Scientist Stories: Catherine Dulac, Molecular Basis of Social Behaviors

Oh my God. Oh my God, I'm just like, what? Look, I'm going— well, after this reception, I'm going to the post-party. So we have a post-party. I'm going to see my postpartum nurse. And then there's a little pizza. And then tomorrow I'm going to come to the gym. And then I'm going to go to the gym. So, I just want to say hi. So it's just like the Hollywood movie. This is just That's— oh my God. Well, then that would explain why you might have gotten an email or a call. So do you get stuff like that? Did they get extra tickets for the graduation? You know, you guys don't answer your emails. Your text. You and you. All right.

Hi, everyone. Welcome to the inaugural symposium of the Zuckerman Mind, Brain, and Behavior. I'm Rui Costa. I'm an investigator at the Zuckerman, and I serve as the CEO. We have high hopes for this inaugural symposium, and we hope the symposium helps us acknowledge So many people that supported this endeavor and that paved the way for us to get here where we are today and also help us to start achieve our goals of doing transformative science to understand the brain by focusing on creating the right environment and by focusing on the scientists at every level from student and postdoc to assistant professor to more established people. So doing science and transformative science by focusing on the scientist. I hope you can see that the program reflects a bit these goals. We have a quiet room, a meditation room. There's going to be storytelling. Next year, parkour, bungee jumping. Who knows? Anyway, to begin our symposium, I'd like to call to the podium Richard Axel, who's the co-director of the Institute. And he'll offer introductory remarks and also introduce our keynote speaker. Richard, please welcome.

It is my pleasure to welcome you to the inaugural symposium celebrating the Zuckerman Mind, Brain, and Behavior Institute. Our architect, Renzo Piano, has described these magnificent structures as factories for ideas. This beautiful illustration of man's early fascination with the brain, the Humani Corporis, was drawn by Vesalius in the 1500s. In this haunting but graceful image, the skeleton is contemplating a second skull. He is studying the brain. Indeed, one of the temptations of having a mind is to try and use it to solve the mystery of its own nature. Historically, scholars have assumed that the nature of thinking can be uncovered by thinking alone. This has been an assiduous pursuit of humans from the earliest moments in recorded history. The brain, however, is the most complex, the most mysterious, and the most interesting structure in our universes. Perception, emotion, cognition, memory, action are represented by the activity of groups of connected nerve cells. How? The president of Columbia, Lee Bollinger, in a prescient moment recognized that this astonishing problem in biology was at the core of virtually all intellectual endeavors at the university. Literature, history, art, law, economics, philosophy, and psychology embrace these problems. Lee conceived— with this recognition, Lee conceived of a Mind-Brain Behavior Institute that integrates the entire university to address this astonishing and profoundly important problem. Problem. The Mind-Brain Behavior Institute represents a new union and is the flagship structure of a new campus. Lee enlisted the help of Tom Jessell, Eric Kandel, and myself. We were then joined by a brilliant and gracious philanthropist Mort Zuckerman, who shared our deep interest in the mind. Tom Jessell was the creative force behind the Mind-Brain Behavior Institute from its conception to reality. Tom was not only a great scientist, but also a brilliant leader. With a magical quality, he defined the cellular and molecular events involved in assembling a motor circuit, revealing how spinal cord neurons acquire specific identity and form selective connections that coordinate behavior. Through his creativity, he constructed biological edifices that have profoundly enhanced our world. This celebration is sadly tempered by his passing last week. His leadership and his creativity will be sorely missed. Let this symposium honor his memory by celebrating science. Now, it is my pleasure to introduce the keynote speaker this afternoon, Catherine Duloc. Catherine is an unusual French neurobiologist. Catherine grew up in Montpellier, the daughter of two French intellectuals, professors in the humanities. She attended École Normale Supérieure in Paris, performed elegant thesis research in neural development with Nicole Douarin, and came to my lab as a fellow over 25 years ago. Historically, French scientists have often elevated ideas above facts. I expected a deeply intellectual theoretical neuroscientist, perhaps a young French Larry Abbott. I was pleasantly surprised. Catherine was an intense experimentalist who demonstrated the unique ability to identify important problems in neurobiology and used creative approaches to affect their solution. Catherine exhibits a deep and abiding passion for science, which she embraces avec élan, with a great spirit. Behavior is a consequence of experience. Experience acquired over long periods of evolutionary time can result in innate behaviors.— whereas experience over the life of an organism results in learned behaviors. Catherine studies innate behaviors, behaviors elicited by collections of sensory neurons that recognize cues emitted by other individuals in a species. She's combined genetics, molecular biology, neural recordings and behavior to provide a fundamental insight into the behavioral circuits that ensure appropriate responses to pheromonal cues. Importantly, through her science and her humanity, Catherine has demonstrated the admirable ability to subvert the limitations that society still imposes upon women. I look to you, Catherine, to help disassemble the remaining barriers to the success of women in science. It has been a great pleasure to watch your science unfold, and we look forward to your lecture, Molecular, Cellular, and Circuit level control of social behaviors.

