Sara Hebert :	I think I'd like to get started with my introduction of Alex Baugh, who's giving today's lecture. I'm so delighted to see that all of you have come out to hear him talk. Alex has just returned from an incredibly productive leave, in which he put out eight papers. Six of them are published, two of them are in review. Included in the authors on these papers are 12 different Swarthmore students. I had a friend who once had to put out a whole bunch of papers, and the way she got herself to just sit down and do it, she actually took a bungee cord and she put it across like the chair, so she bungee corded herself to the chair. I don't know what Alex's method is. I'm going to ask him about that later, but I think what we can take away from this is that his colleagues and his students can look to him as an extraordinary model of efficiency, but also real attention to fine detail and high quality.
	How did Alex find himself in this position? I'll take you back to his undergraduate degree. He went to the University of Utah. He was a major in biology and a minor in chemistry. After that, he went on to do his PHD at the University of Texas at Austin, where he worked in Mike Ryan's lab. What Mike Ryan was doing was working with frogs, and the particular frog that Alex worked on was a timber frog in Panama. I have to say that the name of this frog is onomatopoeia, so this is the call the frog makes. Because Alex said he likes surprises, I'm going to ask him to make that call. 
Alex Baugh:	That's not what I meant by surprises. 
Speaker 3:	Good surprises. 
Sara Hebert :	Yeah. 
Alex Baugh:	[inaudible 00:01:47]
Sara Hebert :	He was looking at the behavior surrounding the calling behavior of the males and the females who were responding to these calls and choosing their mates on the basis of them. That was the frog work, and he's going to come back to his frog work. Then he stuck his fingers in some other pies. He went to the Muskot Institute in Germany for ornithology in Germany. There, he wanted to learn some techniques for studying hormones, because he's really interested in the mechanisms underlying these behaviors. Hormones is a really good place to turn for that kind of knowledge. He worked in the lab of Mikaela Howe, and there he stopped working on frogs for a while.
	The work was done on birds that are very similar to our chickadees. If you have a feeder at your house and you have chickadees come, then you probably have kind of an idea of chickadee personality. If you watch chickadees closely, you'll see that individuals have different ways of behaving, have different sort of personalities. He was interested in that, and also in how hormones are related to that personality. He discovered, in work that he's going to tell us about today, that these different personalities are associated with different hormonal responses to the situations that the birds are in. 
	Today, what we're going to here is sort of the bird phase of Alex's work, before he goes back to working on the frogs. One thing that I think it's important to know about Alex is that he is a first generation college student. He is really interested in supporting first generation, first gen college students in his lab. He's done that beautifully with a couple of students already. I think a really wonderful part of this story is that he has already inspired the next generation to attend college. If you know Alex, his next generation is two and a half years old. His name is Jasper. He's very precocious verbally, but I don't think he's ready for college yet. It's not Jasper. The member of the next generation to be inspired in Alex's family is his mother. Watching Alex do all the work necessary for his PHD inspired her to get her undergraduate degree, and she used that to begin a career in teaching. I just love that. 
	There are so many more things that I could tell you about Alex. I think one of the things that struck me the first time I met him, is that he reads widely, thinks deeply, and is an interesting person to talk to about just about any topic you can think of. You can try that out on him. For now, I'm going to stop talking and I'm going to hand the lectern over to Alex. Let's welcome him and listen to what he has to tell us about his research. 
Alex Baugh:	Wow, thank you Sara, that was wonderful. Can everyone hear me? I'm mic'd because I'm at voice fatigue stage today. This is really wonderful to have a prosthetic. Thanks for that lovely introduction Sara. It's a real honor to be introduced by Sara Hebert Verge today, not only because she's been a really important mentor of mine since coming here to the college, but because she has served our college so well, both in teaching in general and research, and specifically in the field that I'm going to talk about today, behavioral endocrinology. She has taught legions of our students in this field. It's been incredibly popular with her students, and she's done it with incredible success. It's a real honor to be introduced by her. I also want to thank the Associate Provost David Harrison, for organizing this faculty lecture series. It's an honor to kick it off this year. 
	I'm a bit of a split personality, in terms of my research interests. On the one hand, as Sara indicated, I have been studying song birds, but also temperate and tropical frog species, for the past 20 years in the case of birds, 15 years in the case of frogs. With the birds, I've been focused on personality, and endocrinology, and vocal learning. With the frogs, a separate set of questions and interests about sexual hearing, auditory physiology, how females make decisions about who to mate with. These two separate research streams have been pretty separate for most of my career, but there's beginning to emerge a bit of a confluence. Hopefully, by the end of this lecture I'll show you where these two streams meet. 
	Before I do that, I want to give you a little bit of a background on why I do what I do. I'm always interested in hearing what got someone interested in the field that they study. For me, it's pretty straight forward. I study animal behavior because I'm fascinated with animals, because I enjoy working with them, physically interacting with them, finding them in nature. There's a challenge to that treasure hunt that's really rewarding for me. I want to be in touch with them physically. That's why I do what I do. For me, science has, for the most part, merely been a mechanism, a vehicle, for doing that work, for interacting with animals in the natural world. It's been the mechanism that has made those opportunities come alive. 
