 an honorary degree, and we're very fortunate to have Dr. Michael Young with us during this conference. And so I wonder if I could ask the panel to join us, I'm sorry, Dr. Young, Dr. Ferragamo, President Bergman, all to join me on the dais here. And on the panel here, or I'm sorry, I keep calling the panel, on the dais, I have Margaret Bloccazi, the chair of this year's conference, Dr. Mike Ferragamo in the Department of Biology and Psychology who will read the citation, President Bergman, and me. Okay, so, Mike. Today, Gustavus Adolphus College honors Dr. Michael Young, the Richard and Jean Fisher Professor in the Laboratory of Genetics at Rockefeller University, where he also serves as Vice President of Academic Affairs and where his work has been supported by the NIH Merit Program and the Howard Hughes Medical Institute, each granted exclusively to experienced investigators who have a record of superior research. Dr. Young is a member of the National Academy of Sciences and a fellow of the American Academy of Microbiology. The preeminence of his contributions to the advancement of scientific inquiry have been honored by numerous prestigious awards that include, among many others, the Luisa Gross Horwitz Prize, the Mass Reprise, the Shore Prize in Life Sciences and Medicine, and the 2017 Nobel Prize in Physiology or Medicine with co-recipients, Drs. Michael Rospash and Jeffrey Hall. Professor Michael Young's career has been devoted to unraveling the molecular and biophysical mechanisms that organized the daily rhythms of life in the fruit fly, an organism widely used as a model system in the life sciences. He made the remarkable discovery that the activity of genes and the levels of the key proteins that they produce wax and wane to the beat of a 24-hour period. Every one of our cells has a clock controlling its actions. Some cells work best at night and some during the day. This knowledge has had significant implications for medical practice. For example, by allowing precise timing of medications or in mitigating the health consequences of shift work. However, as individuals, we are more than a collection of cells. We also are social creatures. An interaction with others can alter the timing of our daily activities. Very recent work from Dr. Young's lab show that the humble fruit fly can provide insight into this broader human experience, too, as he and his colleagues itemized the cost of social isolation on physical and mental well-being. Dr. Young has illustrated truly that in biology, timing is everything. For his contributions to medicine and physiology in revealing the vital mechanisms of keeping cellular time, Gustavus Adolphus College is both honored and privileged to bestow an honorary degree upon Dr. Michael Young. Upon recommendation of the faculty, Dr. Young is presented for the degree of Doctor of Science, honoris causa. Dr. Young, for your unwavering efforts to advance scientific discovery from circadian rhythms to loneliness, Gustavus Adolphus College is privileged to bestow an honorary degree upon you. And now by the authority vested in me by the Board of Trustees of Gustavus Adolphus College and upon the recommendation of the faculty and approval of the Board of Trustees, I hereby confer upon you, Dr. Michael Young, the degree of Doctor of Humane Letters, honoris causa, with all of the rights, privileges, and honors pertaining thereto. Dr. Young, we now welcome you to share your remarks. So this has been quite a conference. I don't think I've been at a conference that has shown such a diversity of expertise in quite a long time. So it's been truly enlightening to be a part of this. The title of my talk, as you heard, is if I can get this to advance. No, I have to go back. There's a delay. OK. So as you heard, the title of my talk is What Happens to a Lonely Fly. And as you might suppose, the experiments I'm going to tell you about were initiated at a time when many of us were lonely. We were undergoing the social isolation associated with COVID. And the many effects of social isolation that were produced during that time raised questions about whether, once again, we might use Drosophila, which has helped us understand behavior so many times and other features of biology so many times in the past, might provide some information once again. So this is a data that was collected by University College London going from March of 2020 to March of 2021. And the data shows responses from 70,000 U.K., United Kingdom respondents, with regard to their assessments, their personal assessments of their sleep quality. And what you see at the beginning, there's both male and female data presented here. And what you see in the beginning of the study, sleep quality is pretty good, but monotonically declines over the course of a year. And the vertical lines that you see here are just indications of when lockdowns occurred in England or throughout the U.K. And of course, those lockdowns were very much pointed at trying to achieve a high degree of social isolation. Now, it's very difficult to know whether these sleep effects, these effects on perceived quality of sleep, were derived in particular or in part from social isolation being implemented at the time. And it would be very difficult to ask that