Catherine. Thank you, Richard, for the very kind introduction. I'm very honored. I'm also extremely humbled to give the keynote lecture today, as I owe a great deal to you as well as to the Columbia neuroscientists, the Columbia community of neuroscientists, with Tom, with Eric, to transform me from a developmental biologist of the quail and chicken embryo into an aspiring neuroscientist. And so 25 years later, here I am, and I hope you'll enjoy the journey that I would like to take you over, which is our journey to try to understand the control of social behaviors. Why social behaviors? Well, social cues are particularly significant for animals and for humans, significant for behavior, significant emotionally. For humans, it's very clear that social interactions are central to the human experience. We spend a lot of our mental time to think about the people we like, the people we don't like. It's very clear that love, hate are some of the most central themes of human creation in art, in literature, in poetry. Similarly, humans love to be in this large assembly like today or in concerts, in church, in parties. And in the opposite, humans do very poorly when they are isolated. And in fact, one of the most debilitating aspects of mental illness is the inability to engage in fruitful social interaction like in depression or autism or schizophrenia. Schizophrenia. So it's clear to say, it's clear that the human brain is geared towards social interactions. But similarly, animals that are isolated don't do very well, and animals need to interact with others of their species to mate, to nurture their young, to defend their territory. And so over the years, we've been trying to use any type of technology of modern neuroscience from molecular genetic circuit-level approaches to try to gain an understanding, a brain-wide understanding and a mechanistic understanding of the control of social behavior. So we are trying to identify the social cues that animals need to identify in order to recognize other conspecifics. We try to identify the neurons that detect these cues. We're trying to understand the process of social interaction. What is the processing of social cues that lead to this interaction? And we try to identify the neurons that are involved in this processing, identify the properties of the network involved, and finally, how do they give rise to proper social behaviors and behavioral responses. And so today I would like to specifically emphasize one particular set of social behavior, the behaviors of adults with infants. And there are a number of reasons for this. So parenting behavior is a particularly salient behavior, extremely important for both the emotional and physical development of the offspring. Offspring. In human, it involves years of care and sacrifice, but generally in animals, these are often long-term interaction between the parents and infant. In human, there is this very particular type of parenting that involves not only the parents, but the extended family, the friends, the social structure, what is called alloparenting. Now parenting don't always go very well. And a very clear set of illnesses affect the quality of parenting, in particular in postpartum depression that affect around 20% of mothers and 10% of fathers, where symptoms are associated with extreme worry, dread, and feeling of failures that lead to a difficulty in bonding between the parents and the child, and sometimes are associated with or the no action of harming a baby. And here is the testimony of a patient with postpartum depression. I have broken down I don't know how many times, or I get angry about the crying, then sad about being angry, and then guilty, like why do I feel so sad when I have a beautiful baby? The emotions are insane. And these experiences The excruciating feeling of a mother with postpartum depression are quite typical of people with this condition. What is unusual here is the person who emitted this quote is Serena Williams, someone who epitomizes women's strength. And so this is really something I think very striking. Now parenting or interaction with infant can go extremely wrong. In rare cases of postpartum psychosis that affect very few mothers, but in which the obsessive thoughts of harming a baby would require hospitalization in order for harm not to occur. Now in animals, there is a large variety of infant-driven behaviors. Typically, the mother is the primary caregiver of infants, infant, and in mammals this makes a lot of sense since through pregnancy and then nursing, the mothers invest enormously in the offspring and therefore will make sure that this investment comes to fruition and that the infant can grow and thrive. The contribution of father is quite different according to the species, and it varies from very paternal species to species in which one sees neglect or even aggression. So this, for example, is a father titi monkey with its offspring. And in titi monkey, this is a very rare species in which the father is actually the primary caregiver. So beside the specific period of nursing by the mother, then the father becomes the primary caregiver. And in contrast, this langur here has just killed a young infant. And this behavior of infanticide that was actually described for the first time in langur by Sarah Hrdy in the late '70s is thought to be an evolutionary-driven behavior in order for males, foreign males in particular, to gain reproductive advantage by killing the infant offspring of their rivals and also with females that no longer take care of their infant, now becoming sexually receptive again. Now infant neglect and aggression can also be displaced by females, particularly in case of stress, and this is also what I've just described to you about postpartum illnesses. So this fact really illustrates something quite interesting about the interaction between adult and infant, which is the existence of these opposite responses to infant, pro-parenting or anti-parenting, according to the sex of the individual, according to its physiological state, that provide a really interesting naturalistic paradigm to try to deconstruct the control of a particular set of social behaviors. So the first question we ask is how do animals detect infants? And so we're working with mice, and in mice there is a very clear sexual dimorphism in the reaction of males and females towards infants. Females, even virgin females that never had their own pup, are maternal, and they have communal nests and they take turns and take care of their pups. In contrast, virgin males are infanticidal. And when put in presence of pups, they will, after a few minutes of examination, attack them and kill them. So the question we ask is, what are the pup cues that are detected and that lead to these distinct behaviors? For parenting, a lot of classical work, some of it having been done actually here at Columbia many years ago, have shown that that somatosensory cues, olfactory cues, and auditory cues are all important to drive parenting behavior and that the removal of one or even two of these sensory modalities does not affect parenting behavior, suggesting redundancy of the cues that make sure that these behaviors are being displayed. In contrast, nothing is known about the cues that drive infanticidal behavior. That made us really curious to try to decipher the unique cues that drive these virgin males to attack infants. So this work was done by a former postdoc in my lab, Yu Isogai, who wondered what are