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Margaret McCarthy: Brain Sex Differences, Endocannabinoid Biology, Play Behavior, Cannabis/Pregnancy

Updated: Aug 1, 2022

Full episode transcript below. Beware of typos!

Nick Jikomes

Dr. Margaret McCarthy, thank you for joining me.

Margaret McCarthy 3:41

Thanks for having me.

Nick Jikomes 3:42

Can you start off by just telling everyone a little bit about who you are and what your lab studies?

Margaret McCarthy 3:48

Okay, as you said, I'm Margaret McCarthy, but everybody calls me peg or Peggy if you're my mother, and what my lab studies is the developing brain with a particular emphasis on trying to understand how the brain develops differently in males versus females. And that's not, that's not I just want to say that's just not because I'm trying to prove that, you know, girls are smarter than boys or anything like that. But instead because of the enormous gender bias in neuro psychiatric and neurological disorders,

Nick Jikomes 4:17

hmm, yeah, no, on your lab website, your lab website is really interesting. And it's got some some cool stuff on it some facts and some diagrams and things. And you say that the one of the aims of your lab at least is to understand the origins and mechanisms of sex differences in the brain. So as a neuroscientist and a biologist, what does biological sex actually refer to in the context of your research? And can you expand a little bit about why why it's an important area of study?

Margaret McCarthy 4:45

Yeah. But it's, it's also somewhat of a controversial area of study. So I want to just emphasize that I study the laboratory rat. So the laboratory rat does not have gender. It only has a sex, gender. There's a purely human construct that involves one's self perception and society's perception of one's sex. So in our rats, we only have males and females and there's no gender fluidity. The reason it's important to understand it from biologically is that in humans, we know that clinically, there is an enormous bias in what your relative risk is to be diagnosed with a developmental disorder, such as autism spectrum disorders, attention deficit hyperactivity disorders, early onset schizophrenia, stuttering dyslexia, Tourette Syndrome, just about any early onset disorder is going to be more likely to occur in boys, it does occur, they absolutely occur in girls as well as but when we look at probabilities and relative risk, they're more likely to occur and boys. But if we flip it around and look at things that occur predominantly after puberty, like depression, anxiety, compulsion, eating disorders, post traumatic stress disorders, and new inflammatory disorders, those are much more likely to occur in women about twice as frequently as they do to men. That so clearly, there's some kind of a big shift across the lifespan and you're allowed to risk. But in humans of women, by the time that they become susceptible to these disorders, they've had decades of life, living in a gendered world as a woman. And we can't separate out the influence of all of the cultural and societal expectations in that gendered world for both men and women to really get at what's biology versus what has been once lived experience. So that's where the rats come in. It's because we can control all that we don't have to put up with parents trying to raise them a particular way, or just them and speak to them in particular, we can just cut out all of that sort of experiential things and get purely at the biology of the origins of this, this relative risk across the lifespan.

Nick Jikomes 6:53

Interesting. So so there are differences in in mammals, including humans, but but also rats and other things, between males and females in terms of generally, what you just said, is in terms of their susceptibility to developing different psychiatric conditions,

Margaret McCarthy 7:11

right. Now, of course, my rats don't have psychiatric conditions, right? They don't get autism, they don't get schizophrenia, et cetera. So what we do is, we have to look at what we call sort of the phenotypes that are consistent with those disorders in humans. So we look at anxiety, like behavior, say, or depressive like behaviors. But but there's, you know, there's really limitations into what we can really model in the animals. What I tend to do more is, I just try to understand the basic biology of the male versus female brain development and not not so much in terms of, and then I do also try to look at how it responds to perturbations. And when what we find is that the basic developmental trajectory is fundamentally different in particular brain regions, not the entire brain, right? There's nothing I say is about the entire brain, it's really important to keep that clear that there's just different regions, you know, the amygdala develops differently than the hippocampus, which develops differently than the cerebellum, which develops differently than the hypothalamus. You know, people tend to say, oh, a male brain versus a female brain, there's no such thing as a male brain or a female brain right there. Every brain is a mosaic of, you know, a combination of a whole bunch of different regions that work independently. And in trot dependently, which was why neuroscience is so fun and so complicated. I lost my train of thought where it was, was like, my question was answered.