kind of a question in a human population. But we thought maybe we could get some ideas about this from looking, once again, at the simple insect that's taught us so much in the past. So on the right, you see a cartoon essentially that shows a device that we use to follow sleep wake activity in the fly. It's a computer monitor that's hooked up to a plate that carries several vials, each with a single file and fly in it. And those flies can move freely back and forth in that vial. There's food at one end, and the other end is stoppared. So there's a gas exchange, air exchange. So the fly can survive in there for a couple of weeks at a time. And across this set of fly-filled tubes is cast an infrared light beam. And much like a burglar alarm system in a home, when the fly passes that beam, a signal is sent to the computer that's recorded an event, a moving event. So you know the fly is in motion. So the way we think of sleep, ever since work done in 2000 by colleagues Hendricks and Shaw, is to observe the flies and notice that when a fly's been quiet for five minutes or longer, the fly often tends to display characteristics of sleep or rest, a different posture, and often other behaviors that can be associated in insects with sleep. So what we're doing on the left side of this chart is looking at 30 individual records of flies in this kind of monitoring. The blue bars on each line going from one side to the other is a single fly with the blue bars representing times that they're asleep. And one of the things you'll notice is this is during a light-dark cycle, flies sleep a lot. In fact, in these assays, they get up in the morning for a few hours. They take a noon siesta that's quite long and get up a little bit in the evening and then sleep through most of the night. So we've taken the data from all 30 of those flies and plotted it in a different fashion beneath. And you see once again that kind of a curve that indicates a midday siesta followed by fairly consolidated sleep at night. So the social isolation reduced sleep in Drosophila. So does it affect sleep at all for that matter? So the method that we used to ask this question was to allow newly emerged flies after they've developed in their pupae, after they've emerged for three or four days. We put them together, males and females, in a bottle so that they can have some social experience for three to five days. And then we take the individual flies and we move them into vials that contain different numbers of flies. And what you see represented there is a vial of one fly, a vial of two flies, five flies, 25 flies, or 100 flies. And then we hold them in that situation for seven days. And then we move them one at a time into these individual smaller tubes where we can monitor their sleep-lake behavior. And what you see at the bottom of the slide are a series of curves that represent the sleep-lake patterns of all of those experiments. And you'll notice that there's one deviant from the group. Most of the flies show a very similar pattern of sleep and wakefulness, with the exception of the isolated fly. The single fly is showing less sleep during the day and about the same amount of sleep at night, but less sleep during the day. An impressive feature of this that surprised us at first is even another fly, two flies in a vial, will keep this from happening, which is maybe one of the reasons so many pets were bought in New York during the pandemic. Social isolation, is it duration dependent? Does a fly know how long it's been isolated? We knew very little about how a fly perceives time over multiple days. So again, we allowed the flies to gain social experience together and then compared what happens to a group of 25 flies versus a fly that's isolated all by itself for one, three, five, or seven days. And again, monitored in the same way that I described before. And what you see here moving left to right is that the longer the isolation, the more profound the level of impact on daytime sleep. So one day of deprivation, three days of deprivation, five days of deprivation, you begin to see an effect, but at seven days you see a maximized effect, which is held after that time. So somehow the fly is able to determine, perhaps, that things are hopeless after a week, that they're never going to get out of this situation. This is something that we've seen in many different types of drosophila. We've seen it in inbred lines that naturally vary with the amount of sleep that they have under normal conditions. We've got short sleepers and long sleepers, maybe like some of you in the audience that brag about sleeping only four hours a night, others that want your eight or 10 hours a night. There are fly lines that vary in that way. But this kind of an effect occurs in all of those lines. It's a uniform response. It occurs in young and aged flies. It occurs in many other isogenic strains and in many drosophila species, every species we've tested. Now this shows that the maximum effect that we see after seven days is focused on a time early in the morning so that most of the sleep that's being lost is lost from the early part of that midday siesta. So at the bottom of the slide, you see that about 40% of the total sleep expected at that time of day is lost after seven