the signals that virgin males utilize to then recognize pups and attack them. And what he noticed is that virgin males would attack a dead pup, suggesting suggesting that movement or temperature are not important for this recognition. And this drove him to try to establish a reconstituted system in which he designed silicone dummies. The initial making was the wrong color, but this is a silicone cask of a pup, and what he wrote we recognized was that males readily attack these pup dummies and in fact attack them and make bite marks that are absolutely similar to what would be generated by a dead or a live pup. So these attacks also occur in full darkness suggesting that vision is not necessary. And so that led him to try to identify what are the relevant morphological features of a pup. And so he designed this cask with little variations. I should also mention that this dummy elicits attack when they are swabbed with pup pheromones, so extract of salivary glands. And so with a silicone dummy swabbed with salivary extract, he saw attacks by the males. When he took a brick, an object of the same size but obviously very different size, there was no attack. When he reconstituted what he called a blob, which has the shape of the head and the curvature of the body, swabbed with salivary extract, he saw no attack. And in contrast, this hybrid structure that has legs and tails does lead to attack, suggesting that a combination of pheromonal and physical cues are necessary for this virgin male to identify pups and then attack them. So this led us in turn to try to identify more specifically the nature of these chemical cues. And so the work from Richard and my lab and others identified many years ago the receptors that are associated with the vomeronasal system. So neurons in the vomeronasal organ detect pheromonal cues with these two femur receptors, V1R, V2R. These neurons project to the accessory olfactory bulb and then to the medial amygdala, which is an essential processing center, and then to areas of the hypothalamus involved in a number of social behaviors. And by contrast, In contrast, the olfactory epithelium using these OR families discovered by Richard and Linda Buck lead to the cognitive processing of odorants. And so we and others recognized many years ago using genetics ablation of each of these systems that both the vomeronasal organ and the olfactory epithelium need to work in concert to control mating behavior. So both are essential. And the particular role of the vomeronasal system is to gate the sex specificity of social behavior, which is that without a functional vomeronasal system, female mutants for the TRPC2 ion channel display male-like behavior. And so this in turn led Herbert Wu, now a postdoc in Richard's lab, to wonder whether the vomeronasal system was also is also essential in gating the infanticidal behavior found in males. And indeed, Herbert found that animals, males without a functional vomeronasal system are no longer infanticidal, suggesting that this sensory modality is indeed essential to drive infanticidal behavior, and instead these mutants are fully parental. He also confirm an old observation that fathers are also no longer infanticidal and are parental. And this extremely interesting switch in behavior is illustrated in the video here. This is a male that 3 weeks earlier was infanticidal, but after mating with a female and now put in the presence of pups that are not even his own pups are now fully paternal. Maternal. And you can see the male dutifully retrieve these 5 pups one by one. It has built a nest where it retrieve the pups and then does a good job making sure that no infant is left behind. And then come back to the nest and will behave in ways that are completely indistinguishable from the behavior of a mother. So this switch in behavior is quite extraordinary and suggests the existence of parental circuit in the male brain. So let's go back to these cues that are essential for infanticidal behavior. We saw that the vomeronasal organ is essential to drive infanticidal behavior, so we wonder what are the vomeronasal receptors involved. And Joe Isergay a number of years ago developed an assay based on the induction of the immediate early gene EGR1 to identify the cues that are recognized by different vomeronasal receptors. And he found that the majority of the vomeronasal receptors respond to predator or heterospecific cues and then a specific specific set of receptors respond to female cues or male cues. So what are the receptors responding to pup cues? And he was able to identify about 7 vomeronasal receptors that when the animal has been exposed to pup cues, it leads to the specific activation of EGL1 that you see here in green, and then in red is the expression, co-expression of specific vomeronasal receptors. As a whole, these 7 vomeronasal receptors, or 6 that are presented here, represent over 92% of the response. So we think that we really capture the entire repertoire of vomeronasal receptors responding to pup cues and responsible for infanticidal behavior. But to our surprise, none of these receptors are actually specific to pup cues. And we've identified them all already. As responding to adult female cues or male cues or both of them. So this really was intriguing and suggested an interesting possibility that actually the pups do not emit their own cues. In fact, they might try to camouflage and mimic parental cues in order to escape males or perhaps predators. So this was an interesting idea of Vormannisel stealth, if you wish, that we wanted to test further by performing genetic deletion of two of these receptors, the V2R65 and V2R88, that as a whole represent over 50% of the response to PAPS. And when we performed single mutant and double mutant, we were able to show that indeed the double mutant no longer attack pups and either retrieve the pups or ignore them, suggesting that although these receptors are in principle not specific to pups, they are actually involved in the response, in the infanticidal response. So this was intriguing and we were curious to know, well, what are the cues that are recognized by these two receptors? And so I was lucky that Joe was a former trainee from Bob Tijen, so a very accomplished biochemist who found that it was a lot of fun to perform all sorts of biochemical fractionation and ended up with purifying the submandibular gland protein C, so a large salivary gland, as one of the two pheromones, one of the two ligands of our, in our quest, and the second ligand, the second pheromone was really particularly astonishing because we found that it's hemoglobin, so a component of blood. And in fact, the purified fraction, as you can see, has this very clear red tint. We were very intrigued by the presence of hemoglobin and wondered why would blood be present around the pup. As it turns out, we were able to identify blood from the mother several weeks after parturition. So it turns out that during delivery, there's blood around from the placenta and from the mother, and this stays around and might be identified by males roaming around as a possible indication of the presence of pups. So the submandibular gland also is also expressed by the mother and the infant is basically swabbed constantly by this protein, by the licking, from the licking of the mother. So overall, these identified two new mammalian pheromones, this submandibular gland protein and avoglobin, and shows also that somehow the pup identity is is somehow