And so I am really thrilled to introduce today's speaker, Mario Penzo is a rising star in the neuroscience community and we're really excited that he's here today to share some of his work. The doctor pencil received his BS from the University of Puerto Rico. Or if he conducted research on the neuroprotective effects of gaba a receptors and nicotinic receptor activation. He went on to receive his PhD in neuroscience from Albert Einstein College of Medicine under the mentorship of Jose Pena. And during graduate school he carried out some really beautiful studies in one of my favorite systems, the chick embryo in the inferior colliculus. So where he discovered several novel forms of synaptic plasticity and related retrograde signaling. To modulate synaptic events suffer as a postdoc, he moved to Cold Spring Harbor where he continued working in the field of synaptic plasticity. This time in the rodent amygdala. Under the mentorship of boldly, he carried out a series of really fascinating experiments identifying the paraventricular nucleus of the thalamus as a critical presynaptic partner in the regulation of fear, learning and memory and has really become, I'm a leader, a leader in this field. In 2015, Dr. Penzo opened his own research group at the National Institute of Mental Health, where he directs the unit on neurobiology of effective memory. His group has been doing some really elegant studies dissecting neural circuits involved in both the emotional and motivational processes, as well as the susceptibility distress. Recent work includes publications in Nature in Nature Neuroscience, Nature Communications in Journal of Neuroscience. He's won numerous awards for his research on just a few, including the Young Investigator Award from the brain and behavior research foundation, as well as real carp discovery award from Cold Spring Harbor. And actually perhaps as impressive as his research accomplishments are his amazing commitment to mentoring. He's a ton of awards in this area, including the NIH Graduate Student Council, Outstanding Mentor Award, this special Act Award, and the NIMH Outstanding Mentor Award, which is actually 12 times. Just in his short time that NIMH, I could probably go on the book for the full hour for all of his accomplishments, but I will yield the floor to char speaker and welcome Mario tensa. Thank you very much for me for the tiny production. It's really a pleasure to be here with you today. Today I want to talk a little bit about work that my lab has conducted recently, particularly on the role of the paraventricular nucleus of the thalamus in fear related processes. As reinvention. This is an area of the brain that I can interested in when I was a post-doctoral fellow at clothes-pin Harbor Laboratory, where we had some interesting findings about the contribution of this area of the brain and particularly progesterone to the central amygdala in regulating fear memory. And over the last few years in my lab we've been trying to expand on our, on our understanding of how this region of the brain regulates fear related processes. So the focus of my talk today is going to be largely centered on that. Okay? All right, so my laboratory more generally is interested in understanding the neural mechanisms that lead to emotional state and emotional related processes. And to address this question, we tried to undertake on this multidisciplinary approach that branches all the way from molecular processes to cell physiology circuits and ultimately trying to understand how these processes regulate behavior. And as I, as I mentioned in my opening slide, one of the emotions that were particularly interested in studying is a sphere. And of course, we'll address this question by or have been used in addressing this question using animal models, specifically mice. So obviously, you know, when you talk about things like emotion and fear in animal models, um, that can be kind of a heated subject, right? Because we tend to think of emotions, in particular fear from the perspective of subjective reports like feelings and one not so. One of the things that I like to do when talking about is these terms is to try to derive a specific definition and try to define it specifically what we mean when we talk about emotion. And I think from this perspective, one particular framework that I found, we found quite useful in our thinking is that, which was proposed by David Anderson and Ralph aid of which is this idea that Emotions can be considered to be central stay that are triggered by stimuli. The stimuli can be either extra receptors for interoceptive. And that lead to a series of, of readouts of this central emotional state. And these are readouts that we can measure right, in a laboratory setting. So when thinking of applying this particular framework to fear, which is the question that we're interested in. We can, of course, in animal models measure a lot of these readouts of this emotional states with the exclusion of subjective reports, which is still a category that is reserved for, for human subjects. But I'm sure most of you are familiar. In our field in neuroscience. We've come to learn a lot about neural processes associated with fear. Particularly by focused, by focusing on, on, on observed behavior in rodents in particular. And this has been via the use of, of mainly Pavlovian classical conditioning paradigm, which I'm going to describe a little bit more in the next slide. So this classical programs, as you guys are all familiar with, involving association of a rather neutral stimulus, like a tone with a adversive outcome, like in this case a foot shock. This, the use of this paradigm has been very powerful in neuroscience and has led to this particular amygdala centric view of fear. Largely because early studies identify the lateral portion of the amygdala as a region of the brain where these associative memories are formed, Rida, and this is lead to synaptic plasticity in this region and n. So, so this, this is part, this is the view that has dominated the field for, for, for various decades. This idea that the amygdala is like the fear center of the brain. And using this task of course, with, with, with Learn, come to learn a lot about the mechanisms that underlie this associative memory formation, but also the expression of a very specific type of defensive responses is freezing behavior. Since this is the main dominant. Observe behavior in studies of Cabrillo Bian conditioning. Naturally, when you think of this, you realize that this, the use of Pavlovian conditioning is limited in the sense that it doesn't yield a switch of information or any information actually about the brain. Processes that participate in, in the orchestration or modulation of other type of defensive responses. One which we're really interested in my lab is, is avoidance behavior. And much less do we know about the