days in this climate experiment. And there's a progression so that total sleep is lost, but much less severely. Daytime sleep is lost to a higher degree. But that first four hours of daytime sleep is maximally affected. We've tried to ask whether there are exposures that we can produce to these isolated flies that will relieve whatever this stress of social isolation might be. So for example, in the middle of this graph, we show 50 plus 50%, minus 50%, and 0%, where 0% would be the level of sleep loss that is associated with being in a group, which of course, as I've shown you, that's your control value, that's your baseline. And what we're measuring here is we're focusing on that first four hours of daytime sleep, the loss of that early part of the midday siesta to get the most significant insights that we can. So what we've seen is a strong depression. Again, about 40% of that sleep is lost when a fly is by itself for seven days. On the other hand, and we've tried things like leaving a group of 25 flies in a vial, vial number three, for two or three days, and then removing those and replacing them with a single fly. Does that make a difference? It doesn't seem to make a difference. There aren't olfactory cues that are left behind on doing that kind of experiment. We've tried placing a glass slide down the middle of the vial and having 20 flies on one side and one fly on the other side of the glass slide to see if visual stimulation will produce a different effect. But that doesn't work. The flies still do sleep. And we've tried adding netting to separate the single fly and the group of flies, netting of various of different sizes, some that will only permit chemical cues to get across and others that would allow the fly to reach through and perhaps touch flies on the other side of the screen without actually being able to move to the other side of the screen. And what we see in these cases is a further depression in the amount of sleep, almost as if being excluded from that group when you know they're there and you can interact closely with them, but you can't join them, produces an additional stress with regard to the measurement of sleep. We found that if we add a single fly of the species to esophilus semulans, which is a related species to melanogaster, that will be treated as a melanogaster cluster of flies in the same vial. And so that will relieve the stress of being alone. And in two cases, we've tried ladybugs to see if just some other living object in the living being in the tube makes a difference. And in one case, these were from two different vendors. In one case, we got a response that was very much like adding back flies. So it may be, again, I refer to pets as something that many of us ran for during the pandemic if we were alone. Another kind of insect, milkweed bugs, did nothing for this. And we really produced a strong negative response by adding a series of foam balls in the, so unlike the bees that we saw the other day playing with rolling balls around, this didn't produce that kind of interest at all. So how do we go from there? We really did want to understand what was going on in the fly's head in the brain that might be responsible for this response. It's the fly hardwired in some way to have a response to social isolation and involve the depression of sleep. So what we decided to do was to take a sample of RNA, total head RNA, from flies in that critical window when they were losing sleep and just before it, including the time just before it, to see if there were gene expression changes that were associated with a fly that had been chronically isolated versus a fly that had been isolated for only a day where we don't see a sleep defect. And compared to flies that have never been isolated that have been reared in a group. And so you see from top to bottom the plots of behavior of those three different groups. And so what we were interested in are there changes that separate the chronically isolated flies, that is flies separated for seven days, from both those that have been isolated for only one day and those that have not been isolated since neither of those latter two groups are showing a sleep deficit. And what you see from the intersection of these VIN diagrams is that we recognize 274 genes that were uniquely being altered in those comparisons where the comparisons were flies that showed the sleep deprivation versus those that don't. On this slide shows that there are only two categories of gene expression changes that would be of interest in this kind of experiment. We need to either see genes that are going down in response to social isolation, chronic social isolation, seven day isolation, or going up. We're of course not interested in things where all three categories differ. So on this slide category two and category four are the genes that we're most interested in. And you'll see rather few genes shift down in their expression but many genes are shifted up in performing and producing chronic social isolation. On the right is shown a graph of the kinds of genes that are affected. And the majority of these genes are genes that affect metabolic pathways. And you can read the classes of metabolic pathways that are affected at the top. At the top, for example, oxidation