revealed by cues deposited by the parents and particularly the mother. So this really gave us some picture, some really interesting picture of the detection of sensory cues leading to infanticide and then we wondered about the processing of this signal. And so we were very fortunate to establish a collaboration Mark Schnitzer and Venki Murthy to perform mini microendoscopy and use the mini scope with a green lens reaching the medial amygdala. As you may recall, the vomeronasal organ sends input to the accessory olfactory bulb and the medial amygdala is the primary recipient of this input. So this is an area that we think is absolutely critical for the processing of vomeronasal cues. Now the problem with social interaction is that you cannot do experiments, do recording on head-fixed animal. The animal really needs to be moving around and interacting with other conspecifics. So the availability of this microendoscopy was really quite extraordinary for us. And indeed, you can see here when the pups have been put in the cage that immediately the neurons in the medial amygdala start to fire and using computational method that I won't have time to describe, we were able to show that the representation in the medial amygdala of social information is in part displayed by individual population of neurons but is significantly more robust and more reliable at the population level and this certainly was quite surprising surprising for a modality that is thought to lead to instinctive behavior. But on the other hand, it also provides more flexibility for changes in this representation. And indeed, when we looked over time at changes of this representation in animals that are left alone or put in presence of an animal of the other sex and then put alone again, so as you can see, experiment lasts over 3 months and we record every 5 days from the same animal as the animal acquires sexual experience and then is put alone again, we were able to show here in this 2D representation of the neuronal ensemble that at first the representation of male cues in blue, female cues in red, and pup cues in green are at least partially overlapping, but as the animal gained sexual experience, now this representation gets separated. And so the animal, in other words, is able to have way more discrimination between these cues. And interestingly, these changes are very long-lasting, which is 3 weeks later or 1 month later when the animal is alone again, then this increased discrimination discrimination is continued. And as you can see, there's also a difference between what's happening in males and in females, which is in males, as soon as the animal mates with a female, then the discrimination of pups, pup cues from the other type of cues is elicited and then maintained. Whereas in females, this increase in discriminability between between female and puppies and male actually is acquired later, likely when the female give birth. Another really interesting characteristic of this is that we were able to block this plasticity in males but not in females by injecting an oxytocin antagonist. And so what we showed here is this long-lasting modulation of ensemble medial amygdala and the PERP representation in particular due to sexual experience and these mediated at least in male by the neuropeptide oxytocin. So if we are back now to our cartoon, behavioral cartoon, now we recognize that in addition to these infanticidal virgin males, now we have these paternal fathers, these males that have mated with females fathers and females and 3 weeks later are as parental as the mothers and the virgin females. And so these lead us to this really interesting question of the control centers that drive these two behaviors of parenting behavior in fathers and female and infanticidal behavior in virgin males. So the first central control that we looked for and tried to identify are neurons involved in the control of parenting behavior. And this again was initiated by Herbert Hu when he was a graduate student in the lab, and he simply used the ability of neurons activated during parental behavior to elicit CFOS activation. And Herbert was able to identify We could identify this particularly high induction of C-Fos in the medial preoptic area of parental males and females compared to infanticidal males. Now the medial preoptic area does a lot of different behavior. It's involved in parenting, in sexual behavior, aggression, social interaction, thermoregulation, thirst, feeding, reproduction, sleep. All these neurons are intermingled. So it was particularly important to try to to recognize the identity of the cells activated during parenting behavior. And using a candidate search approach, Herbert was able to identify the neuropeptide galanin as marking about half, a little less than half of the neurons activated during parenting. So about 40% of the cells activated during parenting are also galanin positive. And these neurons are also GABAergic, so they are inhibitory. GABAergic neurons. And in the next phase, Herbert used classical optogenetic tools and genetic approaches to demonstrate the role of these neurons in parenting behavior. So first, using conditional diphtheria toxin viruses, he was able to ablate these galanin neurons and showed that the ablation of this population now in compared the display of maternal and paternal behavior and also switched virgin females into becoming infanticidal behavior. The opposite experiment to activate galanin neurons in infanticidal males led to the deduction, the conclusion that these neurons are also, the activation of these neurons induces paternal behavior in phantosettled males. So these phantosettled males no longer attack pups and instead groom them, suggesting that these MPOA galanin neurons are an essential control node for both male and female parenting. Now interestingly, since then, different groups, Lauren O'Connell and Andy Bass, found that galanin expression of galanin is tightly associated with the display of parenting behavior in poison frogs. You can see this paternal poison frog here with a tadpole here on the back. And also in this spawning midshipman fish, which is a paternal fish here guarding his eggs. So in the hypothalamus, the function of neurons are known to be usually very conserved, and it's therefore quite possible that these galanin cells play a role in parenting behavior, not only in mammals but even further in other species. So next, we wondered how these galanin cells played a role in the control of parenting behavior. We talk about parenting as one thing, but in fact, these neurons must receive a number of information about the internal state of the animal, its hormonal state, its gender, also receive input about the presence of pup or other type of input. And in turn, they must control quite a number of behavioral components that overall constitute what we call parental behavior. So for example, the animal need to build a nest, retrieve the pups, groom them, nurture them, And also there are all sorts of hormonal and motivational aspects that change animals now, are driven to interact with pups and no longer interested in interacting with other animals. They have very specific hormonal levels. The saliency of sensory stimuli is modified and they are no longer infanticidal. They have reduced stress level and they are no longer interested interested in other adult social interactions. So basically you have a kid, you stay at home and watch TV and you no longer go to the pub. So