mechanisms that drive the selection between these opposing forms of defensive behaviors. And I think that this is something that's really important to think about because although we started defensive behaviors in the laboratory settings in sort of like reduction is approach. In nature. These type of defensive behaviors are displayed as a function of the spatial, temporal distance from threats in the environment. So when threats are highly imminent, rider pretty close. Animals were engaged in, in behaviors are more reactive. Life fizzy behavior or flight and jumping behavior. Whereas like when threads are much more distance, this is what we refer to as the low imminence part of the spectrum. Animals still engage in others type of defensive behaviors, but the facility, or whether that's actually more flexible and cognitively regulated, like for example, risk assessment and avoidance behavior. So I think this is a this threat immuno, continuum is kind of like a useful way to think about defensive behaviors. So for my talk today, I'm going to focus particularly on, on this behavior, avoidance behavior. And part of the reason we're interested in this is kinda like nicely summarize here and this one chart from a review article that was published a couple of years ago by Chris Kane. Where you can see that while, you know, early on there's a lot of interest in understanding active avoidance behavior. Over the last couple of decades, studies of your condition actually have dominated the literature. And there's been like Canada, like sort of like not so much development in our understanding of active avoidance behavior as it has been. For fear conditioning and its associated defensive accomplish is freezing behavior. So, well, having said this, this is not to say that we don't know anything about active avoidance behavior. So here I'm going to actually just briefly outline how we think about active avoidance behavior, particularly when using these classical shuttle box task or animals or learn to associate shuttled into the adjacent compartment when they're presented with a warning sign to be associated with the avoidance of food shops. So the animals, the training, the shuttle box for the learner every time that there's a warning sign, could be a light or a tone of a shuttle to the JSON compartment. They will prevent the onset of food shock. And they, you know, they learn to do this really well. And this is what that visited type of active avoidance responses we're measuring. So when peoples are using this dancing on people, sort of think about the processes that are taking place as animal and learn the task. And one way that this has been described as this two-stage process in which early on in training, the initial processes that take place is this Pablo be an association? The animal still simply associate in this warning sign, this tone with the subversive outcome, which in our case and most of the literature is a foot shock. And this is consistent with studies of Pavlovian fear conditioning. This is a process that is largely dependent on amygdala circuit. The lateral amygdala, which is important for the association. And in the central nucleus of the amygdala, which is critical for this freezing behavior. Now, oops. Now you can see here is that as with the passage of time as animals undergo additional training, they start to begin to adopt these goal-directed response, which is in this case the avoidance behavior. And the learning of this response is associated with a decrease in the expression of the Pavlovian response, the freezing behavior. And this is associated with a essentially like a remapping of the underlying neural circuitry where the behavior is less dependent on the central amygdala and it comes actually more dependent on the ventral striatum, which is important for goal-directed behavior. Of course, with the passage of time, behaviors can be active avoidance behavior can become habitual and it is thought that this is the circuit you underline these behaviors is resembles security that has been previously described in the context of habitual repetitive behavior like the dorsolateral striatum. And there's actually some newer ongoing work in these, in these interests in this particular question. Now, one thing that I like to bring to your attention here is that you can immediately see that when thinking about transitioning away from this Pavlovian responses to these instrumental active avoidance behavior seems like these two regions of the brain, the central amygdala, nucleus accumbens are particularly important. And in fact, there's been studies in the past and I'm only going to highlight some studies that are particularly relevant to my talk today, but pristine. Many, many studies that have looked into this. But once I do that is that I want to highlight is this study by the group of Joe LeDoux from 2010 where they demonstrated that silencing or in this case actually listening of the central nucleus of the amygdala. Attenuated freezing behavior consistent with the idea that this region of the brain is required for fishy behavior. But this manipulation actually lead to increase active avoidance behaviors in IMO that had this lesion. In a separate study, the group of Gregory quirk, they demonstrated that silencing of the ventral striatum lead to exactly the opposite outcome, which is that these animals show in permanent active avoidance behavior. But they had an increase in freezing responses to the condition or the warning signal, right? So these datas seems to suggest that these two regions of the brain are in this antagonistic relationship for the control of this type of defensive behaviors. We suggested towards, towards that perhaps regions of the brain that communicated directly central amygdala and nucleus accumbens could play an important role in bias in the selection of these different type of defensive behaviors. Now naturally, the first region that comes to mind when we think about this is the basolateral amygdala region of the brain that has been shown to same innervation to both the central amygdala and nucleus accumbens and participates in these behaviors. But studies over time have shown that the necessity of the BLA for the expression of these type of behaviors seems to decrease over time. So this suggest that perhaps other regions of the brain are equally or perhaps more important in regulating. These two type of defensive behaviors and perhaps bias in the selection of these behaviors. And this is what led us to the paraventricular nucleus of the thalamus. An area that I've been interested in the past. Particularly because of his strong innervations of limbic structure and in particular to measure output. Targets are the central amygdala and nucleus accumbens. And in fact, when I was a post-doctoral fellow at cost when Harbor, we show that this projection to the productivity to