Nick Jikomes 8:39

No, I mean, we're just talking about, you know, the susceptibility to different conditions.

Margaret McCarthy 8:44

Yeah, so so. So I tried to understand. So for example, looking at the amygdala, which we know in humans is associated with a variety of social behaviors, fears, and things like that in adults and anxiety. And it has been implicated in a number of neuropsychiatric disorders. And so I just asked the simple question, does the amygdala develop differently in males versus females? And then try to understand and how, how is that differently? And what are the consequences of that difference? Now, sometimes in other brain regions like the cerebellum, I will ask how does the cerebellum respond to an injury early in life? And is that response different in males and females? So there's kind of two different ways to go about it.

Nick Jikomes 9:25

Got it. So one of the things it says on the website, just to give people a little bit more here to anchor themselves to sexual differentiation in the brain of the brain, is a developmental process whereby physiological and behavioral phenotypes are modified to match the Natl phenotype to ensure reproductive success. Can you sort of translate that? Should people that aren't familiar with all those terms?

Margaret McCarthy 9:51

Yeah, absolutely. So a lot of us are familiar with the idea that a male mammal is an X Y chromosome complement and a female is x X chromosome complement. But the only consequence of that is that there's a single gene on the Y chromosome that codes for the formation of a test. It's because there's a little collection of cells, that's just it's by potential exists near the, the developing kidney, and it can become an ovary, or it can become a testis, it's your bio potential. And if there's a Y chromosome with this gene called sry, which is sex determining region of the Y chromosome, that little group of cells will form a test is and I always love to remind people that you know, we didn't discover this gene until the 1990s. Now, for many people, that seems like yesterday, for some people, they're like, Well, I wasn't even born yet. Yeah, but for a lot of a lot of scientists, that's like yesterday, we only discovered this gene in the 1990s, a guy named Robin level bench and London. So if that gene is present, that little group of cells will become a testis. And if it's not present, that little group of cells will become an ovary. That happens stunningly early in development, right? In humans, it's within the first couple of weeks, right. And in the rodents, it's in the first couple of days, in rodents that everybody thinks about the puberty is being the time when you first see steroid hormones, right, that's when testosterone comes. And that's when estrogen and progesterone come. But actually, in mammals, the fetal testis begins to make testosterone while he's still a fetus in utero. And in rodents, that happens about five or six days before the animals are born. And humans, it happens in the second trimester. And there's huge amounts of testosterone that are produced, but very high levels equivalent to adult circulating levels of steroid. And this is after the testis is formed. And all of the reproductive tracts is formed and everything and so as far as we know, the only purpose of this surge, we call it a testosterone surge. I love the Japanese call it the testosterone shower. There's we can tell the only purpose of that is to act upon the developing brain to sort of imprint on it, if you will, the the physiology and behavior that is consistent with masculinization masculine fitness. So in terms of the physiology, you know, the brain controls the pituitary, and the pituitary controls the gonads and there's a feedback loop. We call it an axis, right hypothalamic pituitary gland Atul axis. And that axis functions very differently in males and females because males need to make sperm all the time, so that they're always sexually receptive. whereas females only ovulate periodically in humans about once a month. And then I wrote in about once a week. And so females in most animal species, humans being the notable exception, and most animal species, females will only mate right around the time that they ovulate. And that's the mating behaviors controlled by the brain, the ovulation is controlled by the brain. So if you're going to have those two things be in sync, you have to have the gonet and the brain in sync. And likewise, males have to be able to be ready to mate all the time for their fitness, right. So everything is about evolution, maximizing the fitness of males and females relative to their themselves.

Nick Jikomes 13:16

And so I would imagine that, you know, when we think about something like sexual dimorphism, the the fact that in most sexually reproducing species, you have to do more of that, you know, the female body plan and the male body plan, which correspond to different physical features, different physiological features of the body. When we think about sexual dimorphism. What are some of the ways that the brains of mammals tend to be sexually dimorphic that hold across species? Are there any general things that hold? Or is it really like a species specific thing where you know, when you think about rats versus humans versus something else? It's going to be species specific?