this is, forget about it. So how are all these different components controlled by this one population of galanin neurons? And so Joe Nicole, former postdoc in the lab, decided to dissect entirely the different components of the projection and input of these galanin cells. And what he found was way more complex than anticipated. He found around 20 different input areas to the galanin neurons, and he could hypothesize that some of them, for example, the VTA or the nucleus accumbens, could be involved in motivation, the medial amygdala probably in this vomeronasal input, input, the VMH, maybe in the control of aggression. So we could hypothesize a number of possible functions. One interesting input area is the paraventricular nucleus. This is the area that contains oxytocin, vasopressin, CRH. And to our surprise, we actually found that using RABIES tracing from the galanin cells and then identifying the input neurons specifically using antibodies antibody staining, we found that the majority of the cells in the PVN are not expressing oxytocin but AVP. And so this is a bit unexpected, as you know, oxytocin is always presented as the hormone of parenting. So what about the projection? Well, the projection were also very interesting. To some extent, most of them were also identified in our search for input suggesting the existence of recurrent loops of regulation between these different areas. Here again, there are areas that we can suspect to be involved in the motivational aspect of parenting such as the nucleus accumbens or the VTA, the motor control such as the periaqueductal gray, social behavior such as the medial amygdala or hormonal modulation such as the PVN. But one essential question is to try to understand the logic of this projection. And using pairwise dye injection in different brain areas and just looking at the presence of double-stained cells in galanin-expressing cells, Joni was able to demonstrate that these galanin neurons do not be interrogate, so we rarely, if ever, identify neurons that were labeled by two different dyes when the dyes were injected in different brain areas. And so this is very interesting as it suggests that different galanin neurons form subpopulations or pools that each project to particular brain areas. And therefore, we decided to focus our next this type of studies in 4 different brain areas that we thought could represent different aspects of parenting behavior. The periaqueductal gray for its control in motor control of many social behaviors, the VTA involving motivation, the medial amygdala for its role in social behavior, and the PVN for its role in neuroendocrine regulation. And what we found using RABIES tracing is that from neurons expressing oxytocin, AVP, or CRH, so different neuropeptides, that indeed the MPOA galanin neuron sends projection to all this population. And so we think that this particular pool of galanin neurons is involved in the neuromodulation of parenting. We then looked and manipulated the projection of galanin neurons to the periaqueduct periaqueductal gray, and so we implanted an optic probe on the top of the periaqueductal gray and looked at the influence of either activation or inhibition of this particular projection site when an animal was interacting with pups. And what we found is that when we activate this projection, we see an increase in the motor execution of parenting, suggesting that this particular pool of galanin neurons is involved in the motor execution of parenting. Then we looked and manipulated the projection to the VTA. And so we looked at males and female and activate or suppress the VTA projection from the galanin neurons, and we didn't see anything. We see absolutely no effect. And so we thought about it and then reminded ourselves to how much lengths an animal mother in particular will go through in order to identify and retrieve her pups. So here is a female and her pups are in this deep cup here and she realized that and wants to reach them so is trying to go through the glass and No, can't reach them. So she will see that, oh, there is an opening there. So she will try to reach them that way, but it's pretty tall, so not very easy. But finally, she found a way and successfully retrieved them. So as you can see, she, this female The female is really trying to go through a lot of obstacle to retrieve her pups. And so this inspire this particular behavioral assay where we put some obstacle in between the female and the pups. And indeed, when we stimulate this VTA projection, we found an increase in the probability of the females as well as the males to reach over and gain access to the pups. Now, this experiment actually is really interesting because when you do the experiment in a female, you increase the motivation of the female to reach the pups, and then when she's together with the pups, she starts to groom them and take care of them. But if you do the experiment on a virgin male, it also works, which is that the virgin male will also also have a higher probability to reach over the obstacle. But once it reaches the pups, it kills them. Because what we do here is just very specifically stimulate the projection to the VTA. We're not stimulating the projection to the periaqueductal gray, which is the area that is necessary for the display of parenting. So these animals are still infanticidal, but that particular projection is involved to the motivation to reach the pups. So I think quite an interesting observation. And then finally, when we manipulate the projection to the medial amygdala, we found that we reduce social interaction between adults. So overall, this really provides an interesting deconstruction of the circuit driving parenting in which we have these different pools of galanin neurons that each receive a variety of input that signal pup stimuli, the external state, or external environment. And in turn, these neurons form populations that reach only one target and play one particular role. For example, the motor control by the projection to the PAG, or motivation through the VTA, social interaction through the medial amygdala and various neuroendocrine controls through the PVN. And this really reminded us very much of an organization that was described before by Tom Jessell and Sylvia Arbor of the organization of the motor pool in the spinal cord by different pool of neurons which we think might be also also a motif that is conserved for the organization of social behaviors. And obviously it will be important to see whether other social behaviors obey this rule. So this leads us to then look at another set of behavior, which is infanticidal behavior. One role of the galanin neurons is to inhibit display of parenting behavior in fathers and in other animals. So what is the target of this particular branch of the circuit? What is the control node of infanticidal behavior? And here Anita Autry performed a very similar experiment as Herbert did previously and identified a particular set of neurons in an area that had never heard before called the periphonicle area, and using laser capture microscopy after we were despaired to find candidate genes, she was able to identify another peptide, urocortin 3, as nicely overlapping with the CFOS-expressing cells in the periphonicle area. Now, urocortin 3 is an interesting peptide because it's a ligand of CRFR2, and it's is part of the stress response pathway. And so as I mentioned to you, stress is intimately linked to infant neglect and aggression, suggesting