central amygdala is actually critical for the expression of condition facing behavior is something that was also replicated in rats by February, month and Gregory. But the question is whether the paraventricular nucleus of the thalamus and it's projected to the nucleus accumbens play a role in avoidance behavior. And this is the hypothesis that we wanted to test that actually we think that PBT might be important for, for avoidance behavior be at its projections with the paraventricular nucleus accumbens. And this is a question that a very talented postdoc in my laboratory Zuma, was set to address some years ago when he joined the laboratory. So here I'm just showing you a brief outline of the what my my talk today. So for the first part, I'm going to actually focus on, on, on work that was recently published. This first authorship publication. But by June that was published towards the end of last year, where we find that the paraventricular nucleus of the thalamus seems to shape the selection of avoidance and freezing behavior. This is what I will mainly focus on today. And then for the second shorter part of my talk, I'm going to tell you about some new exciting observations that we've collected since the publication of this other work that relates to the role of prefrontal cortex input to the paraventricular thalamus in the shaping of this type of defensive behaviors. Okay, So when we started this project, he was pretty intrigued about determining whether neurons in the paraventricular nucleus of the thalamus that project to these targets. The nucleus accumbens and the central amygdala actually belong to similar or distinct populations of PVT cells. So to address this question, he injected in, in, in, in wild-type mice retrograde tracers that were coupled to a fluorescent dye so that he could see neurons in the PVT fluorescently labeled that project writers of these two targets, the nucleus accumbens in one color and the central amygdala in another color. So when he sacrifices animals and he looked at their brains, he found that you can see very nicely here neurons in the PVT that predicted the comments and in red neurons that project to the central nucleus of the amygdala. And when he quantify this data, he observed that in general, many more cells in the, in the PVT project to the Canvas compared to the center of the amygdala, which is actually consistent with previous publications. Interestingly, he observed that, that the, the, the, the, the level of quality realization between these two types of PVT cells, central projectors or Acoma projectors. It's relatively low, so perhaps about 20 percent of accumbens accumbens project, it's also subject to central amygdala, but also roughly 30 percent of cells that predicts a central amygdala also project to the nucleus accumbens. Importantly, we find that the vast majority of these neurons that project to either central or amygdala or the nucleus accumbens are positive for or express D2 receptors. This is important because in previous studies we've shown that D2 expressing neurons of the PBT belong to a specific class of pivot in euros that are engaged by adversive stimulus. It makes sense that perhaps this is the population of cells that is important for regulating fear behaviors. So after identifying these, these two, perhaps these parallel streams from the PVT to the comments and the central amygdala. We wanted to assess whether these neurons of the PVT signal active avoidance behavior. So to address this question, June, use the genetically encoded calcium sensor Jiekun six witchy expressing in D2 positive neurons of the PVT of the two green lines. As I mentioned, that most of these projections of the posterior cavity are actually labeled by d2. So he expressed GQM into D2 neurons of the PVT and an implanted an optical fiber right above the PVT. So the hukou could collect bulk changes in, in, in, in fluorescence during the behavior. So again, these animals were then train in the active avoidance task that I mentioned briefly. During my introduction. Essentially animals are placing the shuttle box that is divided by a hurdle and their train such that attorneys play for 15 seconds and after the toys play. A shock is delivered until animals shuttled to the adjacent compartments. Typically shuttle pretty quickly when the shock is delivered. And over time, they'd learned that if they shuttle during the presentation of the tone, this will advert presentation of the shelf. So this is the avoidance response. Here, I'm showing you a short clip where you could see what the signal from this newest looked like during the task. This is an avoidance trial where here you can see that the q star a plane, this is the calcium transient here. And here the animal has about 15 seconds to shuttle or you will get a shock. Can see that when the animal shuttles, there is this strong response, calcium response in this T2 positive neurons of the PVT to after. Imaging animals that have been training this task June. And by the way, you can see here that the behavior actually resembles pretty much what I showed in my introduction, which is that early on, animals are displaying high levels of freezing during the tone presentation. But over time, the freezing diminishes and an active avoidance response increases. So after animals were training this task and June image them for the last two sessions of training. He divided trials between classify Charles into either avoidance trials or failure trial. So the avoidance trust of the trials that I just show you an example of with animals successfully avoided during the presentation or failure trial is a trial in which the animal didn't avoid during the tone and instead receive a shock in which they ultimately SSE k by shuttling across compartments. So this is here you can see the summary data for all the trials that June image here, which is essentially ranked by the latency to avoid. And in general, what we see is that shuttling behavior is nicely associated with an increase calcium signal in this D2 positive news of the posterior cavity. If I here you can see this black line actually depicts the actual expected duration of the tongue, which is 15 seconds, like I mentioned. And here this is just the average of the tones from all these trials in which the animals avoid it. You can see that termination of the tone nicely matches with the increasing calcium signal in this D2 positive PVT neurons. And this is something you can also observe in these individual trials in which each successful avoidance rows associated with this rise in calcium signals from these two positive cells. Now this is all quantify here you can see that indeed, if we look at the area under the curve during the presentation, you can see that in general, for avoidance trials, this was associated with a significant increase in activation of these neurons. Whereas during the