	Konrad Lorenz in 1973, after being awarded the Nobel Prize in Physiology and Medicine, and really the forefather of animal behavior, is quoted saying, "Behavior is the liveliest aspect of all that lives." That's certainly true for me, in that it was animal behavior that captured my attention. I think that's true for a lot of biologists, non-biologists, casual observers, students, children, adults, etc. It's what generates questions immediately for us, and those observations in nature are, for many animal behaviorists and certainly for me, where the science begins. 
	Now I study birds and frogs these days, but I haven't always studied birds and frogs. I've worked with bears. I've worked with sea urchins. I've worked with insects. I've worked with small and medium sized mammals, but I've landed on birds and frogs. That's not random. I don't study those other animals anymore because of a very simple reason, they bite. They charge you, they're scary. These animals are really gentle, and if you know how to handle them, you never get hurt. I like gentle animals. 
	I want to begin by giving credit where credit is due, and that is to acknowledge the really outstanding and herculean effort that my students have done over the past four and a half years. That includes really two enclaves of students. I have the song bird, the bird people and the frog people. They've been, for the most part, pretty segregated parts of my lab. We have, for the past five years, studied a bunch of questions in tropical and temperate frogs, and the work that I'm going to talk about today. I want to begin by shining a light on these projects and acknowledging these five students, Rebecca Sentz, Marion Fairkey, Abigail Lowder, Laura Heider, and Caitlin Rotonsky, all of whom have moved on beyond Swarthmore, are doing great things out there in the world. They worked and put in, clocked hundreds of hours on these projects. They didn't just collect data. These students helped analyze the data, they helped design the experiments, they helped write the papers, they helped publish the papers.
	These are three of the publications I'll be talking about today. The projects have been funded from a variety of agencies. I just want to shout out to Swarthmore College and the Provost's Office that has underwritten the open access charges for all of these publications, which is thousands of dollars. These are all publicly available papers and in the library, so I need to say something. 
	All animal behaviorists ask the same question. We ask, "Why does an animal behave the way it does?" That one basic question is at least four more specific questions that break down in the following ways. Two branches, the proximate and the ultimate. The proximate questions are about what generates behavior in the animal in front of you? What causes behavior to happen? There are mechanisms that give rise to behavior, the brain, hormones, genes, etc., that cause behavior in an animal that we can study in front of us today. You can also study the origins of the behavior ontogenetically, developmentally. How does the behavior come about? Is it learned? Is it innate? Is it an interaction between those two things? You can study the adaptive value of the behavior, or the evolutionary history of the behavior. These are ultimate questions about why behavioral traits arise originally in evolution and what maintains them today. 
	I study behavior at all four of these levels, and arriving at a complete and correct answer about why an animal behaves the way it does is best achieved by integrating across all four of these levels. In the interest of time today, we're going to focus on this branch right here. We're going to look at hormones, and stress hormones in particular, as a potential mechanism that causes behavior in birds. We need a slightly more specific question than why does an animal behave the way it does. The question for today is, how does variation in personality in gray tits come about? I'll tell you more about their personalities in a moment. 
	We're going to be focusing on variation today. Variation is what, as biologists, in some sense most or all of us study. I want to give you an example of what I mean by that with a behavior that I'm not going to talk about today, but a lot of people have studied. This is known as flight initiation distance. This is a typical field behavioral assay that you can do with birds. If we walk out into the krum, we may see a hermit thrush. We could approach that hermit thrush, and then we could see when does it flush. When does the thrush flush? An average hermit thrush, that might be about five meters. We would then measure that, and that would be our flight initiation distance. We could do the same with a male cardinal, and with a cardinal, on average, that might be about two meters. We can get a little bit closer before a male cardinal flushes. We'd record those FIDs, and we could do that across a range of species. Everything from pigeons down to sparrow hawks. 
	What we would find is the following, these are real data, these are average flight initiation distances. Not surprisingly, you can get closer to a dove than you can a sparrow hawk before it flushes. These are really interesting. This is variation across species, averages, and there's lots of interesting questions that come from looking at data like this. They would be difficult questions to answer from a mechanistic perspective, because as well as the different genomes associated with these different species are a host of other variables that differ. Whether they're migratory or non-migratory. Whether they are altricial or precocial with their young. Whether they live at a particular latitude or another latitude. Whether they have a certain day length, etc. Lots of confounding and extraneous variables that make answering the question, why do these species differ, what is the mechanism that gives rise to the differences, really complicated. 