that indeed this could be exactly the node for infanticidal behavior that we were looking for. So one interesting question obviously is whether these neurons are activated by any form of aggression or they're just very specifically for that mediate this particular aggression towards infant. In other words, is there a generic aggressive circuit or is there a very specific set of circuit in the brain that mediate this particular form of aggression towards infant? And what we found is that when we tested animals for male-male aggression, maternal aggression, any type of other forms of aggression, we found CFOS-positive cells but they never overlapped. With urocortin 3. In other words, it seems to be the case that there might be a particular system that has evolved to drive this form of aggression towards infant. So indeed, when we looked at the input of these urocortin cells, we found quite a number of interesting areas, in particular the PVN, the medial preoptic area, the medial amygdala, so all part of the pathway that are expected. And interestingly, when we looked using again rabies viruses from the periphonical area and found the input and the nature of the input in the paraventricular nucleus, we found that vasopressin cells as well as CRH cells were highly marked. And similarly, when we tried to identify identified the input from the preoptic area, we found that these cells were galanin positive. Now this is really interesting because galanin cells, remember, are GABAergic. In other words, when the animal are parenting, these cells are firing and might indeed inhibit the function of this periphornical area that are activated during infanticidal behavior. So the input PAP revealed this link to both the vomit the vomeronasal pathway and neurons involved in social control, stress, and parenting. So what are these neurons doing? Well, when we silence them in males, we found that these animals are no longer infanticidal and take much longer to attack infants if they ever attack them. Interestingly, in contrast to the stimulation of galanin neurons, the silencing of galanin neurons, we found that these have long-term effect, which is that when we silence these cells, the animal become parental and stay parental, which is something we never observed for the stimulation of galanin neurons. So we think these neurons are required for the control of male infanticide. Now, we stimulate them in females either chemogenetically using DREADDs or optogenetically And what we found is the emergence of interesting aggressive displays. So here's a mother and a pup here, and you'll see a little bright dot here when we trigger the activity of these cells. And what you'll see during the stimulation, which is now, suddenly this tail rattling, which is very typical of aggressive displays. Display, and then the females start to roughen the pup a little bit. So again, it seems like the activation of these cells elicit aggressive display and impairs maternal behavior. So as we did with parenting, now we want to dissect how this works. And we found among the projection areas some really interesting areas. So these are the map of projections, and the three that we studied in particular were first the VMH, the ventromedial hypothalamus. This is David Anderson's favorite brain area where he and Daiyulin really established a central role of this locus to drive aggressive behavior. And we thought this has to be it. The stimulation of the periphornical area leads to VMH activation and that drives aggression. We had it all figured out. It turns out that it didn't work that way. As you can see here, when we stimulate optogenetically the terminals of the urocortin cells to the VMH, the female here approaches the pup and sort of hover around the pup and then return to the nest and then come back and then hover again. So seemed to be reluctant to engage in nurturing the pup. But we never saw any aggression. And I kept telling my postdoc, you know, you have to do it again. VMH has to drive aggression. She spent an entire, she wasted an entire year, thanks to me, trying to find aggression. We never found any aggression. Okay, so then we turned, So we think that this projection drives neglect instead of aggression. So then we turn to what we thought would be the second best candidate, the lateral septum, which is involved in anxiety and therefore might drive aggression. So we stimulated here in this video, you see the female will approach the pup and when she approached the pup, we triggered the light And pay attention because the behavior is extremely fast. Here, I'm going to stimulate the lateral septum projection here. Oops. The female immediately flees the presence of pup, goes to the nest, and never reapproaches the pup. So again, no aggression whatsoever, but very clear and sustain pup avoidance. So finally, we looked at the area that is actually the major projection area of urocortin cells, but we had never heard of that brain area. It's called the amygdalo-hippocampal transition area. Never heard of it. If you do a search in PubMed, there's nothing on this, like, you know, whatever, new area. And this time, When we stimulate the projection to this brain area, we found extremely clear sign of aggression, which is the animal, the female grabbed the pup by the belly and carried it around and shaking it exactly as a display of normal classical infanticidal behavior. So here again, I think we reach some really interesting deconstruction of this infanticidal behavior where these different projection areas, the VMH, lateral septum, and the amygdalo-hippocampal area, each drive a particular aspect of PUP-mediated aggression/neglect. Now interestingly, it turns out that in contrast to the galanin cells, these periphornical areas send multiple branches to multiple projection areas. So it's not as simple as distinct pools each doing something different. And we think that the difference in the outcome of these brain areas might also come from the fact that both these urocortin cells as well as the projection areas get input from neurons from the galanin cells involved in parenting behavior. So it's possible that these in part at least modulate some of the response of these projection areas. So overall, we have these really interesting interconnected circuit between these nodes controlling various aspect of parenting and nodes corresponding to various aspect of neglect or attack towards infant. And these two are clearly mutually exclusive. Now interestingly, these neurons are all in the preoptic area, and the preoptic area is extremely functionally diverse, as I mentioned, involved in many different social behavior and also homeostatic function. And so that led us to try to understand a little bit more the organization of this area. And for these, we perform single-cell profiling of the preoptic area using two distinct strategies, single-cell sequencing, so dissociating the cells from the preoptic area and identifying the different population, as well as in a collaboration with— which is this multiplex in situ hybridization that enabled on the a restricted number of genes to identify very precisely the gene profile of cells in situ in tissue section. And so, although the single-cell sequencing gives this unbiased detection of genes but also is not spatially resolved, here we have a technique where we can very quickly scan millions of cells in a spatially resolved way and also in an extremely sensitive way. So this work led to the scanning