failure trials were actually see a reduction in fluorescence in these neurons. And we were wondering as to whether this reduction during the failure trials could be associated to behavior. Because during this failure trials, the animals spend most of their time actually freeze in. I can actually see that here. We quantify the level of facing the time that I was paraphrasing across avoidance trials and favorite trails. In general, they spend much more time freezing during the failure trials. So we wanted to look at this more, more, more closely by looking at the calcium signal as a function of very specific tasks related events. And this is what I'm showing you here in the next slide. And essentially what you see here is that in general, for both avoidance and failure trials, this T2 positive cells of the PVT show an increase in calcium response during the onset of the conditioning stimulus. So the tone and similarly those, the activity of this news was associated with a rise during maximal velocity, which we use as a, as a, as a measurement of the vigor of the behaviors as animal shuttle across compartments. So you can see that from the video that I showed you earlier that this behavior is pretty vigorous and the animal shuttle with a lot of energy. So we look at the maximal velocity as animals shuttling as a measurement of these active avoidance behavior. And you can see that for both actively avoided behaviors or animal avoided a foot shock, these responses look very similar in these neurons. In contrast, when the animals engage in freezing behavior can see here at time 0 is when the episode starts. You can see that freezing is associated with, with an attenuation of, of calcium signal. So this is, this data suggests that these D2 positiveness of the PVT are our signal enduring avoidance behavior. But that their response is actually attenuated touring facing behavior. So naturally the next question that we wanted to address was to, was whether the activity of vision, so it's actually require for active avoidance behavior. And to address this question, we use optogenetics. So in this case, June express the inhibitory opsin, halorhodopsin, specifically on D2 expressing neurons of the posterior puberty. And he implanted an optical fiber above the puberty so that he could silence the activity of these neurons at a very specific time by delivering yellow light to the brain. And here you can see that here's what the data looks like. June train these animals in active avoidance. And after he trained, the animals are fully trained. He subjected them to three additional test sessions. And you can see here that in general, these animals is play pretty high avoidance re both control animals, which are the black line and, and halorhodopsin expressing animals, which is the red line. And here, notice here in the data that's normalized to this first session, that in the halorhodopsin session, in this case, halorhodopsin was stimulated simultaneously with the deliver of each one of the tones. This, this is very restricted to tone presentations. You can see here that when you silence the PVT neurons during the term presentation, this significantly impairs avoidance behavior. This, this reduction and avoidance behaviors associated with an increase in the latency to avoid, we can suppose you can see here and at the same time. Notice here that in these animals that display this decrease in avoidance behavior, they also show significant increase in freezing behavior during the live session. To this, data suggests that not only silence in this neurosis impairing avoidance behavior, but actually facilitating freezing behavior in these animals. And this actually is, is reminiscent of study that I mentioned in the introduction from Gregory course group where they show that silencing of the ventral striatum with new CMO leads to a very similar outcome were active avoidance behaviors in pair and freezing behavior is, is enhanced in these animals. So while this was pretty interesting result and we're actually quite excited by this data. We're also puzzle because in previous studies, we and others have shown that a subset of pivoting use that project to the central amygdala actually is required for driving. Freezing behavior in, in, in fear conditioning are intrigued by the fact that silencing this D2 positive cells actually unmask freezing behavior and attenuate avoidance behavior and activity of business actually decrease during freezing behaves kind of like contradicting perhaps previous finding that we and others have had. So we wanted to look a bit deeper into this. And one way that we thought of it was by imaging the same type of neurons is D2 positive source of the PVT in fear conditioning. So June took a different group of animals, train them in, in, in, in fear conditioning. And then he image the activity of these neurons with fire photometry during the fear memory retrieval test. And here's what the calcium responses look for, all of those trails that he collected. And what you can see here by looking at the average response from these trials is that we didn't see a lot of modulation during the presentation of the tongue, which leads to this fear memory retrieval, if anything, there's a bit of an increase at tone onset, which is somewhat consistent with this idea that PBT is important for, for, for fear conditioning. But, but he took this a step further. And he then classify trials because he noticed that in all of these trials were associated with different levels of freezing behavior. So here as well, what if we, we, we, we try to correlate the calcium responses from, from the different trials with the actual behavior of the animal. What he did here, you can see that here are the files in which animals show high freezing behavior is the trials in which nature of modern freezing on his trust in which they show low facing behavior. And lo and behold, what he observed was that in the trials in which the animals display strong freezing behavior and modern efficient behavior, those trials were associated with a reduction in calcium signals from these D2 positive news of the PVT. Very consistent with what we saw in the active avoidance task in which non-linearity seems to be a sub-population of signaling avoidance behavior, but in other activities being reduced during freezing behavior. So this was. Let's show that the, the, the, the activity of these neurons is consistent or both across both time. So for both types of paradigms. And one interesting thing to point out here is that the similar correlation between the movement of the animal or the lack thereof in facing behavior. And calcium signal is something that emerged after fear conditioning because we didn't notice any relationship between the movement of the animal and calcium signal in habituation prior to fear conditioning. Now this is all, was all very interesting