Sugar on the brain, HIV, and science sex bias
This month on the eLife Podcast we look at how sugar takes away the pleasure of consuming and makes you eat more, we find out what loneliness does to the brain, uncover new insights into how HIV infects females, and explore sex bias in biomedical research...
In this episode
00:36 - Pavlovian training: can pea plants learn?
Pavlovian training: can pea plants learn?
Kasey Markel, UC Davis
Pavlov's dogs learn to salivate the sound of a bell, if the bell was presented alongside their dinner enough times first. We now call this Pavlovian conditioning in his honour, and we know that lots of animals, us humans included can learn like this. In recent years, plant scientist Monica Gagliano has been talking about what she dubs plant cognitive ecology. She's presented some surprising results that suggest that pea plants can be trained a bit like Pavlov's dogs. Chris Smith spoke with UC Davis scientist Kasey Markel, who decided he'd better investigate, and set up an experiment to replicate the results...
Kasey - Me and a fellow researcher were having one of those late night discussions about the nature of consciousness. And we decided that learning was maybe a necessary, but not sufficient condition for consciousness. And so we were wondering which organisms in the kingdom of life had the capacity for learning. And we saw a recently published study that showed learning was reported in plants. And it was very recent and as yet unreplicated, and therefore seemed ripe for replication and expansion efforts.
Chris - And in that particular study, what had they done? And what had they found?
Kasey - What they had done was to adapt the classic Pavlov's dogs experiment for pea plants. So in Pavlov's dogs, you can ring a bell always before the arrival of food. And eventually the dogs will start salivating as soon as they hear a bell. And in this experiment, what they had done was present blue light right before a fan, such that the plants would associate the blue light with the fan.
Chris - So in this instance by pairing the fan, which is one kind of stimulus, with light, something the plants are sensitive to and are always going to grow towards, the argument is that you can train the plants to recognise that there's a fan and it will either be where the light is or opposite to where the light is, depending upon how you've done the experiment and the plants will steer their growth accordingly.
Kasey - Precisely. When these experiments have been done in animals, we tend to forget things after we've been taught them. And so what I was interested in doing was discovering how long the plants could remember this training that had been done in the experiment. We know from the original experiment, they can remember for at least one day, but I suspected it might be longer than that.
Chris - So what did you do?
Kasey - The plan was to run the experiment and design it such that I could test them on successively further out days. So one day after the training, two days, three days, and see if we could get what's called a forgetting curve where there's learning, and then over time, the learning becomes progressively diminished.
Chris - Can you just describe your experiment? What it looked like, what you actually did?
Kasey - Yeah. So my experiment pretty directly followed the protocol of the original study. And these plants are in a capital Y shaped maze. And on the top of each arm, there's a blue light and a fan. And for a period of three days, blue light and the fan will be activated either on the same arm of the maze or on opposite arms of the maze to train the plants. And then on the fourth day for the testing phase, the blue lights are all disactivated and the fans are alone turned on and we try to use the fan to manipulate the direction of growth of the plant based on what it's been trained.
Chris - Therefore one would anticipate if the plants can learn, the fan tells the plant where the light is
Kasey - So that was our expectation. And the plan was to start with that and then test from further and further days out to see how long it took the plants to forget the information. But unfortunately we were unable to actually replicate that feat of training the plants based on the association with fan and light.
Chris- It just didn't work?
Kasey - Yeah. It just didn't work.
Chris - Do you think that's because you did something wrong, or do you think that actually the original observation was just by chance?
Kasey - There are a couple of possibilities there, obviously it's always possible that we did something wrong. I spent a lot of time reaching out to the original authors, but unfortunately they didn't get back to me to kind of help troubleshoot. There were a few things that weren't precisely clear in terms of exactly which equipment they had used or which source they had used for the supply of seeds, things like that. It's also possible, however, that the original study for one reason or another can't be replicated.
Chris - What did you do next then?
Kasey - Well, unfortunately at this point there wasn't all that much I could do. I reached out to a couple of fellow plant scientists and plant physiologists for kind of help with troubleshooting. I reached out more times to the original scientists, but kept replicating the experiments. At some point I'd replicated them kind of as much as I could and run out of time. And so I wrote up my results and started trying to publish them.
