Valeria Gazzola And one of the biggest limitation[s] in particular of the human research is that you can go so far in understanding the brain mechanism. Because we cannot, we don't have tools with the correct in terms of, for instance, understanding what a single cell does, in these processes. And that's why, for many years, we developed, we tried to develop a model in animals where all these techniques are more available that would allow us to investigate similar processes to those in humans. So processes basically that are involved in perceiving the other['s] distress also in animals. And, that's basically the main reasons why we move towards this research and then we started from coming up with the experiment in which we simply measure the distress of an animal in reaction to the distress of another. Which what we call , for instance. And we made it more and more complex in order to reach slowly a degree of complexity that can be comparable to processes in humans. Emotional contagions: you can compare it, for instance, that the level of humans when you are in a crowd, or when you are baby, that as a crowd you are contaged by the emotions are around. And, but then, we wanted to go a little bit of a step forward: okay now I am contaged, what does it mean to be contaged? Can I use this information to, for instance, avoid hurting other people. And that's how we came about, more or less, so. Christian Keysers I mean, one other motivation we have is that in the past we've also been interested in people that seemed to lack the aversion to harming others. I mean, with that , sometimes they're called as well. And so, a couple of years ago, we made this study in which we identified psychopathic criminals in high-security institutions. We brought them to the lab, and we scanned their brains while they witnessed the pain of other individuals. And what we saw was that if they don't have any particular reason to empathise with others, they really have reduced activity in their own pain regions while they witness the pain of others. So, of course, that's interesting and it may suggest that perhaps, the reason why if they can make some money, for instance, they don't mind to harm other people is because they don't activate their own pain much while they witness the pain of others. The problem like Valeria was saying, is that in humans we can see that the brain region is active while you see the pain of others, but you can't easily change the activity in the brain region to see whether that would change the way that the psychopathic criminal would act. I mean, ideally, what we'd like to do is increase the activity of these pain regions in psychopaths and see that now suddenly they would really care to not harm others anymore. Valeria Gazzola Although it's not yet, it's also not yet very clear that we need activity of these regions to avoid the harm, right? At that point, that's how we came about to run the experiment, because the first question was: is it really necessary. The activity of these regions. So, you start with okay, you want to manipulate these regions to reduce psychopathy. But the field could not even prove that this activity was necessary to reduce harm. Christian Keysers And so, that was really one of the strong motivation behind the -experiment, because rats have a very similar to what humans have. It is really a core part of the brain that is involved in feeling your own pain. So, if you don't have a cingluate cortex and you hurt yourself, you still feel that you've cut yourself, but you don't mind. And so you are not going to do a lot to avoid being cut again. So this region exists also in the rats and it is a relatively deep brain region. So therefore, we turn to the rats, and figure that in the rat, we can change the activity in the brain region and see whether that would then change how much the rat would be aversed to harming other rats or not. Christian Keysers Ah. So very briefly, what we do is we give a rat a choice between two that the rat can press. Both of these levers will provide the rat with one little pellet of sugar which they really like. So, they are happy to press these levers to get the reward. In the main experiment, what we do is that one of the two levers is a little bit more difficult to press than the other one. So most of the rats start to prefer the easy lever to press. Valeria Gazzola And that also what we see is that there is variability in the responses of the observers, of the rats that make the choice between the levers. And these variabilities also present in humans: when you do tasks that are rather similar, you see that not everyone acts socially, preferring the social lever. But you also have individuals that would go for the reward more often. So this is also another similarity between the animal world and the human world. And on top of that, I think that the other things we've learned from that experiment is that, the decision is a function, basically. Because, we have a condition for instance where we increase the reward for the animal, for the observer. And when you increase the reward, they are less willing to switch to the other lever. So that also another similarity with human world and shows that maybe that it is a bit of complex, a tipping point, and it is like a function that really weighs the cost and the benefit of that particular context. Christian Keysers Because when Valeria says that we increase the reward, we increase the difference between the two levers. So in the main experiment, the only difference is that one is harder to press than the other, but they both give one pellet. Then in two other experiments, what we did was that now that two levers are equally hard to press; but one of them gives you two pellets, the other one just one pellet. And then rats are still willing to switch away from the two-pellet to the one-pellet to save the other one from pain. But when we made it three pellets versus one pellet, then, none of the rats were willing to give up the three pellets to prevent the shock to the other person. Valeria Gazzola Yeah, and then, the last, I think, result I kind of like in a way is that, as in many human studies, also in the animal world we did not find significant difference between . So both, the female and the males acts in a similar way. And in this type of , is also often the case in humans. Christian Keysers That's true. Valeria Gazzola So that's also interesting. Valeria Gazzola Well, in a way, I would say, to really show — I mean, I always believed it that animal performed these choices too. And that they do it to a certain extent to at least care or at least perceive what goes on in the other, right. It can't maybe specifically speaking of voluntarily care about others, but at least they perceive what goes on and I think this is something that the study shows pretty well that they do perceive, and that based on these perception[s], they make a decision. And I think it is a complex — I mean, to me it was in a way trivial, because I always believed that, you know, if you are a "", whether you are a rat, or a person, we are still animals, right? They must develop behaviours that take advantage, or that they adapt to the fact that it's a social environment that involves more individuals. But [...] the field keeps discussing, right? Whether animals also have emotions, whether animals also can take this sort of decision. And to see, slowly that evidence accumulates in favour of processes similar to what we call or . You know, similar: not exactly the same, but similar processes also are present in animals, to me is sort of reassuring also. And I think it allows us to learn from how evolution evolved as well. Maybe apart from knowing exactly how it works, but we start having some models where we can think, oh, evolutionary: maybe this process was already there, and then it developed further, more complex for the humans, but still is there to a certain extent. Christian Keysers Yeah, and I think the fact that it's really the same brain region as well that seems to be involved kind of gives us some confidence that prosociality in caring about others is really deeply ingrained in our biology. Christian Keysers Yeah so in many