to us, but it's still remain a little bit of a puzzle like I mentioned based on prior literature on the role of PVT in fear conditioning. But we took a step back and try to think of this a bit further. And one thing that I wanted to highlight here is that we are looking at the activity of these neurons using fire for symmetry, which is a, is a bulk kinda like ball calcium imaging technique or where image, where it's actually collecting data from a large population of cells under the optical fiber. And as you recall previously, I mentioned that most, the vast majority of Nursing the posterior P with your accomplice projecting. So it's possible that when we're doing calcium imaging, we are, the signal that we're collecting with fire photometry is largely based on the activity or buyers, rather by the activity of these comments projecting cells of the PBT. So this is the question I wanted to dress directly. And to address this question, what John did was to, in this case, instead of using D2 cre animals. And June, a restricted expression of, of g come to neural activity that projected to the nucleus accumbens by using a retrograde based approach. And essentially to summarize the data that he collected, you can see here the consistent with the previous data from this, the two positive cells, we can observe that shuttling behavior is nicely associated with or avoidance behaviors associated with an increase in activity in these pivoted to accumbens cells. In general, we see a reduction in the activity of these neurons during failure trials. Again, if we look at this, task related events will see that like before. Metrics like maximum velocity or the initiation of scape behavior were both associated with an increase in calcium channels in these neurons. Whereas freezing behavior was associated with an attenuation of calcium in cells. Interestingly, unlike the general DTU based imaging approach in these PBT, a common cells in particular, we noticed that it seems like this. Neurons can discriminate at trial type because the CS was largely associated with an increase in calcium signal in these PVT a common cells only in avoidance trials but not in favor of trials. So this is in contrast with what I showed with the imaging approach. We also try to look at PVT projection to the central amygdala. So here, June image the axons from these PVT neurons over the central locus of the amygdala. And in concert with that I showed you in the previous slide, we found that the CS, in this case was more associated with activating these neurons within failure trials, which is interesting because these are the trouser animals are mostly free scene and not avoiding. And we didn't see any significant change in maximal velocity or sorry, in, in, in calcium signals associated with maximum velocity. So this projection from the ability to central amygdala seems to be a bit different than those that go to the knock-offs, a communist. And so we think that some of this data, it seems to be consistent with our previous hypothesis that perhaps, you know, when we're looking at general D2 expressing source of the PVT, where were we have mix of these two type of projectors of the PVT. So we decided to stick with this approach for the reservoir started to look at and manipulate specific pathways or outputs of the PPT. And this is what John did here, specifically here in this experiment, he tried to assess whether these PVT progesterone to the nucleus accumbens indeed are required for active opponents behavior haven't seen that this neuron seems to be sort of like signaled in avoidance behavior. He then wanted to use halorhodopsin to like you did before. Instead of silence in Nero's directly in the PVT, silence in their terminals over the nucleus accumbens. And he took animals that were trained and then he subjected them to these additional tests, one of which involve silencing of this pathway. And you can see here that consistent with the data that I showed you before, silencing of this PDT accumbens pathway during presentation of the tone lead to a very robust impairment of avoidance behavior that actually persisted a day later. And it's something that I'm going to get two back to in a second. But I also wanted to highlight that interests in which the animals actually avoided. There was a marked increase in their latency to avoid. And similarly, there was an increase in influencing behavior. So it seems like these animals, our candlelight, shifting their, their, their behavioral response to the tone when this pathway is silence. Now, this data was actually of particular interest to us because he was reminded us of a previous publication by for breach of the Montoya and Gregory quirk, where in the context of Pablo be in fear conditioning and measuring freezing behavior. They found that silence in progesterone from the PVT to the central nervous of the amygdala. Attenuated freezing behavior. And these impairments persist. And a day later. In recalling the conclusion from this study, they, they concluded that these data suggested that these PVD projection to the central office of them, of the amygdala seems to be important for the maintenance of fear memory because if you disrupt this projection during fear memory retrieval, you have a long lasting impairment in, in their memory. Here, memory being assessed by how much time the animal spends freezing in response to the tone. And if you look at our data here now in the context of active avoidance and a projection from 50 to the nucleus accumbens. See that it's actually look fairly similar in which you get is impairment in avoidance behavior that persist the next day. And we weren't template to draw the same conclusion that perhaps this suggests that PVT projections to the nucleus accumbens are actually important for the maintenance of this fear, memory or the behavior itself. Now, one particular observation that seem to be in contrast or contradict this initial conclusion is the fact that in these animals that have this lasting impairment in, in avoidance behavior, they also have a lasting increase in freezing behavior in response to the tone. It doesn't seem like the animals are unable to remember the association between the tone and the aversive, our commodity or the sharp. But what seems to be happening here is that they're shifting their overall response strategy to that particular association. So, so this led us to conclude or to try to like, I guess, re-evaluate the contribution of the PVT to fear memory. Because both the Avante Can, an osteon, when I was a postdoc, made statements that the paraventricular nucleus of the thalamus seems to be important for fear. Memory because if you disrupt that, you have these effects on memory like maintenance, for example, as shown here or as suggested by this data that I'm showing here. But we think that, that, that the data that we observe here