	Furthermore, these are averages. While interesting, they may not be what we want to ask. This is a quote from Sir Francis Galton, a fellow of the Royal Society, who in the late 1800s said, "As a statistician, it is difficult to understand why statisticians," and behavioral endocrinologists, that's my addition, "Commonly limit their inquiries to averages and do not revel in more comprehensive views. Their soul seems [inaudible 00:14:22] to the charm of variety as that of the native with one of our flat English counties. There's retrospect with Switzerland was that if its mountains could be thrown into its lakes, two nuisances would be got rid of at once." 
	This was then later referred to as the tyranny of the golden mean. That we're so focused on averages, means, in this case, that we may be missing the interesting biology in this case. The nuisances, to be just very explicit, are these error marks. In traditional experimental work, we might have two treatment groups, placebo and a drug group, and measure some effect of that treatment, a trait. We have averages, and then we have error around those averages. When we get data like that, on average, that's not good. The errors interfere with our ability to detect a difference in the treatment. For biologists, we want something more like that. 
	Perhaps, there's some interesting biology hidden inside those error marks. Today, we're going to revel in more comprehensive views. We need to be more specific. In this case, instead of measuring behavior across species, with all the variables that come with that, we're going to focus in on one species and one population, controlling for all those variables. If we did that for flight initiation distance, this is what we'd see. We'd see variation in this population of gray tits. There are birds that are very risk prone, and will let you approach quite closely. There are individual gray tits that don't let you get very close. Now, the scale of variation is less than among species, but there's still a tremendous amount of variation. Variation is really interesting to us. 
	If we see that kind of variation, there could be a couple of explanations for it. These might be differences between these individuals. Some individuals might be consistently more risk prone than others. That's not the only way you can get a sample of data like this. In order to know whether or not this variation is really something reflecting individual differences, we need to measure it multiple times. We go out and measure it once. We could measure it a second time. They won't be identical, the numbers will change a little bit. If they shift just a little bit like that and the animals stay in the same basic rank order, then we have an indication that we're looking at a repeatable, consistent behavior, IE, we have among individual variation. If we have that, we've met one of two criteria for behavioral syndrome. We'll get more to that in a moment. If, on the other hand, we measured it a second time and we see a lot of variation among individuals in their values, then we would, in statistical jargon, refer to this as variation strictly within individuals. There's some plasticity in each individual. Sometimes this bird gets very risk averse and sometimes he's really risk prone, and everywhere in between. That could happen too. In that case, we wouldn't meet this criterion for behavioral syndrome, the behavior is repeatable. 
	Coping with stressors is not limited to behavior. You and I, birds, every vertebrate has quite an exquisite set of physiological systems to cope with environmental challenges. One of the main players in that system is a stress response, the endocrine stress response that produces cortisol in you and I, corticosterone in birds, very similar steroid hormone molecules. Again, if we look across species, there's a ton of variation, several orders of magnitude in circulating levels of baseline cort. In a marmoset, we're talking about 3,000 nanograms per mil, coursing through their blood. In a lemon shark, one nanogram per mil. In humans, baseline is around 100 nanograms per mil. It's a little bit higher in me right now. That's the kind of dramatic variance that we have in this trade, across species. It's super interesting. Endocrinologists have been fascinated with this for a long time. Very hard to study mechanistically because of all those other bounding and extraneous factors that are playing into this interest specific variation.
	Again, if we focus on one species and one population, we can ask whether or not that variation still exists. It does. It's muted, it's attenuated compared to the cross-species comparison, but there's still several full variation in baseline cort in a single population of gray tits. These are real data that I collected in 2014. If we went out and we measured those animals a second time, and they were similar in their profile, we'd have evidence for a hormonal syndrome, the first criteria of a hormonal syndrome, IE, repeatability and among individual variants in hormones. If we measured them the second time and they were totally different, we wouldn't have evidence for that. We'd have within individual variation cort. 
	The hypothesis that I want to talk about today is that these two things are related. That personality is linked with hormones, and the prediction is that more risk prone animals, bolder individuals, have a suite of behaviors about behavioral inhibition, tend to also have the lower potentiated stress response, and vice versa. This is the idea, repeatable behavioral traits are linked with repeatable hormonal traits. This is one way that the structure of the behavior, the consistency of the personality could come about by consistency in the mechanism that underlies it. It's not the only way, but it's one logical first place to start. 
	The underlying concept here is known as hormonal pleiotropy. If you've heard the word pleiotropy before, it's probably in the context of gene pleiotropy. That idea is that a single gene can impact the expression of multiple traits simultaneously. The same is true with hormonal pleiotropy. The idea is a single hormone, in this case testosterone, can simultaneously influence the expression of multiple behaviors. High testosterone in birds typically results in higher aggression in territoriality. It typically results in the expression of what we call secondary sexual characteristics, the showy morphologies in behavior in the context of courtship.