ways it's a relatively simple study. So we didn't need to use anything very high-tech in this case. Because to alter brain activity, we use the injection of muscimol, which is a GABAA blocker. It is a very simple pharmacological agent. So it's really technologically a very simple study. Nothing fancy. Valeria Gazzola Oh, we brainstormed about it. We started, as I said, we did a lot of research on, since our early ages, let's say in all our scientific career is based on embodiment, and activities in these areas and slowly we kept wondering more and more, on the role of these areas. And then, the way it goes, we normally speak about a topic and include other people and keep brainstorming, and I think, that's how it goes. At least for us. And in humans, we are also developing similar paradigms, so it just came one plus one equal to two. [laughs] Christian Keysers So, Julen Hernandez-Lallement, the first author of the study, was the one that basically made the vast majority of the experiments. And he was helped by the other two: [Augustine Triumph] Attah: who did part of the other animal groups, and then Cindy [Pinhal] and Efe [Soyman] helped to analyse the data. And basically Valeria and me have been kind of help designed this study initially and helped to analyse the data and wind up the study. So we kind of lead the lab, but it's the other people that really manipulate the rats and do all the hard work. Valeria Gazzola Well, it was long. Christian Keysers Yeah, I would say, four years? Valeria Gazzola Yeah, because the behavioural part is the longest one. Because you need to understand the best way to teach the animal, first to manipulate the lever, then to manipulate two levers, and then, before the lever, there is also another action that they need to do; and everything needs to be done within a certain time. So you need to let the animal the time to learn and for you that you run the experiment, you need to learn what's the best way, what's the best way to teach them to do the task? Christian Keysers Yeah, I think that Valeria just said what's really important in these studies is to understand what kind of situation you can create for the rats that they are really understand and which they can in a way, show you what they're capable of. So, kind of fine-tuning all the elements of the task, kind of how long do you let the rats develop the preference for one lever. Because if you give them too much time to get too used to one lever, they are not so willing to switch anymore. So the, most of the time is really involved in trying different versions of the paradigm, understanding what the rats really understand, and then analysing all the behaviour. Christian Keysers Yeah exactly; so the three kind of conditions we looked at was the hard vs the easy lever, and then seeing whether they switch to the harder lever when, to prevent the shock to the other one. And we tried the two pellets versus one pellet and, then the three pellets versus one pellet. And then finally, to test really whether the cingulate is necessary for that, we compare the condition in which we inhibit the activity in the cingulate using muscimol against the condition where we just inject salt water into the same brain region which doesn't have any effect. Christian Keysers Correct. Yeah. Valeria Gazzola Yeah, more or less. I don't know the date. Valeria Gazzola I think it was really again the establishing the behaviour. And in particular because we, at first — so the paradigm itself, we took it from a previous study, that was run years ago, do you remember? Christian Keysers Yeah, in '69, so that's kind of 50 years ago. Valeria Gazzola Fifty years years ago, so the idea of the two levers. And so and at that time, the articles' level of detail were not so many. The articles were not giving as much information as we now have to write down. And so we had to guess some parameters over there. And, the fact that the rats would prefer the easy lever was also not so straightforward. So we thought "oh, they are gonna prefer the easy lever, and that's it." But at the end, you know, some rats, some animals develop a preference on the side; like they prefer their right lever, or the left lever, or others, they prefer the heavy lever. Right? And that was also a bit puzzling at first. We said, okay, so what do we do? We find that a couple of rats prefer the hard lever, is that meaningful or not? So we have to discuss a lot, how to adapt the paradigm to make it work, and this is also why we came about in using this switching index, because for our purposes, at the end, as long as the rat is able to switch a preference, right, whatever preference is developed is irrelevant, right? And before the beginning of this study, we were more thinking: oh, they have to develop the preference for the easy lever. And that's what is bringing us a bit out of the main purpose. And we realised: okay, but, what preference they develop at the end, it doesn't matter as long as we have a preference that we can show they can change because of the shock. So, I think this was one of the longest part[s], I would say. Christian Keysers Yes. Valeria Gazzola Yeah, I would say yes. Christian Keysers For once. Valeria Gazzola [laughs] Yeah, for once, yeah. I mean, there was some evidence around and previous work was already bringing us towards that direction. And, yeah, I mean we could not anticipate the proportion of animals that they would switch. But, we surely were expecting the rats to switch. Christian Keysers We were hoping for the cingulate to be necessary, so. Christian Keysers . Valeria Gazzola We were happy. Christian Keysers Yeah. Christian Keysers I think it's written in the paper, but my guess would be something like, I'd say, 70. Christian Keysers Yeah, so that's interesting, in this study we really find that there is a lot of variability between the rats. So like I was mentioning, we have about 40\% that were ready to switch when they witnessed other one getting shocked. And about 60\% that do not switch, so as a group, we really see that they do switch. But then only a proportion of individuals really show that sensitivity, and others do not. Valeria Gazzola Or did you mean how close they were in terms of familiarity? Christian Keysers So they're not genetically related. They're not brothers or sisters. And what we have is we have, we made one of the experiments with rats that didn't meet each other before, and it [the experiment] works. And we had other cases in which they were living in the same home cage, and so they [would know] each other very well. And the effect was not stronger. So, amongst our rats, we really noticed that they care even about the fate of a stranger. Christian Keysers Correct. Well, mates as in cage mates. Yeah, there was no sign of a difference. We did look at it and it was not the perfect comparison because the two groups were slightly different in the paradigm as well. But, it didn't look like there was a big difference. Christian Keysers So we're actually going to test that very soon. And it is up for grabs. But in the past we've looked at whether a rat shows more freezing when it sees another one get a shock. Which is a form of emotional contagion. And there we compare a kind of rats from different strain,s even. So one of the albino rats against a rat, versus, that had never seen each other before, or ever seen a member of that strain, the one end. And on the other end, really the rats have spent five weeks together and knew each other very well. And we didn't see any difference there. So I think the most robust findings is really that they care even about what happens to a stranger. Valeria Gazzola Yeah, we are testing it again now, in the future. Christian Keysers Exactly, specifically whether a mother would do more for her own kids than for... Valeria Gazzola Kids of somebody else. Christian Keysers Yeah. Christian Keysers Correct. Right. Valeria Gazzola Yeah. Christian Keysers Yeah, what you do is you place a . So you put the animal in a stereotact which is a system where you can go to a very specific position in the brain. We place a cannula, which is like an injection needle just where we know the cingulate to be. Then you let the