suggests that the pivotal role in fear memory is really not in, in, in driving the CSUN, CSUN association, but rather in tying this particular association to specific behavioral outcomes. So if there's something that take-home message from my talk today is that I think this, this, this lead us to think differently, or this, this finding is allowed to think differently about the contribution of, of the pivoted to fear related processes. So to summarize what I've shown you so far, I showed you that based on previous publications, PVT position to the central amygdala seems to be critical for driving condition, freezing behavior in fear condition animals. And today I've shown you that silencing PVT projection to the nucleus accumbens in PR avoidance behavior, but also facilitate freezing behavior during the avoidance does so naturally. A question that came out of this observation was whether when PVT projection to the central amygdala silence which leads to impairment increasing behavior. Does this manipulation also impact avoidance behavior to see boost avoidance be here? And this was actually indeed our provision that he did so so to address this question and actually let me take a step back and give a bit more context as to why this question has precedence. And that is based on, on a stock that I mentioned earlier, my introduction from the laboratory of Jolla do, where they were looking at active avoidance behavior in rats. They found that in general, a sub-population of rats display these low avoidance response, whereby the third training session, they were just like not even breaking the 20th send avoidance mark, right? So they, they refer to these as low avoiders. And what they found is that these rats that were failing to show avoidance behavior actually does play a strong freezing behavior. So they, they, they, they, they, they reason that perhaps the reason that the animals are failing to avoid is because of their strong facing behavior. And since they know this is a behavior that central amygdala dependent, they'd be this lesion of the central locus of the amygdala. And they found that by lesion in the central amygdala, immediately they saw a jump in in the, in the, in the rats avoidance behavior the next session that they tested them. And they, these avoidance actually continued to improve through training. So this is consistent with the idea that suggests that the central amygdala is a surf like antagonizing avoidance behavior. So we wonder whether PVC projection to the central amygdala, which we have shown to be important for this condition, freezing responses are particularly involved in these biasing of defensive behavior. So to address this question, June, use again optogenetics to silence PVT projection to the central office of the amygdala in animal strain inactive avoidance. And to make a long story short, he observed that silencing this projection to central amygdala improve avoidance behavior in, during this session. And this was associated by a reduction in the latency to avoid and also a reduction in fishing behavior. So again, this is consistent with prior literature that since lambda is important for feeding behavior, but it shows that this particular pathway is important for bias in the selection of these two type of defensive behaviors. Because if you get rid of this particular output of the PVT, animals are able to engage in avoidance behavior much more. Again, June to this a step further and his screen for my show, the same type of low avoider phenotype describe bipedal do group specifically screen animals that after three training sessions display very low avoidance behavior. And then he silence this projection from the period to central amygdala across additional test sessions. And what you can see here is similar to what I showed before and to the, actually to the data from the Ledoux group, you can see that these animals improve the avoidance behavior with additional training when this particular pathway was shut down. And we did not observe such improvement in behavior in control animals that instead of the halorhodopsin other control fluorophore expressed in the, in the puberty. So to summarize this part, I've shown you today that, that the PVT seems to be important for bias in the selection of defensive behaviors. Bi's projection to the nucleus accumbens and the central nucleus of the amygdala. With you. Silence or one projection of the OD or the other, you bias you guys behavior in favor of the opposite pathway. And you can see here, you can see this very nicely here and this bit challenging experiment that the June deal where he express halorhodopsin in the purity and the impact that optical fibers bilaterally both above the nucleus accumbens and above the central locus of the amygdala. And he observed that when he silence one pathway, in this case, the nucleus accumbens animals show impairment in avoidance behavior. But the next day, instead of showing this persistent impairment behavior that we've seen previously, if he silenced the central amygdala pathway the next day, these animals show recovery of their avoidance behaviors. Show that manipulating these two outputs within the same subjects can dynamically affect the behavior. So with that, I'm going to summarize this part of my talk is actually going to show you is that this D2 expressing cells of the PVT That scene of active avoidance particularly be added projection to the nucleus accumbens. Interestingly, these cells are also inhibited when the animal engages in freezing behavior, suggesting that there must be some kind of interaction across these two streams are outputs of the PVT. We're actually interested in addressing this question. I also show that optogenetics silencing of these PET projection to the comments not only attenuate avoidance behavior, but also try these lasting impairments in avoidance and a concomitant last an increase in influencing behavior, suggesting that the PVT seems to be important for. Switching the behavioral strategy to spirit to a particular CSU as Association. And now it's kind of like this. The opposite effect when we attenuated PVT protection to the central limiter. Now, I know I don't have much time left and I I you know, I definitely want to leave some time for questions, but I want to tell you very briefly about how we're looking to further these findings. One of the questions that we're trying to address in the lab is how this fear related associations are transmitted to the pivoted to support the expression of these different type of defensive behaviors. And we've done tracing studies in which we use monosynaptic rabies methods to map inputs from different regions of the brain. Mono synaptic input onto, onto projection define Nero's of the pivoting. That is whether they're accumbens project again or central amygdala protecting. We can now identify the specific inputs to these