Chris - And that's the interesting thing here isn't it? Because you say started trying to publish them, what you were trying to publish was a negative result, not just a negative result - a negative result, refuting someone else's positive result. I don't suppose that was an easy path to take, was it?
Kasey - It's been a little bit difficult to publish. I originally reached out to the journal that had published the initial report and spent a long time, over a year actually, in the peer review process there, but ultimately it was rejected. And so I started sending it elsewhere and I'm happy to say it's been accepted at eLife
Chris - Out of interest, when you say it was rejected, did you get some reasoning for why they found your results less compelling than the original observation?
Kasey - Strangely that actually wasn't the claim. All four reviewers ended up agreeing that my experimental design was more rigorous than the original claim. However, because of the lack of the novelty in the results, two reviewers decided it was not worthy of publishing. And two reviewers were strongly in favor of publishing, ultimately that ended up with a rejection.
Chris - And what does that say to you about the practice and process and therefore the reliability more broadly of the results that we see in the scientific corpus?
Kasey - It certainly has been an education to me that it's much safer to trust results that have been replicated in at least a couple of different labs, rather than just looking at one off studies from a single laboratory.
Chris - Well, you know what they say, give peas a chance, although after all those efforts, perhaps Kasey Markel might disagree.
06:39 - Sugar desensitises food pleasure
Sugar desensitises food pleasure
Monica Dus, University of Michigan
Have you got a sweet tooth? If so, the chances are, you've probably desensitized your taste buds, meaning that you're going to have to eat more tasty things more often to get the pleasantly full feeling that accompanies a decent meal. Talking with Chris Smith is Monica Dus from the University of Michigan, with why...
Monica - My lab is interested in trying to understand how some foods change our behaviour, specifically our feeding behaviour. In other words, why it's hard to stick to serving size when it comes to cookies, but it's quite easy when it comes to kale or broccoli! Why do we eat more of some foods over others?
Chris - And why do we? I'm dying to know!
Monica - We look specifically at how the food environment, the amount of sugar in the diet, rewires our brain, our taste system and our reward system to dull the sense of satiety and make us eat more.
Chris - You're saying to me then that if I were to add extra sugar to my tea everyday, I don't take sugar by the way, then what I would be doing is making my cup of tea and other foods I eat potentially less rewarding to me. So I would want to eat more of them to get the same feel good rush.
Monica - That's correct. So we know that the amount of salt and sugar, in this case, in the food, changes the pleasure we get from the food and also dulls the taste or the sweetness we can perceive. The more salt, sugar and fat we have in our diet, the less likely we are to taste those with the same intensity, as of somebody who eats less sugar, salt, or fat.
Chris - There are lots of steps in the chain of taste though aren't there? There's what goes on on your tongue. And then there's the nerve that carries that information centrally into the brain. And then there are the whole series of circuits that control how full you feel and whether you want to eat more. So where in that chain then does this dulling effect occur?
Monica - That's a wonderful question because feeding it's so complex, like you said. It starts with the mouth and it progresses through different brain systems. In a previous study, we found that when we gave animals extra sugar, their ability to taste sweetness was decreased in the mouth. And so in this study, we wanted to know if that decrease is what caused the decrease in pleasure we know animals from flies to humans get when they have a lot of sugar in the diet.
Chris - And how did you do that then?
Monica - In this study, we opened a tiny window in the fly head and then looked at the activity of the neurocircuits that encode for the sweetness, or the pleasure that we get from sweetness. And then we touched a fly mouth with a little bit of sugar and then looked for the activity of these neurons in real time. And we did that in flies that had a healthy diet and in flies that had a high sugar diet. And what we found in the high sugar diet, these neurons did not fire as much to sweetness. And so the pleasure of the flies get is much less.
Chris - Now, is that because the taste sensors having inputs to that bit of the brain are now less sensitive, or is it because the circuits downstream are now detuned, they're less sensitive to sweet inputs.
Monica - It's the first thing you said because the taste cells in the mouth are dulled they don't function as well, the signal that goes through the mouth to the brain, as you said it before, is not as strong. And so that's why these pleasure neurons are not firing as much and telling the brain that the organism has had enough pleasure or reward from the sugar it's experienced.
Chris - And do you have any idea as to how the inputs get dulled by super supply of sweet things?