animal recover with the cannula cemented in place. Then, on the day of the experiment, you basically inject a little bit of muscimol, which is an agent that blocks . Then by doing that, you can reduce the activity of the cingulate for about one hour. And during that hour, you can make the experiment. And then the control group is the same thing, but instead of injecting the muscimol, you inject some salt water. Christian Keysers Nothing really. Because you inject a very small quantity, that should only spread about 1 mm in the brain. So it really just affects the region that you — and you don't see any change in behaviour. So we also tested, for instance, whether they move more or less, whether they press the levers quicker or slower, and there was no difference. The only difference is that they didn't learn to not press the one [lever] that shocks the other. Valeria Gazzola And for instance, when you inject with muscimol, in case you see doing a normal paradigm. So whether you are still shocked to the sound, and then you present the sound. In that case, the rat behaves normally. And it still freezes. So, it does suggest that the muscimol effect is not impairing the normal behaviour, but specifically impairs the social behaviour of feeling... Christian Keysers ...of feeling for others. Valeria Gazzola Exactly. That was also the interesting part of the ACC that we believe to contribute really, to including the other [animal] into your decision-making. Because for a normal fear conditioning paradigm, you don't need to go through the ACC. They are made to be self-sufficient. But, if you do a fear conditioning basically that involves another individual instead of sound, like of pain, then you do need to go to the contribution of the ACC. So there seems to be two slightly complementary pathway for fear-detection. Christian Keysers Yeah, that we didn't measure. So, in that study, the muscimol animals were only in the behaviour with the ACC deactivated and the other ones were only with their ACC intact. So, but this is something that we plan to test in the future as well. To really see whether the ACC is necessary to acquire the aversion versus to maintain it. Christian Keysers We don't know, but I think there is no reason to assume that when the muscimol wears off, they would not become normal again, right? So that's most likely. What we don't know is whether if we had for instance, because there's three days of experiment, right, where we develop the aversion. And what we didn't try is for instance, give muscimol on Day 1 and Day 2 and then stop it on Day 3, and see whether on Day 3, they would then suddenly stop using the lever that gives shocks on the other days. So that we don't know whether it's necessary for learning or for recall; we just know that if you don't have it at all, you don't learn at all. Christian Keysers It's a good question. I would need to look in the paper. If you have the paper there, perhaps you can see it. But I can have a quick look to see if I have the paper somewhere. Yeah, so if you look at the Figure 4c, you can see the behaviour of each individual animal. And what you will notice is that there are some animals like some of the red animals which are the muscimol group that did switch, but the majority didn't switch. So I see there is one and only one animal in the muscimol group that really did switch. But what happens is that when you make those experiments, you never know for sure that when you inject the muscimol, that it has the desired effect. Because of course you have a cannula that's in the brain, and the cannula could become clogged also. So it can be that that one animal switched because the muscimol wasn't effective for some reason. Christian Keysers No. Valeria Gazzola No. Could be nice, but we didn't. Christian Keysers Exactly. Yeah, and in particular, there is a drug called Ecstacy that you may have heard of. So, some people reported that Ecstasy for instance, increases the feeling of empathy amongst humans. And I think that there is somewhere a study suggesting that if you give that to animal, there is also an increase in the sensitivity to what happens in another animal. So we didn't test that. Christian Keysers Yeah, no so we didn't do that. That is a very old experiment from . I think in the 50s, he did that experiment kind of with rats and with monkeys. And he sees that the rats will use the lever less. So even if you just have one lever, they'll use it less if it causes pain to others. But in monkeys, that was a very strong affect. Some of the monkeys really would rather not eat anything than to shock another monkey. So it's an interesting experiment, but we haven't done it. Christian Keysers Yeah, what you see here is the two lines: the dark green and the light green that represents the males and the females. For harm aversion, for that you should look at how much it changes from the baseline through the three shock sessions. And the change is exactly the same in males and females. But the difference between the two curves that you see is the fact that the females started off with the higher preference than the males did. But the reduction in preference is same across groups. Christian Keysers Between which rats? Rats, you mean, in general in the experiment? Figure 3b of the study. In general, like the rats, which were on the extremes, how extreme were they on the case-by-case basis? Christian Keysers Well, that you can see, if you look at Figure 3b in particular, you can see that some examples on the top of 3b of rats that had started off with a very strong preference for that were around 100 on the baseline session. And it stayed at 100. So they absolutely didn't care at all. And in the other case, if you look at the bottom panel, you have some animals that initially had a preference around kind of 80\%. And by Day 3, they really go down to 0\% of the shock lever used. So it's really a very substantial difference; some changes much as they possibly could and some don't change at all. Christian Keysers Yeah, it's a good — if we knew that, we would be very happy. I think the things that we did look at and didn't play major difference is actually the reaction of the demonstrator. So the first thing we figured perhaps you have demonstrators that really squeak a lot, and others, that don't give a lot of pain signals, and that could could explain the difference. Then we quantified the reaction of the demonstrators, we saw there were no major differences there. Where we did see really, big differences is in the reaction of the observer. So we saw that there is some observers for instance, when they realised that they had shocked the other one, they really stopped to do anything for a while, and took longer to go get their food and ate the food very quickly. So they really showed that they were disturbed by these shocks. And those were the ones that switched. And then were other rats that didn't seem to mind at all, they just kept eating their food as if nothing had happened. And those ones were the ones that didn't switch. So we feel that there's really just a striking difference in how much attention as well the agent paid to the harm signals of the other person. Valeria Gazzola But the causes are not known. The causes of these are not: could be different brain activity, it could be the relationship between the distress that the observer perceives from the other that stops them to do anything, or actually motivates the other. I mean, there are a lot of factors that could play a role. And we are trying to think of it: how to test and untangle this aspect apart. But it is not easy, but it is definitely a topic we are interested in. Christian Keysers Yeah, well we don't know that much. So from a kind of biological point of view, kind of it's only over the past, I would say, 10-20 years, that evolutionary biology is really paying a lot of attention to kind of and . Because if you read kind of traditional and evolutionary kind of books, they underline the selfishness of individuals. So the idea that you mainly need to survive yourself, and that other animals are really competitors. And then more slowly, people started to say, "well of course, you care about your