	The primary role of testosterone is to support the maturation of sperm in the testes, another really important part of reproduction. It's an anabolic steroid that regulates growth, and it underlies courtship behavior. All of those things simultaneously. Likewise, it has negative impacts on the immune system. It suppresses the immune system. It impairs provisioning of young parental care. You can't have it all. If you're going to have high testosterone, there's going to be a cost that's paid. This is referred to as antagonistic hormonal pleiotropy. The basic idea though here, is that a single hormonal system could drive the expression of suites of behavioral traits, organizing them into personalities. 
	Here's what we're going to do, we're going to evaluate whether or not we have evidence of behavioral syndromes in this bird, whether we have evidence of hormonal syndromes, and whether those two level of the phenotype talk to each other. There's two criteria, stable individual differences in single behaviors, and behaviors coming together as packages, tendencies arising in suites. Graphically, what does that look like? A behavioral syndrome, IE, a personality, is two things. It's among individual variants, IE, consistency in individuals, and among individual co-variants, that's the traits as packages part. 
	If we measured three birds, bird A, B, and C, for a single behavior, say a risk taking behavior, multiple times throughout their life, say seven times each, and this is what we found. Every single time, bird A was higher on this wider step, it's risk taking. B was intermediate, and C was low. We have pretty good evidence for consistency. We have individual differences in risk taking behavior. That's the first criterion. If we measured a second kind of behavior, say exploration behavior, and we again found consistency in that, A has higher exploration than B, than does C. That's the X intercept. These two things were related, correlated positively in this case. Bird A is both the most risk taking and the most exploratory. Then we would have met the second criterion of the among individual level, suites of behaviors. 
	We can measure that variance within individuals. We can measure it among individuals. If the among individual variance is bigger than the within individual variance, that's a good sign that we're looking at a repeatable trait. We estimate repeatability, R, by simply taking that variance among individuals and dividing it my total variance. A large enumerator, small denominator, we get a large R value. R values vary between zero and one. I'll be talking about R values today. I want you to know that perfect repeatability, an R value of one, would be if you measured the same thing in individuals over time, with every individual it'd be the same numerical answer each time, not shifting at all in their rank order. That never happens for a basically innate trait. Even if you measured height, you wouldn't get a repeatability of 1.0. Zero repeatability means that it's just random. Every time you measure each individual, you get a different value. That sometimes does happen, but typically we're somewhere in between. We talk about repeatabilities around 0.3 as being moderate for behavior and hormones. Values above 0.5 as being relatively high. That kind of gives you a bit of a calibration. 
	We're interested in the repeatability of traits, not only because it tells us something about personality, but because typically, repeatable traits are inheritable traits. Not always, but typically. Inheritable traits have the potential to evolve, and so they're interesting for a variety of reasons. 
	The three behaviors that I want to talk about today are the following, exploratory behavior, a neophobia assay, and a risk taking during foraging. We're going to evaluate whether or not there's consistency in these three behaviors. I'll show you the first one. This is a novel environment exploration assay. It starts out with some soundtrack. This is a bird that was collected in the field, allowed to climatize overnight into a novel environment with some volition. There's my doppelganger and postdoc advisor Kase, who's measuring through a one way mirror, the movement of this animal. Each trial lasts from two to ten minutes. What we're looking at is how much of this novel room does that bird explore. About three quarters, pretty high tech. At the end of the trial, we needed to get that bird out of that room. If you haven't worked with birds before, you have no idea how to do that, a real mess, but it's that simple. Turn off the light, and birds will freeze when you turn off the light. You can just walk in, pluck it off of its perch, and put it back in its cage. 
Speaker 4:	All birds do that? 
Alex Baugh:	All songbirds do that. That's the exploration of a novel environment. These are the novel object, neophobia assays. This is an example of a bird with high neophobia that's going nowhere near this novel object that's been added to its home cage. This is a bird with low neophobia that's interacting with that novel object, which happens to be a Pink Panther toy. It doesn't really matter what that novel object is a lot. These are sort of the two ends of the spectrum. These are wild individuals that we're looking at here [inaudible 00:26:40]
Sara Hebert :	Alex, how do you quantify this? 
Alex Baugh:	Yeah, I can go into some detail afterwards if you want to talk about that. It's actually a related complicated calculation involving 18 different variables that go into this. Regardless of how you do it, you basically get the same result, but we validated these measures extensively in wild and captive reared populations, and artificially selected populations. How you measure it depends on where the population comes from, wild, captive reared, and artificially selected, because they're influenced differentially by time of year, which you have to correct for. I can tell you more about that calculation later if you'd like. 
	The third behavioral assay is the risk taking. This is the one I want to focus on more today. This is done in semi-natural outdoor enclosures in Germany. This is the work that Abby Lowder did several years ago. This involves going into the animal's semi-natural enclosure, putting a platform down. It's never seen this platform before. On that platform rests a dish full of mealworms. These birds are fed ad libitum, but they're fed a grain, and egg, and vegetable, and fruit diet, which they love, but not as much as they love mealworms. When the bird, which is perched up in a tree up here sees these mealworms, it will eventually come down to get a mealworm. We'll record what that latency is. That initial latency tells us something about the boldness of the bird. This is basically a novel object test. 
