of influence or induce torpor-like states?

So, I mean, I certainly don't know, and I'm not sure to what extent that has been studied. I think there are examples where of humans that are able to meditate and lower their heart rate, I think, really dramatically, and respiration rate, perhaps to some extent body temperature as well. But to be honest, I'm not very familiar with that literature to know to what extent.

Yeah, I think it's a fascinating question. It may be something to be studied. There's a kind of quick question of how long you were fasting the mice in order to induce the torpor-like state. State?

Yeah, so I was kind of surprised by this, to be honest. So it's, if you start the fast at the beginning of their dark cycle, which is when they're active, it usually takes 8 to 10 hours of the fast in order for mice to enter torpor. We almost never see them entering it within the first 4 to 5. Now, if you were to start the fast sooner in the light cycle, we actually You almost add the hours of the light cycle to it roughly. So it's— and the reason for this is that torpor is in part circadian regulated. So the deepest bouts of torpor are between the transition from the dark to the light cycle. And you kind of want to, you know, want to be in a properly fasted state at that time in order to enter it. So it's not just the duration of fast because you might recall that eventually the curve comes comes back up. So eventually, although they're still fasted, their body temperature recovers, and at the 24-hour time point, they're much less likely to be in torpor.

Yeah. And so relatedly, Monty asks whether the multiple rounds of torpor that you see in hibernating animals might be used to probe the environment and know when it's sort of safe to come back out of hibernation.

It is quite possible. So I think for— I mean, so hibernation aside and, and what it's causing those states, but in mice it is quite possible that that's playing a role in it. It's interesting also that the male and female mice have different patterns, so the male mice tend to enter shallower torpor and more bouts, while female mice kind of hang out longer and then enter deeper ones. So there's a part of me that wondered if you have a colony of animals, if it actually makes sense to have somewhat asynchronous patterns so that there would be a surveillance mechanism But that's— yeah. So I don't know much beyond that, though, to be honest. And I think if we figured out what are some of the neurons that regulate arousal from torpor, which could be doable as one of our next steps, we could think about when they're active and what is the range of situations. Yeah.

Now, you were telling us about mammalian torpor, of course. But there's a question from Michael Sheehan. Asking whether these processes in mammals are similar to those observed in hibernating amphibians.

Yeah, that is an area that I would be— that I definitely need to learn more about. And I am interested in thinking, but I don't know how similar they are and how different. I think amphibians, many— I'm not sure if there are actually exceptions to this rule, but but generally don't regulate body temperature through thermogenesis, I believe. So I think some of the mechanisms are going to be distinct from the standpoint of how the process is regulated to be initiated. But at the end of the day, they're cells which are also at different temperatures, and a lot of the cellular processes that allow them to live at those temperatures may be more conserved. And I guess to relate to this, right, this idea of evolution and how did this whole process evolve in mammals, is it actually a remnant of the fact that we evolved from cold-blooded animals and some of those mechanisms stayed behind? So in many ways, I think mammals are relatively new evolutionarily, and a lot of the ways in which cells survive at lower temperature may be conserved and more.

Yeah, and that organisms have had to respond to stress since their origin in a way. Some of these same pathways then get drawn on for new adaptations. Really fascinating.

Beyond and thinking even about plants, and of course they don't have the option to move around and hide from the weather. So there's— yeah, yeah.

Anthony Komarov asks, when nutrient deprivation or cold activate the torpor nucleus, what might be the molecular mechanism that activates those neurons, and what are the mechanisms that deactivate those neurons during arousal?

So those are good questions, great questions, actually. So if we think about nutrient deprivation, so we are trying to figure this out in part, and some of that is sensed through other specialized populations in the neurons, populations of neurons in the brain, for example, in the arcuate nucleus, there's these AGRP, POMC neurons. So we think that they actually play a role in sensing the nutrient state state of the body, whether via leptin or a variety of other factors, and perhaps conveying that information to the neurons that regulate torpor. There might be other ways in which that's done, by the neurons themselves sensing the availability of certain amino acids and states, but we don't actually know about that. That's just a hypothesis. How would, how would a population of neurons prevent or cause a arousal. It is just possible that there is a population of neurons that reacts to, let's say, stress, to follow up on Monty's question, or some other environmental stimulus, which, which then simply inhibits the neurons that are activating the torpor circuit. And, and that, in addition to stimulating other types of movements and so on, should in principle be sufficient to cause arousal. Back out.