kids, because they have a lot of your and therefore, that makes sense". And it's only quite recently that people start to really see that even if you are not really related to another animal, but kind of you help each other, your whole kind of herd or group ends up better-off. And the individual ends up being better-off. Because often, rats actually, or animals have a sense of mutualism. So if someone was kind to you at some point, you're more likely to be helped by that person in the future. So that helping others is a way to facilitate being helped by them in the future. And so, it still is a relatively young field of research that really acknowledges the fact that kind of in social animals, that live in groups. And individuals will really do things for others kind of and that this is beneficial for evolution. So it's really only starting to be explored. Christian Keysers Yeah, so what we know is that for instance, in monkeys, the cingulate seems to be necessary as well to, for instance, decide to work, to give juice to someone else. So, in the they have some paradigms where you see a symbol on the screen that means that if you now do something, there can be juice for yourself. And a different symbol if you work will give juice to another monkey, and a third symbol if you work that one, there'll be juice that's delivered in an empty glass. And what you see is that monkeys that really work hard for themselves, but they're also willing to work a little bit to give juice to another monkey. And that's more than they are willing to work if the juice is just gonna get wasted in as glass. And there as well, the ACC seems to be important to motivate the monkey to work for another monkey and that's the other example that we know. In mice, there is some evidence that if you disrupt the cingulate, then the mouse will no longer freeze when it see[s] another one getting shocked. But that's it. As far as I know, there's no other animal that has been explored. Christian Keysers Yes, of course! So, we ourselves, of course do not particularly like to have to give shocks to one animal. So the only reason we really do these experiments is that we feel that harm aversion is such an important phenomenon. Because in human society, when people lack harm aversion, they really cause a lot of damage to society. Both because they harm people, and they end up in prison and that has a huge cost; but as well for the fact that these individuals really erode trust and they harm in a way the interaction that all of us have with each other. Because we are always afraid that the person we have in front of us might be someone that will take advantage of you and harm you. So I think, in understanding how that works in the brain is really really important. And because of that, we feel that it's important to really understand how it works. And currently, working with animals is the only way that we can really modify brain activity and see if it changes behaviour. Christian Keysers Yes, we might do it in mice. So, of course, for other people, rats and mice are pretty much the same thing, but kind of there is a difference between them. And ultimately, we hope we can do this in humans. But for that, we need techniques in which we can modify brain activity in deeper brain regions. The insular lobe is highlighted in red. Valeria Gazzola You can do it once in a while in patients that already suffer from and normally the surgeons inserts electrodes to measure, to find out the source of epilepsy, and you can at times stimulate to see whether the region is essential in some of the normal behaviour. So at times, you can have access to these patients and ask them to participate in our task and we have a couple of collaborations, that we hope that will help us to tell something more about the role of the ACC and the in these type of tasks. And we are also currently trying to develop methods that are human-friendly, in the sense that they are not invasive, and to be used in active participants to try to disrupt the activity of these regions as well. Nowadays it's not easy to do because the TMS that [is] normally used to simulate the brain in humans, the , is not very well suited to reach deeper structure. Like the ACC goes really inside and it's a bit curved as well. You can reach with TMS, but it's not the cleanest way. So we are also trying to see whether we can use, for instance, stimulation to do it and test it directly in humans. Of course, it will also make use of data that comes from patients that show damages in the ACC. And it was a study from Ralph Adolphs in the past that did confirm that some of these regions like insula for instance, are important and essential for discriminating emotions for instance, so, you can also test in these patients: use our tasks and test these patients. So there are bit, some ways, to do it in humans, but it's quite small so we are investing, but the results take longer. And of course, when you use patients, you always have the problem of reorganisation of the brain structure that you are looking at, possibly, so. Valeria Gazzola Yeah, that's the question we are really interested in, and we don't know. And that's why we are interested in finding out whether a particular region like the ACC is necessary for the development, or for the behaviour to occur. Because once we know, we can confirm that in humans as well, what you could think are several ways. You can manipulate plasticity in that region, so, ask the participants to go do certain tasks where they learn something. And while you are modifying for instance, the {w|neuroplasticity|plasticity}} of the brain with current or magnetic simulation. Or you can think of targeted therapies, for instance like , when you project the activities of the areas where the participants doing a task and the participants can directly modify through visual feedback the activity of these areas coming up. So that if I now project on the screen the activity, real-time, the activity of the particular regions, I can tell you "okay, try find a way to modify that region". So you would come up with thinking of something, you know, it's just a random process, but you could come up with a task saying like, "Okay, if I think of how my friend is feeling now, I see that manipulated region". And then you can practice and practice to the point that it becomes automatic kind of things and that activity of that region now is modified based on your exercise and your activity. 350px|Representation of a neuron Valeria Gazzola Ah, yes there are a lot of plans and, roads, let's say. One of the plans we have is to really understand a little bit even more in details what is happening in the brain, at the cellular level and which are the inputs of these are involved, what type of neurons are involved and all these aspect[s], we are also interested in trying [to] understand the developmental curve, what is happening, for instance, if you are exposed to a very stressful life when you're young, what kind of consequence it has on harm aversion later in life. And we are interested in seeing how it is also transmitted through generations, like if you have these variabilities between individuals, with some individuals having higher propensity to be prosocial, or whether, you know, you select individuals that are mostly prosocial, would this selection be brought forward in the next generation. So we also want also to see how much is learned and how much is more genetically encoded. So all these aspects, we are trying to tackle with different experiment. And let's hope we — of course the other aspects are very early at this stage. Valeria Gazzola [laughs] You want them all? No, okay, so as I mentioned from the animal's point-of-view, we are doing, we want to do on the, a bit more of the physiology, and up the genetic to discover the role of the ACC and each single cell of the ACC, as I mentioning before. So, is it for instance, let's say, what type of cells is involved in mapping the other pain into my own pain. And where the information comes from, so what is the circuit that is involved, and how the other things change the information: this is one aspect of one of the experiments we're gonna run. Christian Keysers Yeah. Valeria Gazzola Yeah. Christian Keysers Yeah, so we basically are both of us department heads, so that means that we each have a a group of people we basically manage. And of course we really work very very closely together. So our two groups kind of together form what we call the Social Brain Lab which is basically investigating how our brain processes the emotions of others and reacts to them. Christian Keysers Okay. Very good. Christian Keysers No. Valeria Gazzola No, I think it was quite thorough. Yeah. Christian Keysers You are welcome. Valeria Gazzola Thank you. Christian Keysers Thank you. B-bye. Have a good day. Valeria Gazzola Bye.