	This is what's going to happen. Abby, in this case, is hidden about 30 meters away and trying to hide. She's holding onto a piece of monofilament attached to this mousetrap. This female bird comes down, she's going to alight on this platform. She's going to take a tasty mealworm, fly up to the perch. We record that latency. It was 74 seconds in this case. Now she wants more, so she's going to come back. She's going to come back a little bit more quickly, but she's not going to get that second mealworm. Right before she takes it, we startle her. It doesn't hurt her, but it scares her. The question is, does she come back and if so, how long does it take her to come back? That's our startle latency. This is the high risk moment in time. You can see more vigilance behavior. She's hanging out. She's doing a lot of really interesting behavior here that I'm not going to talk about, signal of the raven. Things are about to get scary. She jumps up, she gets her second mealworm, and flies away. 
	We record those three latencies. That's her risk taking assay. This female had a 93 second startle latency. We can then test a bunch of males and females. This is a subset of the 26 animals. A lot of variation. As I mentioned before, we're studying behaviors that we know are hypervariable. Some birds come back quickly after startle, 25 seconds. Some never come back. After 20 minutes of elapsed time, the ceiling of the trial is over, and they don't come back, and everything in between. 
	That's our first trial run. Now we need to replicate it, because we need to know whether or not it's consistent. We replicate it a second time, and a third time, and the numbers change a little bit. For the most part, the rank order of the individuals is preserved. That's a good indication that we're looking at a repeatable trait. The repeatability varies between 0.3 and 0.5. It's always statistically significant. The variance here in the R estimate is just a product of how you model that statistically. 
	Next, we saw that not only are these behaviors repeatable, but they're related to each other. Birds that come down quickly initially for that mealworm, come down quickly after startle consistently. This is an among individual correlation. We're looking at the average latency for each bird. Every bird has three points, but we're taking the average. This helps us rule out the possibility, for instance, that birds that come down quickly are really hungry, and they come down quickly after a startle because they're really hungry still. One, they're fed ad libitum, and two, we're seeing this every week for three weeks in a row, reducing the likelihood that we're just dealing with a hungry bird right here, and a full bird up here. 
	For the purpose of simplicity, I am going to be labeling these behavioral phenotypes, talking about the ends of the acts, the ends of the spectrum. Here we've got risk prone birds down in the lower left. These come down quickly for the initial latency and they come down quickly after startle. They're risk prone. At the other end, the other quadrant, we've got risk averse birds. I want you to know that none of the analysis that I'm about to talk about is done on dichotomous data. All of the underlying statistics are done on the continuous data set. 
	Here we have evidence of repeatable risk taking behavior and it's linked with other behaviors, not only within the assay itself, but across different types of behavioral assays. I don't have time to talk about all of those. Here's the outcome. Exploratory behavior is repeatable. Object neophobia is repeatable. Risk taking behavior is repeatable. These are interrelated. Birds that are more neophilic tend to be more risk prone. They tend to be more aggressive. They tend to be more routine forming. If you give them some task and then you interfere with that task by providing a reward or punishment, they're less likely to change their behavior and do the task differently, compared to their neophobic peers. This is purposefully dichotomous in the labels, but really there's an underlying continuous distribution here. We have two ends of the spectrum, the risk averse, neophobic, behaviorally inhibited birds, and at the other end, the risk prone, behaviorally uninhibited, neophilic birds. We have evidence of a behavioral syndrome. I'm not going through all of the behavior. There is a lot of work that went into demonstrating and validating these personality measures. 
	The second question is, are there endocrine syndromes? As I mentioned, coping with stress is something that goes beyond behavior. There are physiological systems for that. In this case, the hypothalamic pituitary adrenal axis that produces adrenal hormones like cort, is a main subject of interest and a main player in the study of stress. It's important to know that stress is a very, endocrine stress response is a very important adaptation in all vertebrates, it's widely conserved. The way the HPA axis works is, under non-stress conditions, this hypothalamus at the top of the HPA axis, the basil brain, is secreting a hormone known as CRH. That hormone binds in the pituitary and results in the secretion of another hormone known as ACTH. That hormone goes into circulation and it binds the receptors in the adrenal cortices above the kidney, and that results in the secretion of corticosterone and cortisol. That goes into the blood, and that's now the hormone that we're studying. Under non-stressful conditions, it's relatively low in concentration of the blood. When it's relatively low in concentration in the blood, it's binding, because it's circulating throughout the body and all the tissues, it's binding to receptors in the brain, including the hypothalamus. It's regulating its subsequent secretion through negative feedback, so it keeps that basil rhythm. 