And right now we don't know what that kind of arousal nucleus is, right?

We don't. Yeah, we don't. And I'm not sure it's going to be super simple, but we don't. Yeah.

But a really fascinating question. We have a question about whether there are any drugs that are known to interact with this specific region of the brain and induce sleep or lowered body temperature, torpor-like states?

It's a great question. I, I, so I don't exactly know. So there's definitely drugs that, that are, that, that induce fevers, which are also regulated by the same region of the brain, of the brain through, through prostaglandins. And there is drugs that, that inhibit that process in some sense. But I, in humans, I don't know of drugs that directly target neurons and target the kind of torpor or thermoregulatory neurons that are lowering the body temperature. Again, for fever, I think we know some of them. In mice, there are certain substances that you can inject into mice to lower their body temperature. So adenosine receptor blockers, receptor agonists do it. We don't think— it seems to be mediated through the brain. We don't exactly understand how. And yeah, there. Yeah. So I don't, I don't know.

I don't know if there is some hope through research and development to be able to kind of generate chemical modifiers of these states.

Yeah, I definitely think so. And I think it is possible to— I mean, we are beginning to do a lot of this molecular profiling of neurons and understanding receptors that are expressed on them. And you come up with receptors that are orphan or that we don't exactly know how they function, but some are really specifically expressed. And of course, we're just talking about one node, and then there is also the neurons downstream of it. So there could be quite a few areas that one can target with regards to changing the body temperature set point or thinking about lowering temperature or metabolic rate.

Yeah. From Paul Soloway, are the energy sources used by animals changing when they enter torpor from glucose to glycogen or fatty acids?

Yeah, so they appear to be changing to fatty acid metabolism really rapidly. So I think, yeah, I think in the literature people have looked at this in the steady state. We've actually seen that when you stimulate these neurons within within a really short period of time— I mean, half an hour or less— there is a rapid change in the metabolism. And it's unclear which organs are all responsible for that, but there appears to be a pretty large systemic effect.

Yeah. Ruth asks whether there are cells that don't respond to torpor and that function normally even if the other cells are slowed down.

I don't know. I'm guessing— I'm sure there must be some cells that don't. So I'm thinking about— I'm thinking about there is neurons in the brain that— there's neurons in the brain that are supposed to set body temperature set points and certain set points with activity. But temperature changes so many things. So it would have to be temperature independent. Dependent. I'll think more about this. I don't know. It's a good question.

Yeah, like maybe a challenging thing to assess overall a cellular function in a whole animal.

Yeah.

We have time maybe for just a couple more questions. Can hibernating animals form any memories during their arousal periods?

I don't know an answer to that question. I think whether hibernating animals retain memories and to what extent is somewhat controversial There's some examples in which they do, some in which it's not as good. But I don't know if— I don't know of a study. Maybe there is one that's tested specifically that. It's a good question. Yeah.

And then from Boshia at NYU, considering all of the different animals that enter hibernation, are there geographical differences that distinguish hibernators from non-hibernators?

So a lot of hibernators out there do enter hibernation during the cold season, winters, on either hemisphere. There are also animals that enter states of torpor that are in the tropics where it isn't cold-induced. So lemurs in Madagascar is an example of that, and some of that has to do with, with being faced with seasons of drought and lack lack of water, presumably also food. But so they are, they are kind of, I think, animals that are entering on either end of the extreme.

Thank you so much, Suneesha. Then looking at the time, that really concludes today's presentation. I want to thank everybody for joining us today, and a special thanks to Suneesha for sharing your work with us. I hope you'll stay involved with Whitehead and join us on future webinars. Have a good day, everybody.

Yeah, thank you all. That was fun.