 And one of the biggest limitation[s] in particular of the human research is that you can go so far in understanding the brain mechanism. Because we cannot, we don't have tools with the correct in terms of, for instance, understanding what a single cell does, in these processes. And that's why, for many years, we developed, we tried to develop a model in animals where all these techniques are more available that would allow us to investigate similar processes to those in humans. So processes basically that are involved in perceiving the other['s] distress also in animals. And, that's basically the main reasons why we move towards this research and then we started from coming up with the experiment in which we simply measure the distress of an animal in reaction to the distress of another. Which what we call , for instance. And we made it more and more complex in order to reach slowly a degree of complexity that can be comparable to processes in humans. Emotional contagions: you can compare it, for instance, that the level of humans when you are in a crowd, or when you are baby, that as a crowd you are contaged by the emotions are around. And, but then, we wanted to go a little bit of a step forward: okay now I am contaged, what does it mean to be contaged? Can I use this information to, for instance, avoid hurting other people. And that's how we came about, more or less, so. I mean, one other motivation we have is that in the past we've also been interested in people that seemed to lack the aversion to harming others. I mean, with that , sometimes they're called as well. And so, a couple of years ago, we made this study in which we identified psychopathic criminals in high-security institutions. We brought them to the lab, and we scanned their brains while they witnessed the pain of other individuals. And what we saw was that if they don't have any particular reason to empathise with others, they really have reduced activity in their own pain regions while they witness the pain of others. So, of course, that's interesting and it may suggest that perhaps, the reason why if they can make some money, for instance, they don't mind to harm other people is because they don't activate their own pain much while they witness the pain of others. The problem like Valeria was saying, is that in humans we can see that the brain region is active while you see the pain of others, but you can't easily change the activity in the brain region to see whether that would change the way that the psychopathic criminal would act. I mean, ideally, what we'd like to do is increase the activity of these pain regions in psychopaths and see that now suddenly they would really care to not harm others anymore. Although it's not yet, it's also not yet very clear that we need activity of these regions to avoid the harm, right? At that point, that's how we came about to run the experiment, because the first question was: is it really necessary. The activity of these regions. So, you start with okay, you want to manipulate these regions to reduce psychopathy. But the field could not even prove that this activity was necessary to reduce harm. And so, that was really one of the strong motivation behind the -experiment, because rats have a very similar to what humans have. It is really a core part of the brain that is involved in feeling your own pain. So, if you don't have a cingluate cortex and you hurt yourself, you still feel that you've cut yourself, but you don't mind. And so you are not going to do a lot to avoid being cut again. So this region exists also in the rats and it is a relatively deep brain region. So therefore, we turn to the rats, and figure that in the rat, we can change the activity in the brain region and see whether that would then change how much the rat would be aversed to harming other rats or not. Ah. So very briefly, what we do is we give a rat a choice between two that the rat can press. Both of these levers will provide the rat with one little pellet of sugar which they really like. So, they are happy to press these levers to get the reward. In the main experiment, what we do is that one of the two levers is a little bit more difficult to press than the other one. So most of the rats start to prefer the easy lever to press. And that also what we see is that there is variability in the responses of the observers, of the rats that make the choice between the levers. And these variabilities also present in humans: when you do tasks that are rather similar, you see that not everyone acts socially, preferring the social lever. But you also have individuals that would go for the reward more often. So this is also another similarity between the animal world and the human world. And on top of that, I think that the other things we've learned from that experiment is that, the decision is a function, basically. Because, we have a condition for instance where we increase the reward for the animal, for the observer. And when you increase the reward, they are less willing to switch to the other lever. So that also another similarity with human world and shows that maybe that it is a bit of complex, a tipping point, and it is like a function that really weighs the cost and the benefit of that particular context. Because when Valeria says that we increase the reward, we increase the difference between the two levers. So in the main experiment, the only difference is that one is harder to press than the other, but they both give one pellet. Then in two other experiments, what we did was that now that two levers are equally hard to press; but one of them gives you two pellets, the other one just one pellet. And then rats are still willing to switch away from the two-pellet to the one-pellet to save the other one from pain. But when we made it three pellets versus one pellet, then, none of the rats were willing to give up the three pellets to prevent the shock to the other person. Yeah, and then, the last, I think, result I kind of like in a way is that, as in many human studies, also in the animal world we did not find significant difference between . So both, the female and the males acts in a similar way. And in this type of , is also often the case in humans. That's true. So that's also interesting. Well, in a way, I would say, to really show — I mean, I always believed it that animal performed these choices too. And that they do it to a certain extent to at least care or at least perceive what goes on in the other, right. It can't maybe specifically speaking of voluntarily care about others, but at least they perceive what goes on and I think this is something that the study shows pretty well that they do perceive, and that based on these perception[s], they make a decision. And I think it is a complex — I mean, to me it was in a way trivial, because I always believed that, you know, if you are a "", whether you are a rat, or a person, we are still animals, right? They must develop behaviours that take advantage, or that they adapt to the fact that it's a social environment that involves more individuals. But [...] the field keeps discussing, right? Whether animals also have emotions, whether animals also can take this sort of decision. And to see, slowly that evidence accumulates in favour of processes similar to what we call or . You know, similar: not exactly the same, but similar processes also are present in animals, to me is sort of reassuring also. And I think it allows us to learn from how evolution evolved as well. Maybe apart from knowing exactly how it works, but