	Under conditions of stress, say the presence of a predator, areas of the brain that are detecting and perceiving that threat, visual areas of the brain for instance, communicate with the hypothalamus, increase the secretion of CRH, which results in an increase in the secretion of ACTH, which results in an increase in the secretion of cort. Cort starts to build up in the blood and the plasma. That increased concentration of cort saturates all of those MR receptors in the brain that are high affinity receptors. That begins to bind to GR receptors. They're lower affinity receptors, they start to bind at a higher concentration of cort, and they help shut off the stress response after the stressor has been removed. 
	The stress response is really an important and good thing. Too much of a good thing is a bad thing. Elevated levels of cort need to be brought down back to baseline. The reason is that this stress response facilitates a really important thing in animals. That is the distribution and availability of glucose to tissues. At a moment when it's time to escape a predator, is not the time to invest in reproduction and the immune system. It's a time to shunt that glucose from those tissues to skeletal muscle and the brain, so that you can live to breathe another day. I'm going to talk about MR and GR. After GR's role of binding cort and negatively feeding back on the system and down regulating cort, and back to a basil level of cort. It's going to become important in a second. 
	The work that I'm going to talk about now is work that I did with wild animals in this area of Central Holland known as the Valu. This is a really gorgeous part of Central Holland, forested. It's called the Dutch Highlands. That's really an apt name. It is technically a couple years above sea level. This is work involving these birds in the field. This is a population that's been worked with a lot. I went to Europe because this population has been studied as extensively as it has and has been ringed. All of these animals in this population, virtually all of them have been banded and ringed. We know a lot about these animals. That's super important. 
	This is how we studied stress in wild animals and wild birds. We go out, we erect miss nets. We wait for birds to fly into them. We hide. They can't see the miss net and they can't see us. We want them to not start their stress response until the instant they hit that net. Once they hit that net and that stressor is perceived, the clock is ticking. You don't have very much time to get a uncontaminated baseline estimate of cort. You have about three minutes or less. Here I am bleeding a bird really quickly after it hit the net. I'm going to hold onto it for 30 minutes in a cloth bag, restrain it, and I'm going to take a second blood sample at 30 minutes, to try and estimate what its maximum stress hormone levels are. Then I'm going to release it, and I'm going to pray to my atheist God that I capture it again, because it was a lot of work to capture it the first time and get that blood sample. It does me no good if I don't capture it at least a second time. In this case, I caught a couple hundred birds. You have to overproduce in order to get your sort of target population sample size. That resulted in getting around 58 birds that I got more than once, two to three, or four times.
	This is what it looks like. We've got, in our first replicate, our first capture, we've got birds that are at one nanogram per mil in baseline, birds that are at 28 nanograms per mil, everything in between. Capture them a second time, a third time, and sometimes a fourth time, we can estimate repeatability. It's all over the map. There's no repeatability. The R estimates are very low. They're not significant, despite having quite a bit of statistical power. We don't have repeatability. If you don't have among individual variants, IE, repeatability, there's no point in looking for among individual co-variants. You can't have co-variants if you don't have variants. That was disappointing because the next couple of years of my grant money all of a sudden didn't have any basis. That's not what I expected to find. 
	It's difficult working with wild animals, so I took a break. I started some other projects, and as I was doing that, I was reading more widely and I came across this work by Michael Romero, who's an alum of Swarthmore, graduated in the eighties, is that right? Yeah. Michael Romero developed a method in another songbird for probing the dynamics of this HPA axis in more detail. After talking with him and working through several months of validating this new technique, I decided I'll come back to this. The absence of evidence is not evidence of absence. Perhaps I'm not looking at the right hormonal traits in the right way. 
	What I set out to do was take into account this dynamic nature of this HPA axis, and probe those dynamics, the temporal dynamics as my estimates, instead of these static measures. This is the method that I used. It's a four time quant stress series. Capture the bird, rapidly take that baseline sample before, uncontaminated by the experience of capture. Allow the animal to exhibit its natural stress response after a restraint, in this case 15 minutes instead of 30 minutes, so that we get the increasing phase of that stress response. Then inject the animal with a synthetic corticosterone called dexamethasone. This mimics corticosterone. To the bird's blood, and to its receptors, and its tissue, it's the same thing as cort. This jump starts negative feedback. It's really high levels of cort. Now we're going to induce shutdown of the stress response. When you do that, the stress response goes down, and now we can inject the upstream secretogogue, that ACTH molecule that's going to bind to the adrenal cortices and result in the secretion of cort. We can ask, "Now that you're back down to baseline, how good are you at mounting a second stress response?" We call that adrenal capacity. Baseline, natural stress response, negative feedback, adrenal capacity, all in one capture. We get some more data in more fine detail about the animal's potentially informative snapshots of the HPA dynamics. 
	This is work that we did in an outdoor enclosure. It's where we can control the social environment and the nutritional environment, but allow all those other environmental variables to fluctuate naturally. Here's what we found. Do that four time point HPA assessment on the first replicate, you have variation. Baseline cort, for example, replicated a second time and the numbers change a little bit, but not that much. Here are the repeatability values, those R values for each of those elements of the HPA response. In this case, all four of them are repeatable. We actually get the exact same estimate of repeatability from the earlier study. The only difference is that it's technically statistically significant now, even though it's still very low.