reduction processes. So a great number of genes that affect metabolic pathways. The open bars represent the probabilities that those would occur by chance with the longer bar being the lower probability. And the black bars are just indicating the numbers of gene expression changes that fall in that category. Now if we focus on the right side of this graph, of this chart, we see the top 20 changes in gene expression. And once again, these are almost all metabolic changes in the fly's head. And if you'll note the arrows that are on the far right side of this chart, those are all arrows that show the positions of genes whose expression was most altered in flies that had been starved for 24 hours for software in another study, an earlier study. So this profile of gene expression changes strongly resembles flies that have been starved. And of course, these flies have had full access to food. The only thing they haven't had access to are have been other flies. So this was an interesting puzzle to see. So what we thought we should ask is whether or not we've been looking at sleep, but maybe we should look at something else. Maybe we should look at feeding and appetite. And to do this, we had to modify the behavior assay that we were using somewhat. We used a video assay and a feeding paradigm in which we put liquid food in a capillary tube and provided that through a hole in a stopper at the top of the tube carrying a single fly. And we can look at the response of a single fly that's been group reared or reared for seven days in isolation using a video camera to look simultaneously at the change in the amount of food in the capillary versus over 24 hours versus the movements of the fly. So we can plot both of these simultaneously. We can mark the meniscus of the food with a die so that the video recording will very accurately tell us how much food is being consumed by the flies. And what you see, if you look at the bottom, you see that there's a substantial difference in purple of the amount of food being consumed by the fly that's been socially isolated. So seven days of social isolation produces flies that consume about twice the food that a fly that has been reared in a group consumes. And again, you've got a fly that's been in a group for seven days, a fly that's been alone for seven days. You put them in this monitor for a single day and you see the after effects of that social isolation. Now if we just look at the bottom of this slide in purple and green, we see these changes in food consumption with different measures, different lengths of daytime. So total food consumption is on the bottom left. Food consumption during the daytime is shown in the second panel. The third panel is nighttime consumption. And the last panel is that food consumption during that first four hours of the morning. And what you see is in every case at least twice the amount of food is being consumed by the lonely fly, by the fly that's been in isolation. The top just reinforces our earlier conclusion that sleep is also being lost. But I think the change in feeding is so striking that I wanted to concentrate on that. So this reminded us of another study, again, in humans, which took place during the same period of the pandemic. This involved a little over 3,000 adults in the US, well, mostly adults in the US. And as you see, many different groups are being surveyed here. Two thirds of the adults in this study either had a significant weight gain or a significant weight loss, with most of the changes on the weight gain side. And some of the gains can be quite substantial. For example, in millennials, maybe that's surprising. We get an average of 41 pounds of weight gain over that year of time. And that's for ages 25 to mid-40s. And you can read the rest out. But it's a fairly consistent pattern where there was a very strong tendency for weight gain in this human population of about 3,000 individuals. So this fly is doing two things that we've seen in previous studies. This study was from the American Psychological Association. The initial study I showed you on sleep was from University College of London. But in both cases, we see this striking parallel of our own behavior. And the behavior of these much simpler, as we view them, flies Drosophila. Now, when we look through that list of gene products or gene activities that are most changed, there are a few things that stand out, especially since we've now seen that food consumption is so strongly affected. Limistatin, which you see in the center of this slide, is an appetite-stimulating hormone. And what it's doing in these socially isolated flies is it's lifting and its abundance very significantly. On the other hand, Drosophilkinin is a satiety signal. It's a signal that's produced when the flies had all it wants to eat and doesn't want to eat anymore. And what you see is that that's depressed in our socially isolated flies. So we're seeing something very consonant with the observation that chronic isolation is producing a change in the amount of food consumed. So we thought this might be a pathway into understanding where in the brain these responses, what part of the brain might be responsible for these responses to social isolation. So this slide shows on the left the