Margaret McCarthy 13:50

Yeah, yeah. No, I would say that we have some, some generalities. And some, we haven't been able to ask the question yet. But probably so. So a lot of times, let me just reframe your question for an earlier question. Because a lot of times people will just ask me, What are the sex differences in the brain? And that seems like that should be such an obvious question to answer right, I should be able to say, Oh, well, ABC and D. But it's actually it's a lot more difficult question. So the brain can differ by the size of a particular region, right. So it can be bigger or smaller in males versus females. And that's what we call sort of a macro difference in humans, it can be the amount of white matter versus the amount of gray matter, but then it can also be at the more micro level, it can be the number of synapses that are formed within a particular region, the number of axons that project from one region to another region can vary enormously. The the type of the neurons, right, they could be more inhibitory GABA ergic or more excitatory glutamatergic and the relative amount of each can vary so and then you could have the exact same cell We'll population but then the genes that they express can differ between males and females. So we can really go for a big sort of macro, you know, the hippocampus is bigger down to the cellular exactly what you express difference in males and females. Now, there are some generalities that we know about one of the most famous is what's called the sexually dimorphic nucleus of the preoptic. area, which is was named for its discovery. It's a, it's a really fascinating kind of story, I think, a scientific story of how it was discovered, there have been attempts to look for sex differences in the brain in the 1960s. And you know, if you just hold the brain in your hand, you can't tell it male from a female, they don't come and pink and blue, right? You literally do not know what it is. And two sets of scientists one is a guy named John faff at Rockefeller, and then another apparently Raisman. In fields, they looked at very precise, exactly like using electron microscopy to look at really like they weren't looking at trees for the forest, they were looking at the bark on the trees, right, really, really, really, really detailed. And they found very small and subtle. sex differences, one of them only in response to an injury, like they did a lesion. This was such huge news that it was published in Science, both both papers were published in Science, because nobody had reported anything different in the male and female brain before. And then it's sort of like nothing happened until the 1970s. When a guy named Art Arnold who was also at Rockefeller as a graduate student, was thinking a lot about the brain. And he was thinking a lot about behavior. And he was studying birds. And he was thinking about Canaries, in which the males sing this extremely complex, beautiful song. That's why everybody wants a male Canary and the females just tweet, right? They just they don't sing. And he speculated that that had to be controlled by the brain. And so he took male and female Canary brains and he section them and just stain them to just you know, like, we call it a nissel stain, right just to visualize them. And he saw this huge nucleus in the males that was related to the song that was just tiny in the female. So it was it was available, you could see it with the unaided eye, it was such a big sex difference. So that was also a science paper that he published along with Fernando not Abom. That stimulated a guy named Roger Gorski, who was at UCLA who had been studying rat brains to understand that difference in the control of the gonads I was talking about earlier, the ovary versus the testis, and ovulation versus spermatogenesis, to go back and look at his brains just from a distance. And then all of a sudden, this little tiny nucleus popped out at him that was much bigger in males and females. And so and it was at the macro level, right, so he named it the sexually dimorphic nucleus. And it's happens to be located in the preoptic area, which the preoptic area has nothing to do with vision, it just happens to be a region that's preceded the optic chiasm in the brain, that was in the 1970s. And that really sort of stimulated just a huge amount of interest. And you could almost say, you know, founded the field of sex differences in the brain. That nucleus exists in just about every some variant of it, and about every species that we've looked at, including humans. And there's an analogue in humans, that's considered a sexually dimorphic nucleus. It's called the interior nucleus of the interstitial hypothalamus. And it was reported by Simon LaVey in the 1980s, that that it's larger in males and females in heterosexual males and females. It was right around the time to the AIDS epidemic, when a bunch of brains were becoming available from homosexual men who died of AIDS. And that nucleus was smaller in homosexual men than heterosexual men, it was in between heterosexual women. And it has since been discovered in sheep in rams, a guy named Chuck Roselli, has been studying them out west in which there's about 10% of the rams are naturally occurring, preferring to meet with other rams, as opposed to meeting with us. And the sheep, again, is sexually dimorphic nucleus is different in size. And he finds it in the males that prefer to mate with other males, the nucleus is smaller, and more on the size of the females. So so that one really, really cuts across quite a few species. There are other examples as well. But some things like a lot of these extra things I've discovered are things like the number of synapses, right, or the morphology of the astrocytes and things like that. And those are much harder to do in a human brain. Because we don't have the sort of, we can't do the kinds of manipulations and microscopy and stuff in humans or people just haven't done it. I see.