we start having some models where we can think, oh, evolutionary: maybe this process was already there, and then it developed further, more complex for the humans, but still is there to a certain extent. Yeah, and I think the fact that it's really the same brain region as well that seems to be involved kind of gives us some confidence that prosociality in caring about others is really deeply ingrained in our biology. Yeah so in many ways it's a relatively simple study. So we didn't need to use anything very high-tech in this case. Because to alter brain activity, we use the injection of muscimol, which is a GABAA blocker. It is a very simple pharmacological agent. So it's really technologically a very simple study. Nothing fancy. Oh, we brainstormed about it. We started, as I said, we did a lot of research on, since our early ages, let's say in all our scientific career is based on embodiment, and activities in these areas and slowly we kept wondering more and more, on the role of these areas. And then, the way it goes, we normally speak about a topic and include other people and keep brainstorming, and I think, that's how it goes. At least for us. And in humans, we are also developing similar paradigms, so it just came one plus one equal to two. [laughs] So, Julen Hernandez-Lallement, the first author of the study, was the one that basically made the vast majority of the experiments. And he was helped by the other two: [Augustine Triumph] Attah: who did part of the other animal groups, and then Cindy [Pinhal] and Efe [Soyman] helped to analyse the data. And basically Valeria and me have been kind of help designed this study initially and helped to analyse the data and wind up the study. So we kind of lead the lab, but it's the other people that really manipulate the rats and do all the hard work. Well, it was long. Yeah, I would say, four years? Yeah, because the behavioural part is the longest one. Because you need to understand the best way to teach the animal, first to manipulate the lever, then to manipulate two levers, and then, before the lever, there is also another action that they need to do; and everything needs to be done within a certain time. So you need to let the animal the time to learn and for you that you run the experiment, you need to learn what's the best way, what's the best way to teach them to do the task? Yeah, I think that Valeria just said what's really important in these studies is to understand what kind of situation you can create for the rats that they are really understand and which they can in a way, show you what they're capable of. So, kind of fine-tuning all the elements of the task, kind of how long do you let the rats develop the preference for one lever. Because if you give them too much time to get too used to one lever, they are not so willing to switch anymore. So the, most of the time is really involved in trying different versions of the paradigm, understanding what the rats really understand, and then analysing all the behaviour. Yeah exactly; so the three kind of conditions we looked at was the hard vs the easy lever, and then seeing whether they switch to the harder lever when, to prevent the shock to the other one. And we tried the two pellets versus one pellet and, then the three pellets versus one pellet. And then finally, to test really whether the cingulate is necessary for that, we compare the condition in which we inhibit the activity in the cingulate using muscimol against the condition where we just inject salt water into the same brain region which doesn't have any effect. Correct. Yeah. Yeah, more or less. I don't know the date. I think it was really again the establishing the behaviour. And in particular because we, at first — so the paradigm itself, we took it from a previous study, that was run years ago, do you remember? Yeah, in '69, so that's kind of 50 years ago. Fifty years years ago, so the idea of the two levers. And so and at that time, the articles' level of detail were not so many. The articles were not giving as much information as we now have to write down. And so we had to guess some parameters over there. And, the fact that the rats would prefer the easy lever was also not so straightforward. So we thought "oh, they are gonna prefer the easy lever, and that's it." But at the end, you know, some rats, some animals develop a preference on the side; like they prefer their right lever, or the left lever, or others, they prefer the heavy lever. Right? And that was also a bit puzzling at first. We said, okay, so what do we do? We find that a couple of rats prefer the hard lever, is that meaningful or not? So we have to discuss a lot, how to adapt the paradigm to make it work, and this is also why we came about in using this switching index, because for our purposes, at the end, as long as the rat is able to switch a preference, right, whatever preference is developed is irrelevant, right? And before the beginning of this study, we were more thinking: oh, they have to develop the preference for the easy lever. And that's what is bringing us a bit out of the main purpose. And we realised: okay, but, what preference they develop at the end, it doesn't matter as long as we have a preference that we can show they can change because of the shock. So, I think this was one of the longest part[s], I would say. Yes. Yeah, I would say yes. For once. [laughs] Yeah, for once, yeah. I mean, there was some evidence around and previous work was already bringing us towards that direction. And, yeah, I mean we could not anticipate the proportion of animals that they would switch. But, we surely were expecting the rats to switch. We were hoping for the cingulate to be necessary, so. . We were happy. Yeah. I think it's written in the paper, but my guess would be something like, I'd say, 70. Yeah, so that's interesting, in this study we really find that there is a lot of variability between the rats. So like I was mentioning, we have about 40\% that were ready to switch when they witnessed other one getting shocked. And about 60\% that do not switch, so as a group, we really see that they do switch. But then only a proportion of individuals really show that sensitivity, and others do not. Or did you mean how close they were in terms of familiarity? So they're not genetically related. They're not brothers or sisters. And what we have is we have, we made one of the experiments with rats that didn't meet each other before, and it [the experiment] works. And we had other cases in which they were living in the same home cage, and so they [would know] each other very well. And the effect was not stronger. So, amongst our rats, we really noticed that they care even about the fate of a stranger. Correct. Well, mates as in cage mates. Yeah, there was no sign of a difference. We did look at it and it was not the perfect comparison because the two groups were slightly different in the paradigm as well. But, it didn't look like there was a big difference. So we're actually going to test that very soon. And it is up for grabs. But in the past we've looked at whether a rat shows more freezing when it sees another one get a shock. Which is a form of emotional contagion. And there we compare a kind of rats from different strain,s even. So one of the albino rats against a rat, versus, that had never seen each other before, or ever seen a member of that strain, the one end. And on the other end, really the rats have spent five weeks together and knew each other very well. And we didn't see any difference there. So I think the most robust findings is really that they care even about what happens to a stranger. Yeah, we are testing it again now, in the future. Exactly, specifically whether a mother would do more for her own kids than for... Kids of somebody else. Yeah. Correct. Right. Yeah. Yeah, what you do is you place a . So you put the animal in a stereotact which is a system where you can go to a very specific position in the brain. We place a cannula, which is like an injection needle just where we know the cingulate to be. Then you let the animal recover with the cannula cemented in place. Then, on the day of the experiment, you basically inject a little bit of