type of cells. And this is helping or informing our ongoing studies of how a specific inputs to the, to the paraventricular thalamus shaped this defensive behaviors. And one input that we've been interested in, a particular context of this transmission of associative information to the PVT has been to look in the middle prefrontal cortex, largely going to be seen in previous studies, we found that the prefrontal cortex sends power projection to the, to the paraventricular thalamus. In particular, based on our retrograde stories, we see that the dominant channel from the prefrontal cortex project into the posterior PBT is the prelim big region. So because it's the part of the PVD that we've been focusing on. We became interested in investigating the function of the limbic projections to the paraventricular thalamus in the context of defensive behaviors. And, and again, there's precedent for this that the publisher, the mountain and quick study that I mentioned earlier also look at the contribution of the limbic cortex projection to the PBT in the context of fear conditioning. If under this projection is actually require for the retrieval or the expression of, of, of condition freezing behavior. Similarly, in additional work from, from Gregory quirks to group, the US is pretty cool. Platform base avoidance task with animals like pressing for food here rats. But they learned during presentation of the tone they can jumping this platform and avoid a show. So this is like a more of a conflict, motivational conflict task because animals are pursuing reward and balancing the pursue of reward with the threats in the environment. But what they found also is that silencing of the PL with new CMO also impress the avoidance behavior in this task. So, and of course, more recently a study from Jill Harris group using optogenetics, which is more, allows for more better time precision in manipulating this area of the brain. They showed that silencing of the limbic cortex, particularly good and the presentation of the tone, as you can see here, also attenuated avoidance behavior in a shuttle box task is similar to the one that I show you today. So altogether, this the, you know, the, the, this, this is, there's a lot of evidence that the limbic cortex, you know, It's important for the expression of the defensive behaviors and its role in decision-making processes. Were interested in investigating whether peel projection to the, to the PVT were actually important for driving avoidance behavior and whether they may play a role in, in the biasing of defensive behaviors that I mentioned in the first part of my talk. So I'm not going to because of because of time, I'm not going to get too much into this, but I just want to show you briefly some of our more recent fire photometry imaging data where June use against fire photometry tool monitor the activity of neurons within the limbic cortex that specifically innervates the paraventricular thalamus here using retrograde methods, you can see that these animals learn avoidance very well and that over time they're freezing behavior or the amount of time that they spend freezing is attenuated. Unlike the previous study that we publish. In this case, June wanted to see how these responses in this region of the brain evolve over time through training. So he recorded both in day 1, day 3, and the 5. Here, essentially the summary data for all the trials that he collected. And you can see the summary of that data here. Something that I like, I like to point out was pretty interesting to us is that with the course of training, you can see here the one is implied between blue and the five in red. You can see that during the avoidance trial there is this increase in the responses of these PVT, oh sorry, pl to PVT neurons doing avoidance trail that is not occurring in failure trial. So you can see here that towards the end of training, there's no significant change in the activity of these neurons during the CS, actually during the, during the whole duration of the CS. But here you can see that there's a persistent change or increase in activity of this PL neurons that project to the PVT specifically for avoidance trials, of course, they suggest that perhaps these peel to pivot in euros may discriminate trial type. So we look at it a little bit more closely into this data using the same type of analysis that I showed before. And you can see that indeed if we know, if we look specifically at the onset of the tone, you can see that indeed there is a significant increase in activity in response to the tone. By the end of training in avoidance trail that's not observing failure trials. But if we look at specific aspect of the behaviors like maximum velocity, we don't see much of a change towards the end of training, either during avoidance or failure trials suggesting that perhaps these particular part pathway is not important for the motivational aspects of the behavior, but it may be important for the more associative aspect that, that instructor, the behavioral decision-making aspect of the behaviors. Interestingly, with the ops, we did see that when the animals initiated their escape, this was associated with a reduction in activity of these PL project NPV teen years. That again was clear for the avoidance behavior, but not for the escape behavior during failure. And in contrast, we see that the activity of these nerves is increasing towards the end of three into freezing. So it seems like this, this newest do seem to perhaps be important for the behavior selection process. And you know that, um, the, the behavior that animal engages in is nicely tie with dynamics in, in this particular pathway. But this is something that we're exploring further and we're trying to use other methods like single-cell calcium imaging with mean is Coke so that we can get more which dataset and care and can try to address this question about the dynamics within this particular pathway in the context of, of, of, of learning, but also in, in behavioral selection. Now, I think I'm out of time here, so I'm just going to mix up here and I'll be happy to take any questions. Thank you so much for that really lovely talk. There was a very everything he did. It was beautiful. It was really him. Very well, well explained throughout. We have a couple of questions in the chat. And so it's going to say that once he might want to unmute yourself and ask a question, I think he had a question that popped up during the first part of the talk with the EBT2 DeCamp enriching. Or if he's not available, then as I think I think I can see the question here is, is the question about the percent of trials or failure guy. So he asked about the percent of failure trials. Yeah, So with this actually varies. It depends on the level of learning the animals. We try not to train animals for too many sessions because with, as you can see