using MERFISH of the entire preoptic area using 155 genes that were partially identified using the single-cell sequencing. And so this technology enabled to see in situ various markers that each characterizes various cell types. And I think one of the extraordinary advantage of the MERFISH approach also is the extraordinary sensitivity of the approach, which is that genes that are typically not detectable by in situ hybridization, such as neuropeptide receptors, ocytocin receptors, are very clearly identified using MERFISH. And so this really provides this mapping of all the cell types in the preoptic area. These are just the very large cell types, the big category of cell types. But for us, obviously what is interesting is to try to see now to relate this work with the cell population we know that are involved in social behavior. And in particular, what about galanin cells? Well, to our surprise, we actually identified 11 subtypes of galanin cells, which each a distinct molecular and spatial profile. So here is I7. So this is a cluster that has a very particular gene profile, and as you can see, a very particular spatial expression. This is I11, I14, et cetera, et cetera. So all these 11 types can be now mapped very precisely on the preoptic area. And even more interestingly, we can also identify their function by looking at C-Fos expression when the animal has undergone a particular behavior. So here's a typical MERFISH image with all the RNA, and now C-Fos that tell us, reveal to us, which of these populations are actually involved in a particular behavior, for example, parenting behavior. And so this led us to identify a number of populations involved in parenting in ways that are, I think, extraordinarily interesting. First, we found 6 populations of cells involved in parenting. One of them expressed galanin. That's the cluster I14. And this is, we think, the initial population that Herbert had identified, this 40% of cells that are galanin positive and C-Fos positive. These are cells of this I14 cluster here. They are activated in mothers, in fathers, and in virgin females. So all animals that are parental activate this particular cell type. But interestingly, there are additional cell types that are not positive, but that are found activated only in mothers or only in fathers or both in mother and in father. So we now have a more sophisticated view of all the cell populations that are involved in parenting. And we also found two populations that are involved in infant-mediated aggression in virgin males, with one of them, this cluster E28, corresponding to these neurocortin cells that we had identified before. So we now have not only one population, but actually six populations of preoptic area neurons involved in parenting in both males and females. And these core MPOE-galanin cells now are narrowed down with very high specificity to this one cluster, I14., and then mother and father recruit other population. So I think what we have now is really an extraordinary tool that identify cells that are behaviorally relevant with extraordinary high granularity and cellular resolution. And that really, I think, will transform the way we now will look at behavior. And I would like to give you two examples of these. This is work in progress. But I think at least I found quite interesting. So one is that now we are imaging using microendoscopy to look at animal engaging parenting behavior and try to identify the function of the various population that we have identified molecularly. Now this is not a molecular identification. All the neurons express GCaMP, but when we have this female here interacting with pups. So in a minute, or in a few seconds, you see here is the pup. And as soon as the mother starts to interact with the pups, we can see calcium transient in specific population of neurons. And interestingly, when we look at the number and the type of population and their association with behavior, we found that there are indeed many neural populations that associate with different aspects of parenting. So we found, for example, that one cluster is very tightly linked to nest building, another one to the retrieval of pups, and another one to pup grooming. And so now we are trying to link this particular population to their molecular makeup. So I think this is really enlarging our view of to the control of parenting in animals with different physiological status. But to go back to this galanin population, which we now can narrow down to this very particular cluster called i14, these are these i14 cells in this area called the striohippocampal area, and then here the MPN. First, now all of these cells, Over 90% of these i14 cells are activated during parenting. So this is really our parenting neurons very clearly. And now we can perform molecular studies and try to see how gene expression changes in these cells or their morphology or their synaptic activity or their synaptic contact between animals that have different parenting display. So just as a teaser, is some really interesting gene expression difference between mother and virgin males. And one gene particularly interesting is the progesterone receptor. This is interesting because it was reported many years ago that the progesterone receptor mutant males are unable to become paternal. And so we found that indeed these genes is highly expressed in virgin males but no longer expressed in mothers. Another really interesting gene is one of David Anderson's favorite genes, Tac2, which is involved in aggressive behavior. And so in addition, we also know that there are profound changes in activity in galanin cells between parenting and non-parenting animals. And indeed, we found profound changes in the expression of certain ion channels. So I think just to summarize, But I think at this point we've been able to provide some circuit deconstruction of infant-mediated behavior. We found this circuit that rely on specific sensory cues which we were able to identify and that have this very interesting logic of organization which enable the display of very specific specific components of parental behavior, but also that enable them to be modulated by the animal physiological state such that not only these behaviors are sex-specific, but they also vary according to the physiological state of the animal. We also have now reached this exquisite spatial molecular and functional resolution of cells involved in behavior. Behavior control, and these give us access both to molecular tools as well as physiological tools to understand how behavior is controlled. And we hope that this will lead us to a better understanding of the control of this behavior, maybe in other species, and also when things get wrong in diseases. And on this, I want to thank the people who performed the work. The work on parenting was initiated by Herbert, who I mentioned several times. He's now in Richard's lab. Joni Cole, Anita Autry worked on parenting and infanticide circuit. The microendoscopy was performed by Ying Li and now Mustapha Rahman and Kelsey Clausen. Single-cell analysis was done in collaboration with Xiaowei Zhuang by DJ Bamba Mukhu and Eric Vo, and now by two new members of the lab. And I'm very I'm very grateful to all my collaborators, Venki Murthy and his member of his lab, Alexander Mattis and Vik Kapoor, Mark Schnitzer and Benny Groove, Li Chunlu and Naoshida and Benedict Babayani's lab, and then finally people in Joshua Elkington's lab. Thank you for your attention.