pattern that we get in the central brain. On the right side of this slide is shown a cartoon of a cross section of a Drosophila brain. And in the center of that image, you'll see something called the FSB for the fan-shaped body. This is a densely populated. It's full of neural projections. And that region of the brain is known to be involved in a number of very complex behaviors, ranging from sleep to orientation to olfaction. And one of the great benefits of this region of the brain is that Genalia, part of the Hughes Institute, has published a complete connectome of this region of the brain. So that every neuron has been mapped and every synaptic connection for those neurons have been mapped. And a vast connectome that was produced by looking at serial electron micrographs from hundreds of investigators who pulled their energy to make this extraordinary resource. So on the left side is shown a pattern of immunostating that comes from using an antibody to limistatin. Remember, limistatin is turned way up in these socially isolated flies. And if we look at which cells in the brain are producing limistatin, we find it limited to this group of neurons. On the top are shown the positions of the projections of those neurons on the bottom in purple are shown where the cell bodies, they're at some distance away from the fan-shaped body. And so they project into the fan-shaped body where they make these many synaptic connections that I was talking about. Now the trick that we wanted to take advantage of is we've got many, many tools in Drosophila that have been created to allow us to specifically produce activity of a gene of our choosing in a specific cell type, in a specific neuronal type, in this case. And so we identified one of these tools, which is labeled P2-Gal4 in this slide, which specifically produces a gene activator, which is in these cells. And in this case, what you see is that tool P2-Gal4. Gal4 is the activator. P2 is the cellular response element that gives it specificity in just this group of cells. And this is driving the expression of a fluorescent protein, green fluorescent protein. So now a specific group of cells will respond to that tool by creating a green image. And what you'll notice is that that green image overlaps with our limostatin immunostaining. And if you superimpose the two images, the immunostaining image and the fluorescent image, you see that you get white overlap essentially everywhere that you saw the limostatin cell bodies and their projections. So this is a very useful tool then in probing the function of this specific group of cells that is responding by producing limostatin at high levels in response to social isolation. So what can we do with a tool like that? Well, what we can do is we can instead of expressing a fluorescent protein, we can express a channel, a protein channel that allows ions to flow into these neurons. So we've chosen to use the P2 tool to drive instead of the fluorescent protein, a protein called Cure2, which is a potassium channel, a specialized potassium channel. Overexpression of that channel in these cells will produce hyperpolarization of those cells so that they can no longer fire. So essentially what we're asking is, what happens if we now have identified the cells that we're interested in? What if we silence those cells with a special channel? And what you see on the top right is that this normalizes the sleep pattern. The sleep pattern now we don't have that deficiency in daytime sleep that we saw before. On the other hand, if we have just the tool or just the channel but not in the same fly, we get the responses that are seen on the lower two panels where once again we see a return of the loss of sleep. So putting these two things together abolishes the sleep problem that we have by maintaining a fly for seven days to give them chronic isolation. And again, at the top is just showing the overlap of the limit statin expressing cells and the cells that are being driven with this tool. This is the result that we see in that experiment when we look at appetite rather than sleep. And what you see on the left are the two elements of this construct, which by themselves should not relieve anything about chronic isolation. But on the right, we've combined them. And we see now inactivation to hyperpolarization of those cells. And you now see the purple trace of appetite collapse on the appetite of group reared flies. So we can eliminate both components of this response to social isolation by turning off this specific dedicated group of cells in the central brain. Now, this is a little more complicated experiment, but it's an important one. And I'm just going to focus on the right side and the rightmost set of experiments, which are indicated in red. What we have here is the same kind of experiment where we're driving in the same neurons with the same tool. We're now driving, instead of something that will silence those neurons, we're expressing a channel which will activate those neurons when we raise the animal at slightly higher temperatures. We're using a channel called trip A, which was discovered because it's the channel in your mouth that recognizes chili pepper is hot. But it's the same channel that recognizes hot temperature and