Monica - Actually, we do. That was part of the study we did last year and it was quite interesting. It's a metabolite of sugar that caused the taste cells to be dulled or decreased. The sugar molecule itself changes the physiology of the taste buds.
Chris - Therefore, if what we're seeing is a reduction in the input from the sweet detectors in the mouth parts, this then produces a smaller signal in the parts of the brain that process satiety. So therefore you feel less happy. So that means you then demand more sugar input to get the same pleasure centre activity that you would have had originally in the brain. Is that why the intake is effectively escalated?
Monica - That's certainly one interpretation of it, but some people think that on top of being pleasure, this sweetness is also used as a cue to predict how much we eat in order to get full. And so if now we cannot taste the same cue as we did before, right? Because it's more dull. It's not as intense now. We essentially make a bad prediction about how filling that food will be.
Chris - And if the satiety does lie downstream of a reduction in the activity of these particular groups of nerve cells in the brain that you've been studying, if you increase artificially the activity of those nerve cells, does that reverse the phenomenon? So in other words, in a fly, that's very habituated, very used to eating very sweet things that would normally overeat. If you boost the brain activity, does that put it back to normal?
Monica - That's also correct. And that's what we showed in the study. In the study, we use a light to turn on these particular cells, even in flies that had a high sugar diet, and the flies now ate normally and were sated. What we think is happening is the cells are very likely controlling the eating rate. And during the course of the meal episode, we start eating really fast because we're hungry. But then towards the middle, we have to slow down. We can't wait for our stomach to be full, that would take a lot longer. And so the sort of cues we get during the meal are very important to start fast and slow down. And we think that the dulling sweetness prevents the feeding rate for decreasing towards the end of the meal and so you end up overeating,
Chris - Is this reversible? I should think there's probably lots of people now panicking thinking I had ice cream with my dinner, and I've got quite used to that, I quite like it, am I therefore destined to overeat for the rest of my life? Can you wind the clock back?
Monica - In flies we find that this process is partially reversible, but not entirely reversible. In humans certainly there have been studies where people decrease the amount of content of sugar in their diet, and what they found is that their sensitivity to sweetness increases. So I think the process is very likely reversible, how much we don't know and that's what we're going to study next.
14:03 - How social isolation changes the brain
How social isolation changes the brain
Elena Dreosti, University College London
During the coronavirus pandemic, we've mostly had to isolate ourselves as much as possible. But what effect could this be having on our brains? Researchers at University College London have been trying to answer questions like this using teenage zebrafish. The advantage of zebrafish is that they're transparent, so you can see what is changing in the brain, as Eva Higginbotham heard from Elena Dreosti...
Elena - So zebrafish are the fish that we use in the lab, and they're very tiny fish. And I'm sure that everyone has seen them because they are usually bought as pets and they are kept in aquariums. Like many fish they're very social. So they all swim together and they like to stay together. And they establish a lot of different hierarchies within their own groups. So what we are interested in our lab is basically to understand what happens when we're deprived of other humans, and in fish clearly deprived of other fish. As you know, in a normal population, we have people that are very shy and people that are very easygoing and bold. And that's exactly what we found in a normal zebrafish or fish population. And usually what we have is that 10% of the fish are very shy and very aversive to the other fish.
Eva - How do you know if a fish is aversive or social?
Elena - Yeah, what we do is very simple. So we have a simple arena where a fish, they can choose between two different sides of this arena. They can either stay near other fish and they see this fish through a glass slide, or they can decide to be on the opposite side of this arena, where there are no other social cues. And depending on how much time they spent on one side or the other, we divide them into very social fish or a fish that are aversive. And what we did is basically we isolate the fish for 24-48 hours, so a very short period of time, but also for a much longer period of time. And then what we did, we took the brain of this fish, and then we put it under a microscope and we imaged the whole brain and we could see at the level of single cells. And so we have a proper, very high resolution. And at that point we could perfectly understand what are the areas that were different between a fish there were either social or non-social, and then the differences between the fish that were isolated.
Eva - And what did you find?
Elena - So what we found is that when we isolate the fish, what happens is that this behaviour of being aversive increases and there is a much larger population of fish that become aversive to other fish. So the beauty of using zebrafish is that they are transparent, fully transparent, and we can see through their brain. And then we can actually record the activity of each single cell, each single neuron, of the brain with a very, very high resolution. What we saw is that some areas that are either very specific and involved in social interaction were very different compared to aversive fish and social fish. And then there were also areas that were involved in the reward. For instance, one of them was the hypothalamus and these areas were also very different compared to isolated fish versus normal fish.