	What's really important, what I want to draw your attention to are these last two components. The strength of the negative feedback, the ability to turn off the stress response physiologically, and the ability to remount that stress response, are highly repeatable. That's really interesting. Moreover, there is an among individual correlation for those two really repeatable traits. If you can turn off the stress response really well, back down to baseline levels, you're going to have a hard time mounting a second stress response. This is probably a pretty interesting and important trade off. It's good to turn off the stress response, but it's also good to be able to mount a second stress response. You can't have both, statistically. We have evidence of the second criterion here, that there are, in this case, suites of hormonal traits that come together as a package, ability to turn off the stress response and turn on the stress response are linked. 
Speaker 5:	Alex, real quick. It seems like you were saying they were anti-correlated, but then your plot here shows a positive. So far they've plotted in a different way than ...
Alex Baugh:	Yeah, so this is post-dexcort. Lower post-dexcort means ability to turn off the stress response better. If we look at this figure again, run through my little animations, so having a high level here means you have high cort after dex injection, which means you can't turn off the stress response well, but you can turn it back on well. 
Speaker 5:	You didn't get down very far. 
Alex Baugh:	A little bit confusing, thanks for pointing that out. 
Speaker 5:	Thanks. 
Alex Baugh:	This is nice evidence of an endocrine syndrome. We've got birds that are on the low end here, are pretty unpotentiated. They're just low cort birds. They have low baseline cort. They have low natural stress response. They have the ability to turn off the stress response well, but they can't turn it on again, and vice versa on the potentiated side of things. 
	Furthermore, these potentiated birds, and I'm not showing you the data here, just in the interest of time, are able to turn on their initial stress response faster as well. They can turn it on in less than two minutes after capture. The birds that are unpotentiated, take three to five minutes to turn on that initial secretion after a capture. We've got repeatable and co-varying hormonal traits. We've got an endocrine syndrome. That's great. 
	Are these two things related to each other? Are endocrine syndromes correlated with behavioral syndromes? They are. Those birds that are unpotentiated, that have low stress in general, low stress hormones in general, they're the fast explorers. They turn their stress response on slower. They reach about the same levels, and they turn it off faster. These are the low stress cort birds, low cort birds, and they're the bold, faster explorers, more risk taking birds. Those slow explorers, they turn on their stress response faster. That's probably a good thing, but it is enduring. It stays elevated for hours, and that's not a good thing. That [inaudible 00:45:22] natural feedback at an early onset and a late offset of the stress response, take the area under the curve here, means that those slow explorers are experiencing a greater amount of integrated total cort over the period of the stressor. We think that's probably ... If you're going to measure one thing about the stress response, that's probably the one that has consequences in the long run, that total exposure to chronically elevated levels of cort. Slow explorers, aka risk averse personality birds, have a more potentiated HPA axis. 
	Hormones don't cause behavior, the nervous system causes behavior. What we really need to do now is go up a level in this HPA axis and ask what's happening in the brain. What relationship does the brain and its distribution of these hormone receptors tell us about the secretory patterns in the plasma that tells us something about the behavior? My student Rebecca Sentz spent a couple of years and hundreds of hours with me at the Roslyn Institute at the University of Edinburgh, slicing these brains up, doing in situ hybridization and estimating the concentration of these two receptor types across 48 different brain areas in 26 animals, which was really a herculean effort. 
	In the interest of time, we're going to talk about these two regions of the brain, the hippocampus and the paraventricular nucleus of the hypothalamus. Remember the H in the HPA is hypothalamus. The PVN is really at the epicenter of controlling the entire axis. The distribution of these two receptor types and the balance between them, may tell us something about whether you can predict who's potentiated and who's not potentiated in their plasma hormones. With the two levels of the phenotype then, the neuroendocrine, as well as the endocrine, you may be able to predict the behavior. 
	First, what we found was that there's some evidence of a so called neuroendocrine syndrome. Sorry, I didn't mean to blast you with so many syndromes today. If you look at the distribution of these two receptor types across the two different areas of the brain, they're strongly positive and co-linear. If you have high MR in your hippocampus, you're going to have high MR in your hypothalamus, you're a high MR bird, and likewise with GR. This is good. This is some evidence that maybe there are structure to the receptors in their distribution that are giving rise to structure in the secretory patterns in the animal, maybe giving rise to structure in the behavior. 
	This is a figure I'm not going to make you understand, but it's the full sweep of data that we collected. Neuroendocrine across two different receptors, two different brain areas, four time points, four stress series in the endocrine, and then the behavior, the multiple behaviors. I'm going to reduce that to the following model. The idea is that receptors and their density in these two areas of the brain influence and drive the expression of the secretory patterns in the blood. These two levels of the phenotype impinge and effect behavior mode. What we did in this case was collapse all these variables into principle components. We're dealing with a simpler system. That's, in this case, justified, and we'll go into why. We did what's called a confirmatory path analysis. The hypothesis is that the neuroendocrine syndrome drives the endocrine syndrome, which drives the behavior. 