with additional train, they can get to very high avoidance rates. So we want to train them to a level in which we get like a nice mix of trial types so that we can assess activity in this pathway for both types of trials. But people have, a lot of people have looked into specific aspects of the task in terms of like the into trial interval and duration of the tone. That can actually drastically alter the level of avoidance performance in the animal. And I'll be happy to talk a little bit more about that. But in general, in our cases, we get mostly avoidance trial, but I think at least typically will get TE percent of the trials to be failure trials. Okay. And then there's a comment from Chile trial, I'm Jill, you're welcome to unmute yourself or if you're unable to read the comment for the audio. I will, I will just read the comment for the audience, which is that this is very reminiscent of marriages work has shown that the nucleus accumbens is an effective keyboard in an app actually keyboard, excuse me. At one end you can activate working for reward. At the other end you can get defensive baring. Yeah, that's an interesting point. I think. We've actually look test this same pathway, this puberty projection to the nucleus accumbens in the context of the war seeking. And we actually see a pretty similar dynamics. Of course, we have not done an exhaustive assessment of the diversity, projection, diversity of Nero's from the PVT that innovate the nucleus accumbens. We have, we and others have found some interesting differences where there are classes of purity saw that innovate more the core where the shell, etc. But in looking at this pathway inputs with the type of manipulations that I've, that I've shown you today. We see that these projections are also signaling rewire approach. So we think that this, this, this highlights a general role for these telomeres triad or projections in, in driving goal-oriented behavior, perhaps Vietnam by, by engaging with motivational circuits. So it's kind of interesting to think further about how these pathways I've done this particular stream that seems to be important for both rewire approach and goal-oriented defensive behaviors like avoidance may participate in biasing behavior. I made motivational conflict where there's the possibility of searching for, for a reward one. There's also threats in the environment. I'm, and I think there are multiple labs that are interested in addressing this question. What wonderful. But people can feel free to put questions in the chat, but I actually have a, have a few that I would love to ask you about. The first is some in the, in the calcium imaging experiments where you were looking at the, the calcium signal during the failure trials. I actually noticed, of course, like as soon as the tone finishes and I suppose a shock is delivered, you know that there's that large calcium transient during the period when the shock is on. And I was curious whether or not that large calcium transient is something that is always present during training or it's something that emerges over the course of learning. Like the actively avoided maybe, or whether or not you think that activation is driving any kind of plasticity. Probably know that is always present actually. So one of the things that I enter if I fell to point it out when I showed that particular data. But one thing that I was, I had mentioned at the beginning is that we and others have shown that the posterior purity and in particular these D2 positive news, are particularly specifically activated by aversive stimuli. In fact, early studies on the paraventricular thalamus branded it as a stress sensor. The brain because this region D, this particular, these posterior part was or is a strongly activated by negative stimuli like Charles or restrain, stress or tell suspension. Yeah, so, so, so this is something that we always see that an aversive stimulus leads to strong activation of these neurons. And that's why you see that strong response in the failure trials when you actually get the shock on solid and c. Um, and then I had one other question which was on, I'm fascinated by this result that you have like kind of the the lasting, you know, toggling between the avoidance and the freezing behavior even one day after the halorhodopsin stimulation. And I'm curious whether or not, you know what the activity maybe during or maybe more importantly after a halo stimulation looks like. I think that a lot of us who've used halorhodopsin see very high firing rates upon release from halorhodopsin stimulation. It's, I was just curious if, you know, it made me think like, oh, maybe perhaps released from that halo stimulation is maybe even activating those PBT neurons after the tone that could drive some kind of plasticity that supports the freezing behavior. I was just curious if you have any credit for that is a fantastic question. We have not look into that, but that's a real possibility actually. Because of how these thalamic cells respond, right? And they have these strong IH current hyperpolarization activated Quran. And they have these T-type calcium channels. Both, both of which will lead to this type of posts inhibitory rebound phenotype. So it's a very, it's a very interesting question. And trying to look at this from a different perspective, which is that we are. So we recently identify actually that engagement of gaba receptors in these neurons is critical for the avoidance behavior. And it's kinda interesting towards, because engineers are, zeros are actually activated during avoidance behavior. But it seems like at some point, gaba receptor function is actually pretty important for, for driving the behavior. Yeah, Actually, if we suppress gaba receptor function in the puberty, we see impairments in avoidance behavior in well trained animals. But if we actually enhance postsynaptic our responses in these neurons, they actually have an increase avoidance responses in these animals. So we have some preliminary data that suggests that perhaps there's inhibitory inputs to the PVT. There are actually time in this, these behavior, meaning that they lead to suppression of the PVT and potentially a rebound of activity right after. So, so it's an interesting question that you posted. I mean, we have now look at this is true that in the context of our experiments, the light is stopping right at the end of the CS, right? So one wonders whether that could be like reinforcing the freezing behavior, something like that. That's an interesting question, but, but, but, but, but, you know, although I, I cannot address that Guido thing that perhaps you know, this, this, this circuit is an activities, this circle activity within the circuit is largely shaped by inhibition. Actually. I am sense it another time and so am. I do want to thank you again for just a wonderful talk on virtual round of applause.