Eva - In what ways were they different?
Elena - In the case of the normal population, the hypothalamic area is either very activated in case of prosocial fish, whereas the loner fish are not, they don't have this increase of activity. And this is probably because these fish, they do not have this experience of reward from the presence of other fish. But what we did see in the isolated fish in this case is that the areas that were mostly activated were areas associated with stress and anxiety, and this was not the case in a normally raised loner fish that are normally aversive.
Eva - I see. So the naturally shy ones don't have the decrease in activity in the hypothalamus, but the isolated fish that have been sort of made to become shy, they did see the change.
Elena - Exactly. So what happened is basically that if you isolate the fish, you have an increase in activity in areas that are activated with anxiety and fear. And this is not the case in fish that are normally aversive to the presence of other fish.
Eva - Did you find that they could recover from being isolated?
Elena - Yes. What we saw is basically that if we treat the fish with a drug that reduces anxiety, then the fish, they do recover almost fully their social behaviour. The drug that was used is called buspirone and it's a drug that is used in humans to reduce anxiety. What it does it basically increases the level of serotonin and that's the way that in humans it acts to reduce the anxiety.
Eva - This study seems very appropriate considering the coronavirus lockdown times that we've all been living through for the last few months. Do you think that there's anything we can learn from this study as to how humans react to social situations after they've been sort of deprived of social contact for awhile?
Elena - Yes. I think definitely. Because what we saw is something that we thought it was at the beginning counter intuitive. In fact, if we put fish in isolation, we would expect that they were more likely to be willing to be near other fish. But instead what we see is that there is an increase of anxiety and fear in response to social cues. And what I think could be the take home message is that, especially right now that we all are experiencing this lockdown, is probably we're going to be a bit more anxious when starting to return to our normal social lives. But I think what we should do, and what we learned from zebrafish, is that we should just start to go out and start to meet other people and trying to get over this anxiety, this first level of very stressful response to the presence of others.
Sex bias in science
Nicole Woitowich, Northwestern University
A decade ago, a landmark study analyzed a slew of scientific publications, and it showed a bias in the sex of the human or animal subjects that were considered by those research papers. So 10 years on, have things got better? Chris Smith heard how Niki Woitowich took it upon herself to reroll that original study and find out...
Niki - About 10 years ago, this study came out that looked at sex bias in biomedical research. And what this study found was that overwhelmingly biomedical researchers tend to use males in their research. And I thought to myself, you know, this is coming up on the 10 year anniversary of when this data was collected originally. I just wanted to know simply had there been a change. And so I reached out to the original authors, Annalise Berry, and Irving Zucker. And I asked if they would be willing to share their original data with me and would be interested in doing this 10 year follow up.
Chris - Now, when you say there was a male bias, do you mean as in animals, just humans, where was the bias?
Niki - The bias was actually across all research that used mammals. So from male mice, rats, primates, dogs, cats, and humans.
Chris - Wasn't legislation passed though in the interim that this shouldn't be the case? Because people did acknowledge. Look, there is an issue here. We may be missing important biomedical facts by not studying the right groups of individuals and actually it became law.
Niki - Yes. In the United States in 1993, there was a law that was passed that requires the inclusion of women and underrepresented groups in clinical research. So that accounts for human research. However, no such law existed for basic science research. So in 2016, the National Institutes of Health in the U S created a policy requiring investigators to consider sex as a biological variable.
Chris - Right, so given that we've now got legislation there and it doesn't just apply to humans, what one would hypothesise is that, if you do compare the situation in 2019 with the situation in 2009, there should have been an improvement?
Niki - The good news is that compared to 2009, many more studies are including both sexes. But one of the things that we found, which was a bit disheartening to hear, was that while inclusion has increased, the number of studies which analyse data by sex has not changed in 10 years.
Chris - Can you just clarify what that means in practical terms then, what you just said?
Niki - So when scientists have a study and they use both males and females, one might imagine that they would analyse their data based on the sex of their subjects. But what we're finding is instead most studies combine their data and look at it as one homogenous group. This really becomes an issue. If we're trying to determine things related to health and disease, we know the influence of sex impacts health and disease from disease severity, disease progression to incidence. So, you know, I think it's really important that if we're doing these basic science studies that are designed to be the early steps in, you know, the development of drugs and treatments and therapies that even in those early stages, we are considering the influence of sex at that early stage.