the reason that chili pepper tastes hot is because it's the same channel. So we're using that channel to say, what happens if we activate these particular neurons in the fly brain with temperature? So we have on the right side, you'll notice at the right bottom, you will see flies that are marked as 22, 22, 28, and 28. And the 22 refers to the temperature being 22 degrees, which does not activate this channel. And 28 represents the temperature sensitive response of that channel so that we get activation of that channel. We get activation through 28 degree temperature. And what you notice is that the purple response goes up fairly dramatically over on the right side relative to both the 28 without that channel and the 22 with or without the channel. And an important point to make here is that we don't see a response if we just activate those cells in group red flies. The fly has to have some social isolation but only a single day. So now the fly is interpreting a single day of social isolation as if it's been chronically isolated, isolated for seven days. So somehow, not only did these neurons control a response to social isolation, but there's some measurement function involved so that the fly is able to use these particular cells, fire these particular cells in a fashion that reflects how long that social isolation has been. And now what we've done is we've fooled these neurons and therefore fooled the fly into thinking it's been isolated for a full week. So why would there be a convergence of sleep and appetite in response to social isolation? Again, we've seen it in humans as well. And it doesn't have to be the same mechanism, but in the fly we can explore this pretty directly. And we know from other studies that there are convergent projections to the same area of the brain coming from well-studied sleep-promoting neurons in the dorsal brain. And those are shown here. There is a tool that will mark those particular neurons called R23E10. So when we express that tool along with that green fluorescent protein product that I was speaking of previously, and we look using anti-limistatin at the antibody to limistatin versus this new sleep-promoting tag, which is going to be tagged with fluorescent green, we see a strong convergence once again. But this time the convergence is in an overlap. It's a direct juxtaposition on the right of the projections of the sleep-promoting neurons and the projections of the limistatin-expressing neurons. So it's possible that the sleep response or the appetite response is due to the fact that you can't both sleep and eat at the same time. And therefore you're measuring a consequence of having two behaviors that are mutually exclusive. But it's also possible that we have a wired system that deliberately generates a response in both sleep and appetite in response to social isolation. And some evidence in that favor is that there are abundant synaptic connections between these two groups of cells of neurons, the projections from the sleep-promoting neurons and the projections from the limistatin neurons. So let me just finish up by giving you some conclusions. First, we never really thought of Drosophila as being highly social, but we're impressed that a fly does care whether other flies are around. And in fact, we have a good deal of evidence now that they can distinguish long periods of isolation from short periods of isolation acute from chronic social isolation. As I've shown you, chronic social isolation activates central brain neurons that regulate appetite. The increased feeding, the starvation brain state induced by chronic social isolation appears to broadly reset expression in metabolic proteins throughout the brain. So we believe that now is beginning to make sense. Since we've stimulated appetite, we've stimulated consumption, we might expect to see metabolic ramp up in genes handling higher metabolic rates. As I said a minute ago, the drive to feed may indirectly inhibit sleep. But again, these connections, these tight synaptic connections, abundant synaptic connections between these two groups of neurons make us entertain another possibility, which is this is really a deliberate wiring of these two sets of behaviors together in response to social isolation. And then finally, the hyperactivity of the P2 neurons, which alters the duration of social isolation that's needed to get to reduce sleep and to elevate appetite. Our ability to manipulate that by stimulating these neurons again suggests that those neurons also have some capacity for determining how long the fly has been isolated to distinguish chronic social isolation from a brief period of social isolation since we can convert one to the other by prematurely activating these neurons. So this is a bit of a ways from our original interest in circadian rhythms, but I think it was a fascinating journey that was stimulated by a huge health problem in human populations, which we thought might be much more difficult to study in human populations. And maybe we could get some insights from Drosophila. And so I think once again, Drosophila has proved itself to be a useful model for asking questions. We're gaining insights that would be difficult to have in any other model system. So thanks very much. And that's it.