Thank you, Catherine, for a wonderful talk. So we have time for some questions. If you have a question, please raise your hand, and then there are people with mics at the edges, and the mic will come to you wherever you are. So there's no central aisle, but we'll wait here until there are questions. Oh, we have a question here. We'll get one of those.

Thank you. Always amazing work. After 3 weeks of a bred male, will he then retrieve the hybrid plus pheromone block, or are more cues needed for retrieval compared to attack? Sorry, I didn't understand the beginning of your question. Start again. After 3 weeks, will a bred male who formerly was attacking the hybrid block, will he then retrieve the hybrid block plus pheromone, or are more cues needed for retrieval versus the attack? You know, we've not done the experiment on animal after mating. So we've done only on virgin males, so animals that will attack. And by the way, animals that are parenting, Maybe that answers partially your question whether females or fathers don't care about these dummies. In other words, those dummies do not elicit parenting behavior. It's very clear.

That was very nice. Do you find any evidence for homosexual behavior? Yes.

Tripsy 2 mouse. The mice we have, the mutants we have, are, I would say, bisexual. They would mate indiscriminately with males and females.

So it's going in that direction. Principally, it could be possible, yeah. It would be interesting to see whether by behavioral manipulations alone, not genetic manipulations, you could move somebody from being primarily interested in one sex to being interested in another.

No, so we don't see this. But what we see, however, is another part of the equation, which is the sexual identity of the animal. And this is, I think, extraordinarily interesting. Because we have no idea what provides the identity of oneself as a male or a female. And what we found is that in the mutant, it's something in between, which is the animal now also behaves like a male. And so this might have an impact, maybe not on the understanding of homosexuality, but maybe transsexuality. And in fact, as you know, Ben Barris and other people were really interested in this data because of this reason. So you've beautifully shown us selection between behaviors, but in a sense it's almost binary. There is aggression or there isn't aggression. There's maternal behavior or there isn't, and so on. How many neurons are involved in the circuits that you show us that actually determine these behaviors? Obviously, clearly the motor output is going to require large numbers of neurons, but the choices themselves could almost be made by single neurons as you diagrammed it. And so the question is, how many neurons are involved in each of these layers to make the behavioral choice? I would say— well, we don't know for sure, but I would say several hundred. In probably several hundred. But I'm not sure it's entirely binary because there's neglect also, and that's also part of the negative reaction towards infant. I'm over here, hi. So I'm a psychotherapist and a parenting coach, so all of your research is really interesting to me in terms of how that translates to humans. And earlier when there was that little video of the mother kind of hovering over the pups, It got me thinking about helicopter parenting, which we talk about so much nowadays, kind of hovering over children. And I was wondering if you've noticed any, whatever we would define to be helicopter parenting with mice, if you've seen that kind of behavior in your research. Well, so I actually have a different interpretation of that result. I think what's happening is that the female is trying to reach the pup. Wants to be parental, and when we stimulate neurons that drive neglect, then she avoids them, and then come back, and then avoid them, and so it's like a conflict, right, between the drive to be parenting and the stimulation of that particular population that tells her not to be parenting. So I would not say, I don't think it's helicopter parents, but maybe a woman who is postpartum depression who tries and can't do it and comes back and forth and have difficulty of taking care of a child. One last one. Do you have any insight into what's happening in the female brains when they're undergoing infanticide for their first litters, for example? Yeah, yeah. So, you know, when females have their first litter, they are very stressed.. And we think that this is the component that tilts the balance, activates these urocortin cells. In fact, we've done the experiment. When we stress females chronically, we see high activation of these urocortin cells, and as a result, a reduction in parenting behavior. So we think that this is where stress acts. And in fact, because these cells receive these massive input from CRH neurons that are involved in the neuroendocrine aspect of stress, that makes a lot of sense.

So now before you all go, first of all, I would like to thank the team that has been putting this together, all our staff, the people in the advisory committee that included students, postdocs, faculty, staff members. And I'd like to invite Kelly Remole, our director of programs, to come up and explain a bit the organization of the symposium and what we need to do next. So thank you for everyone that helped make this possible.

And thank you, Kelly. Thank you, Rui, and thank you for all of you— to all of you for your attention here. We're doing great things and we're so happy that you're meeting us here. We'd like you to continue the conversation online on Twitter at the hashtag #ZuckermanSymposium. Also, as you can see in your program, we have a poster session across the street as you exit at the auditorium, you'll go down the stairs or down the elevator across one block and join us on the 9th floor. And at 8 o'clock, if you're willing to stick around, we're showing you how we do things differently here with a live storytelling event, also in the same building, the Jeromel Green Science Center on the ground floor at Dear Mama. Tomorrow, please join us at 9:00 AM for David Anderson's talk. Keep your badge, keep your materials, and come early as we're expecting another full house. Thank you.