Chris - Do you think then that what you're seeing is just a nod to the rules to get the legislators off people's backs? Or do you think that actually there's something going on that can be fixed because it's an oversight?
Niki - That's a really good question. I would say, I think it's a combination of both. I do think that there are researchers who are, you know, stuck in their ways, thinking that this is the way I was trained. This was the way it's always been done. This is the way my colleagues do it. They might see these policies as simply another box to check in order to get grant funding. Alternatively one can say that maybe there's a lack of education and awareness about the influences of sex on health and disease. Because for many years it was assumed that there were no differences between males and females outside of the reproductive tract, but we know that to be false yet some of these behaviours and beliefs might persist within the biomedical research community and impact the way we conduct science today.
Chris - We've mentioned that the rules apply in the US. Are there equivalent rules governing other countries?
Niki - So I must admit, I am less familiar with the policies. I know there are similar pushes towards sex inclusion in Canada, but I am less familiar with the European research policies.
Chris - Where I was going with that is if your sample did not consider exclusively US data subject to that legislation, then it could be that the American side of things is performing beautifully, but actually it's other people who are not feeling the pressure legally to comply that are diluting the message.
Niki - Ah, yes, that's a very good point. And I think we can take the countries of origin of where the research studies are coming from. I think that's something I personally would like to look into in greater detail, but I think because of the high degree of international collaboration among scientists, I think this can't be a single country issue. I think the entire biomedical research community needs to come together and hold ourselves to higher standards. And ultimately I think that comes from journals themselves. Publishers, editors, and peer reviewers can really ensure that we have rigorous science being conducted by ensuring reporting and analysis based on sex.
Chris - You've highlighted with your study an important deficiency that is in current practice. So that's the challenge. How do you think it should be approached? Or what is the solution to this so that when you come back in 2029 and you do this again, what will you hope to see has happened?
Niki - So I think there are three strategies I would like to see happen. Number one is that all funders can be a gatekeeper and they can stipulate that you will not receive funding unless you provide justification for the use of single-sex studies. In addition to funders, publishers have a responsibility. All of these studies reviewed in this paper were from peer reviewed literature. And then ultimately I think it's a matter of education. This may not be on the radar of certain scientists, and if we cannot change the hearts and minds of seasoned investigators, then I think we can start with educating our trainees in the biomedical sciences.
27:39 - How HIV infects females
How HIV infects females
Nadia Roan, University of California San Francisco
Say the word "pandemic", and most people immediately think of COVID-19. But while the coronavirus outbreak may be the worst pandemic we've ever faced in economic terms, it's certainly not the worst in terms of human cost. In the coming years we're going to recognise the 40th anniversary of the discovery of HIV, the virus that causes AIDS. 40 million are already dead, and about the same number are currently living with the virus. We still have no vaccine. It's surprising, then, that in 2020 we don't actually know the answer to a critical question about the disease, as Chris Smith heard from UCSF's Nadia Roan...
Nadia - The purpose of our study was to understand how HIV establishes infection in women. Surprisingly as a field we still have a very limited understanding of what happens during those initial early vital stages. This process occurs predominantly through the female reproductive tract as HIV is most often sexually transmitted. And so in our study, we wanted to essentially better understand this process.
Chris - Now the reproductive tract, isn't just one single entity, is it, especially in women, it's quite complicated, there's quite a lot of different anatomical areas. So which of them is the dominant player in the transmissions, do we think?
Nadia - Yeah. So it's thought that HIV can be transmitted through both the lower reproductive tract, as well as the upper reproductive tract. Now we decided to focus on the upper reproductive tract because it's lined by a single layer of epithelial cells that doesn't provide much of a barrier for pathogens, such as HIV to traverse. And so we think that this was a main portal of entry for the virus. And so we wanted to understand what are the cells specifically in the upper reproductive tract, specifically, cells of the endometrium that are most susceptible to HIV?
Chris - How do you think the virus gets there though? Because sperm can swim, but viruses can't. So how do you think the virus accesses the endometrium?