	Here's what we found. If you have high MR in the brain, that is going to positively predict a more potentiated endocrine axis. You're going to be that bird with high levels of cort if you have high MR, and vice versa with GR. If you have high GR, you're going to have low cort and be an unpotentiated bird. That's exactly what we predict from basic theory. The GR is intimately involved in this negative feedback regulation. That's nice. The endocrine level, how potentiated your HPA axis is, predicts your behavior. If you have a really potentiated HPA axis, IE, a lot of cort, you're going to be a more risk averse bird. You'll be stressed out, when you kind of think about it that way, really risk averse, and vice versa. Then these two levels of the analysis also have explanatory power. If you have high MR, you're going to be risk averse. You have high GR, you're going to be a risk prone bird. These all follow from the basic predictions of if you can turn off your stress response and regulate it at low levels, you're going to be more risk prone. You're going to take risk more often. 
	These are all significant. All of these paths are significant, and they explain a lot of the variants in these [inaudible 00:50:25] the HPA and the behavior. We can attach our error terms on top of that. The take home message is that low levels of GR, high levels of MR, predict a very stressed status at the endocrine level in birds, and predicts a risk averse personality, and vice versa. The GR is really of paramount importance. It explains a very large fraction, a shockingly large fraction of the behavior, as far as I'm concerned. I'm not sure I believe it yet. We'll talk about that more in a moment. The other thing I want you to know is that not only do we have high explanatory power here, we're predicting a lot about the animal's personality, a lot about its endocrine status, knowing very little about the animal. This is also robust. The model is robust to its exact structure, whether we're using collapsed variables or raw variables, how we design the analysis doesn't really matter. 
	We have evidence that endocrine syndromes are correlated with behavioral syndromes, but that's not the title of my talk. I didn't ask whether they're correlated. I asked whether endocrine syndromes underlie behavioral syndromes, whether they cause it, and correlation is not causation. This is where the talk gets less riveting, because I don't know whether there's any evidence whatsoever for this HPA axis underlying personality. We have correlation. It's certainly intriguing. What we'd really like to be able to do is now experimentally manipulate endocrine status and see whether or not personality is impacted, but that, for very good reasons, is extremely challenging. 
	I hope that I convinced you by showing you that it's the dynamics of this axis that are important, in terms of explanatory power and personality, but convinces us that manipulating those dynamics is probably what we need to do, and that is not simple. We can implant or inject an animal with cort, for example, and acutely effect its hormone status, but that's probably not going to one, be naturalistic, and two, be the variable of interest. The static levels don't predict much about the behavior. It's about the ability to turn on and turn off the axis. That's really challenging. The temporal details, the devil is in the details. 
	What we can do and what I can say is that if we take a different approach to this problem and say perform selection on behavior, select on personality, and ask whether or not what piggybacks with it is HPA axis reactivity. We see that, that's in fact the case. If we take these birds from the wild and we breed them artificially, and we test them for personality, and then we breed shy birds with shy birds, and bold birds with bold birds, and we do that over several generations, we see this divergence. Bold birds become bolder, and bolder, and bolder, and vice versa with shy birds, so that's great. That's actually the evidence that we're looking at an inheritable trait. Now what we can do is we can take say that fourth generation of selection and say, "What's your endocrine status?" What we find is that, as I showed you with the other types of data, that these shy birds, these risk averse birds, tend to have higher levels of cort. That's consistent. 
	That doesn't demonstrate causation either. In addition to this HPA axis variation piggybacking on the behavioral selection that we're doing, a variety of other things are undoubtedly piggybacking along with it. Those other things, those other potential physiological explanations, they may be what causes the behavioral variation. We don't know that from this data. It's certainly worth studying in more detail. 
	I want to end there in the interest of time. I want to just indicate before I do so, that addressing these other levels of this paradigm may bring us closer to that question of causation. One particularly productive line of research, one that I'm pursuing moving forward and have recently just finished in this bird study, is asking whether or not we can program individuals early in life by manipulating their early life environment, including their in utero environment, including the environment in the egg hormonally, to program them behaviorally later in life. That's one lever that we can pull in order to do this experimental manipulation, and ask whether the endocrine system that we're particularly interested in, is involved causally in the organization of personality. 
	I just published with a colleague, a study showing some evidence for that in these birds, looking at yolk hormones and how they effect behavior, and how behavior effects them, the deposition of the hormones in the yolk. Moving forward, this is what, one of the questions that I'll be asking in frogs, is can we phenotypically engineer animals early in life, at the egg stage, at the tadpole stage, in order to generate a phenotype of interest behaviorally later in life. 
	I'll end there and take any questions. I'll leave this pair of timber frogs to wow you with their foamness construction. Alright, thanks for your attention.