muscimol, which is an agent that blocks . Then by doing that, you can reduce the activity of the cingulate for about one hour. And during that hour, you can make the experiment. And then the control group is the same thing, but instead of injecting the muscimol, you inject some salt water. Nothing really. Because you inject a very small quantity, that should only spread about 1 mm in the brain. So it really just affects the region that you — and you don't see any change in behaviour. So we also tested, for instance, whether they move more or less, whether they press the levers quicker or slower, and there was no difference. The only difference is that they didn't learn to not press the one [lever] that shocks the other. And for instance, when you inject with muscimol, in case you see doing a normal paradigm. So whether you are still shocked to the sound, and then you present the sound. In that case, the rat behaves normally. And it still freezes. So, it does suggest that the muscimol effect is not impairing the normal behaviour, but specifically impairs the social behaviour of feeling... ...of feeling for others. Exactly. That was also the interesting part of the ACC that we believe to contribute really, to including the other [animal] into your decision-making. Because for a normal fear conditioning paradigm, you don't need to go through the ACC. They are made to be self-sufficient. But, if you do a fear conditioning basically that involves another individual instead of sound, like of pain, then you do need to go to the contribution of the ACC. So there seems to be two slightly complementary pathway for fear-detection. Yeah, that we didn't measure. So, in that study, the muscimol animals were only in the behaviour with the ACC deactivated and the other ones were only with their ACC intact. So, but this is something that we plan to test in the future as well. To really see whether the ACC is necessary to acquire the aversion versus to maintain it. We don't know, but I think there is no reason to assume that when the muscimol wears off, they would not become normal again, right? So that's most likely. What we don't know is whether if we had for instance, because there's three days of experiment, right, where we develop the aversion. And what we didn't try is for instance, give muscimol on Day 1 and Day 2 and then stop it on Day 3, and see whether on Day 3, they would then suddenly stop using the lever that gives shocks on the other days. So that we don't know whether it's necessary for learning or for recall; we just know that if you don't have it at all, you don't learn at all. It's a good question. I would need to look in the paper. If you have the paper there, perhaps you can see it. But I can have a quick look to see if I have the paper somewhere. Yeah, so if you look at the Figure 4c, you can see the behaviour of each individual animal. And what you will notice is that there are some animals like some of the red animals which are the muscimol group that did switch, but the majority didn't switch. So I see there is one and only one animal in the muscimol group that really did switch. But what happens is that when you make those experiments, you never know for sure that when you inject the muscimol, that it has the desired effect. Because of course you have a cannula that's in the brain, and the cannula could become clogged also. So it can be that that one animal switched because the muscimol wasn't effective for some reason. No. No. Could be nice, but we didn't. Exactly. Yeah, and in particular, there is a drug called Ecstacy that you may have heard of. So, some people reported that Ecstasy for instance, increases the feeling of empathy amongst humans. And I think that there is somewhere a study suggesting that if you give that to animal, there is also an increase in the sensitivity to what happens in another animal. So we didn't test that. Yeah, no so we didn't do that. That is a very old experiment from . I think in the 50s, he did that experiment kind of with rats and with monkeys. And he sees that the rats will use the lever less. So even if you just have one lever, they'll use it less if it causes pain to others. But in monkeys, that was a very strong affect. Some of the monkeys really would rather not eat anything than to shock another monkey. So it's an interesting experiment, but we haven't done it. Yeah, what you see here is the two lines: the dark green and the light green that represents the males and the females. For harm aversion, for that you should look at how much it changes from the baseline through the three shock sessions. And the change is exactly the same in males and females. But the difference between the two curves that you see is the fact that the females started off with the higher preference than the males did. But the reduction in preference is same across groups. Between which rats? Rats, you mean, in general in the experiment? Figure 3b of the study. In general, like the rats, which were on the extremes, how extreme were they on the case-by-case basis? Well, that you can see, if you look at Figure 3b in particular, you can see that some examples on the top of 3b of rats that had started off with a very strong preference for that were around 100 on the baseline session. And it stayed at 100. So they absolutely didn't care at all. And in the other case, if you look at the bottom panel, you have some animals that initially had a preference around kind of 80\%. And by Day 3, they really go down to 0\% of the shock lever used. So it's really a very substantial difference; some changes much as they possibly could and some don't change at all. Yeah, it's a good — if we knew that, we would be very happy. I think the things that we did look at and didn't play major difference is actually the reaction of the demonstrator. So the first thing we figured perhaps you have demonstrators that really squeak a lot, and others, that don't give a lot of pain signals, and that could could explain the difference. Then we quantified the reaction of the demonstrators, we saw there were no major differences there. Where we did see really, big differences is in the reaction of the observer. So we saw that there is some observers for instance, when they realised that they had shocked the other one, they really stopped to do anything for a while, and took longer to go get their food and ate the food very quickly. So they really showed that they were disturbed by these shocks. And those were the ones that switched. And then were other rats that didn't seem to mind at all, they just kept eating their food as if nothing had happened. And those ones were the ones that didn't switch. So we feel that there's really just a striking difference in how much attention as well the agent paid to the harm signals of the other person. But the causes are not known. The causes of these are not: could be different brain activity, it could be the relationship between the distress that the observer perceives from the other that stops them to do anything, or actually motivates the other. I mean, there are a lot of factors that could play a role. And we are trying to think of it: how to test and untangle this aspect apart. But it is not easy, but it is definitely a topic we are interested in. Yeah, well we don't know that much. So from a kind of biological point of view, kind of it's only over the past, I would say, 10-20 years, that evolutionary biology is really paying a lot of attention to kind of and . Because if you read kind of traditional and evolutionary kind of books, they underline the selfishness of individuals. So the idea that you mainly need to survive yourself, and that other animals are really competitors. And then more slowly, people started to say, "well of course, you care about your kids, because they have a lot of your and therefore, that makes sense". And it's only quite recently that people start to really see that even if you are not really related to another animal, but kind of you help each other, your whole kind of herd or group ends up better-off. And the individual ends up being better-off. Because often, rats actually, or animals have a sense of mutualism. So if someone was kind to you at