Nadia - Yeah. So that's a great question. There's something called peristalsis, basically movement of particles from the lower tract into the upper tract. And so that's one potential mechanism. There's also some suggestions that viruses such as HIV might even be able to somewhat hitch a ride along the sperm cells.
Chris - So this would ferry at least a proportion of the virus to this site, but what's there that the virus is going for then? Because these viruses are tropic for cells in the immune system.
Nadia - Yeah. So the primary targets of HIV are CD4 positive T cells, a type of immune cell also called a helper T cell. And so these are the main targets of HIV. And indeed in our study, we did find that these were the main targets, but interestingly not all CD4 positive T cells were infected. In our study we compared genital T cells to blood T cells and quite strikingly we found that genital T cells were about a hundred times more susceptible than their blood counterparts to infection. And the more susceptible genital T cells exhibited some distinguishing features, including high levels of a protein called CCR5 which HIV needs to infect cells. And they also exhibited some other defining features, including a heightened state of activation and a higher state of differentiation.
Chris - How did you actually do the study though? Blood that's easy to get hold of, but what about the endometrium? Did you actually get samples of that to look at what T cells were in there then?
Nadia - We did. Yeah. So as you might imagine it's very difficult, in fact, pretty much impossible to study the details of what occurs during actual HIV transmission in women. So we studied it in the lab by exposing endometrial biopsy specimens from uninfected women to HIV.
Chris - And what comes out of this is you see this population of T cells, which is there's something special about it in the endometrium that means they're much more likely to pick the virus up and they're much more likely to proliferate the virus compared with the source of T cells that are washing around in the blood.
Nadia - That's correct, but not only that, we also found that after HIV has infected a cell, that cell looks very different from any original uninfected cell. And so this phenomenon has a name. We call it viral induced remodelling, and consider these cells as quote unquote remodelled because they have changed quite a bit from their original state. And when we looked more closely at exactly how HIV was changing the cells, we found some really interesting results. And so first HIV seemed to modify the cell in ways that made it more difficult for the T cell to function properly and do its job. And so it did this by disrupting what's called signalling through the T cell receptor. Second HIV also seemed to modify the cell in ways that could make it leave the genital tract and migrate to lymph nodes where it's known that there's an abundant source of additional cells that are highly susceptible to HIV. And we think potentially this is a way for the virus to spread to the rest of the body. And finally, we also found that the virus increased levels of a particular protein called survivin, which as its name implies, promotes survival of the cells, and this could potentially serve to keep the infected cell alive long enough so that it can spread to the rest of the body to infect other cells.
Chris - Is there an equivalent to this in men?
Nadia - So we haven't studied for example, rectal mucosal cells, but that's something that we plan to do. I would guess that something similar would happen in a male derived cells.
Chris - I'm just wondering if, because circumcision is very powerfully protective against HIV infection, whether there are similar interesting subsets of T cells in that bit of the anatomy, which would facilitate infection via that route?
Nadia - Yeah, absolutely. So you're alluding to the fact that circumcision protects men from sexual transmission by about 60%, which is very effective. Interestingly, the mechanisms aren't well understood. It's thought that cells, perhaps in the foreskin, might be highly susceptible to infection. There's also thoughts that the microbiome might play a role, but that's a line of investigation that's actively going on at the moment.
Chris - And do you think that what you're doing in the dish is an accurate and faithful representation of what's really happening in the body?
Nadia - It's quite difficult to study what occurs in women, but there are in vivo animal models, in nonhuman primates. And so one important future direction will be to confirm what we found here in a MEVOL model.
Chris - And there must be some profound implications of this because if this is the main route by which the virus accesses the body and establishes itself, does this give us any insights into what other sorts of interventions might work?
Nadia - Yeah, so we think that our characterisation of the way the virus modifies cells may reveal some new targets for HIV prevention strategies. And given that HIV seems to change the infected genital cell in ways that promote its ability to spread to the rest of the body and to survive, we are interested in whether drugs that block these processes might limit the likelihood that HIV establishes that initial beachhead of infection. And so in our study, we actually found that there's a drug that specifically targets the survivin protein that HIV regulates to promote its survival, and that that drug preferentially kills HIV infected genital cells. And so we're very interested in whether that drug, when combined together with traditional antiretroviral drugs, could that be more effective in preventing HIV transmission in the context of microbicides.