some point, you're more likely to be helped by that person in the future. So that helping others is a way to facilitate being helped by them in the future. And so, it still is a relatively young field of research that really acknowledges the fact that kind of in social animals, that live in groups. And individuals will really do things for others kind of and that this is beneficial for evolution. So it's really only starting to be explored. Yeah, so what we know is that for instance, in monkeys, the cingulate seems to be necessary as well to, for instance, decide to work, to give juice to someone else. So, in the they have some paradigms where you see a symbol on the screen that means that if you now do something, there can be juice for yourself. And a different symbol if you work will give juice to another monkey, and a third symbol if you work that one, there'll be juice that's delivered in an empty glass. And what you see is that monkeys that really work hard for themselves, but they're also willing to work a little bit to give juice to another monkey. And that's more than they are willing to work if the juice is just gonna get wasted in as glass. And there as well, the ACC seems to be important to motivate the monkey to work for another monkey and that's the other example that we know. In mice, there is some evidence that if you disrupt the cingulate, then the mouse will no longer freeze when it see[s] another one getting shocked. But that's it. As far as I know, there's no other animal that has been explored. Yes, of course! So, we ourselves, of course do not particularly like to have to give shocks to one animal. So the only reason we really do these experiments is that we feel that harm aversion is such an important phenomenon. Because in human society, when people lack harm aversion, they really cause a lot of damage to society. Both because they harm people, and they end up in prison and that has a huge cost; but as well for the fact that these individuals really erode trust and they harm in a way the interaction that all of us have with each other. Because we are always afraid that the person we have in front of us might be someone that will take advantage of you and harm you. So I think, in understanding how that works in the brain is really really important. And because of that, we feel that it's important to really understand how it works. And currently, working with animals is the only way that we can really modify brain activity and see if it changes behaviour. Yes, we might do it in mice. So, of course, for other people, rats and mice are pretty much the same thing, but kind of there is a difference between them. And ultimately, we hope we can do this in humans. But for that, we need techniques in which we can modify brain activity in deeper brain regions. The insular lobe is highlighted in red. You can do it once in a while in patients that already suffer from and normally the surgeons inserts electrodes to measure, to find out the source of epilepsy, and you can at times stimulate to see whether the region is essential in some of the normal behaviour. So at times, you can have access to these patients and ask them to participate in our task and we have a couple of collaborations, that we hope that will help us to tell something more about the role of the ACC and the in these type of tasks. And we are also currently trying to develop methods that are human-friendly, in the sense that they are not invasive, and to be used in active participants to try to disrupt the activity of these regions as well. Nowadays it's not easy to do because the TMS that [is] normally used to simulate the brain in humans, the , is not very well suited to reach deeper structure. Like the ACC goes really inside and it's a bit curved as well. You can reach with TMS, but it's not the cleanest way. So we are also trying to see whether we can use, for instance, stimulation to do it and test it directly in humans. Of course, it will also make use of data that comes from patients that show damages in the ACC. And it was a study from Ralph Adolphs in the past that did confirm that some of these regions like insula for instance, are important and essential for discriminating emotions for instance, so, you can also test in these patients: use our tasks and test these patients. So there are bit, some ways, to do it in humans, but it's quite small so we are investing, but the results take longer. And of course, when you use patients, you always have the problem of reorganisation of the brain structure that you are looking at, possibly, so. Yeah, that's the question we are really interested in, and we don't know. And that's why we are interested in finding out whether a particular region like the ACC is necessary for the development, or for the behaviour to occur. Because once we know, we can confirm that in humans as well, what you could think are several ways. You can manipulate plasticity in that region, so, ask the participants to go do certain tasks where they learn something. And while you are modifying for instance, the {w|neuroplasticity|plasticity}} of the brain with current or magnetic simulation. Or you can think of targeted therapies, for instance like , when you project the activities of the areas where the participants doing a task and the participants can directly modify through visual feedback the activity of these areas coming up. So that if I now project on the screen the activity, real-time, the activity of the particular regions, I can tell you "okay, try find a way to modify that region". So you would come up with thinking of something, you know, it's just a random process, but you could come up with a task saying like, "Okay, if I think of how my friend is feeling now, I see that manipulated region". And then you can practice and practice to the point that it becomes automatic kind of things and that activity of that region now is modified based on your exercise and your activity. 350px|Representation of a neuron Ah, yes there are a lot of plans and, roads, let's say. One of the plans we have is to really understand a little bit even more in details what is happening in the brain, at the cellular level and which are the inputs of these are involved, what type of neurons are involved and all these aspect[s], we are also interested in trying [to] understand the developmental curve, what is happening, for instance, if you are exposed to a very stressful life when you're young, what kind of consequence it has on harm aversion later in life. And we are interested in seeing how it is also transmitted through generations, like if you have these variabilities between individuals, with some individuals having higher propensity to be prosocial, or whether, you know, you select individuals that are mostly prosocial, would this selection be brought forward in the next generation. So we also want also to see how much is learned and how much is more genetically encoded. So all these aspects, we are trying to tackle with different experiment. And let's hope we — of course the other aspects are very early at this stage. [laughs] You want them all? No, okay, so as I mentioned from the animal's point-of-view, we are doing, we want to do on the, a bit more of the physiology, and up the genetic to discover the role of the ACC and each single cell of the ACC, as I mentioning before. So, is it for instance, let's say, what type of cells is involved in mapping the other pain into my own pain. And where the information comes from, so what is the circuit that is involved, and how the other things change the information: this is one aspect of one of the experiments we're gonna run. Yeah. Yeah. Yeah, so we basically are both of us department heads, so that means that we each have a a group of people we basically manage. And of course we really work very very closely together. So our two groups kind of together form what we call the Social Brain Lab which is basically investigating how our brain processes the emotions of others and reacts to them. Okay. Very good. No. No, I think it was quite thorough. Yeah. You are welcome. Thank you. Thank you. B-bye. Have a good day. Bye.
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