Nick Jikomes 19:33

So you can get sexual dimorphism in the brain differences between males and females at sort of every level of resolution. Some of them are big enough that you can see them with the naked eye and some of them go all the way down to the level of synapses and molecules and genes and things. And are there any general rules for when things are strongly dimorphic between the sexes? So for example, is it fair to say or is it reasonable that most of the day morphisms have something to do With brain regions that are related to reproductive behavior, or is it not limited to reproductive behavior?

Margaret McCarthy 20:05

Yeah, that's an excellent question. And at the beginning of the field, the assumption was that they were only related to reproductive physiology and behavior. And in fact, that's sort of relegated the fields is sort of a sideline of neuroscience, right? It's considered sort of niche right and not not particularly important. And then in the 1990s, a woman named Catherine wooley, who at the time was a graduate student of Bruce McEwen published a series of papers a building on Maya Frankfort and other people that love showing that in the hippocampus, extra dial across the estrus cycle would change the number of synapses across the estrus cycle up to about 30%. And that was considered an absolutely crazy heretical idea that that could happen. In fact, they had to prove it many ways from Sunday, but she and Bruce McEwen are both outstanding scientists, and eventually the community community had to accept that this was true. And this had nothing to do with reproduction, right? It had to do with with learning and memory. And with stress responding, which is what the hippocampus does. Now, that wasn't a sex difference, right? That was a hormonal modulation that she found. But that sort of broke open the idea that there could be sex differences, and other regions of the brain that were related to other functions, then then reproduction, and many have been found. I myself, I still kind of tend towards thinking that they're more related to reproduction. But I'm often chastised and corrected on that by a colleague named Larry Cahill, who was absolutely adamant that the magnitude of the sex differences in the hippocampus and subcortical regions are just as great as those that are related to reproduction. And I will say that by and large, with the exception of the sexually dimorphic nucleus, which is really big, bleep different, big ly different, it's about it's three to five times bigger than male rats and female rats and humans, it's only about one and a half times bigger in males versus females, most sex differences are on the one to two fold level. And I think that that's by design, I think nature catalyzes sex differences to keep the two sexes really separate, but not flying off in completely different directions, right. So so you don't want them to be so divergent that it's, you know, females are from Venus, and males are from Mars kind of thing, because they reproduction requires such delicate coordination. And plus, also, a lot of times the two sexes are solving the same problem, right? Both sexes have to learn, right, but both sexes have to feel pain, both sexes are susceptible to habit formation, and hence, addiction, you know, both sexes have to be stressed, and etc. So, so they're not most sex differences are not, you know, huge, but they're, but they are what I would call reliable, right? You know, they're usually not a continuum in my, my world. And the endpoints that I look at, you tend to have you got the male and the female, and you don't have a lot in between.

Nick Jikomes 23:06

I see. So so if you, let's say that you just sort of picked a random chunk of brain in a random mammalian species, maybe it's a rat, maybe it's a human. If you sort of had infinite data, if you could go in and like count all the synapses, or look at the gene expression patterns? Would you tend to find some kind of difference between males and females on average? Or would that be exceptional? Or would it you know, happen half the time?

Margaret McCarthy 23:33

That's a fascinating question. It would be a great experiment to do, because of course, we don't do things randomly, right? We always have a reason. And I would say, Well, yeah, I would say in my experience, I tend to look at a lot of fairly different regions. And for example, when we look at the cerebellum, we don't see any sex differences at all. It's developing, it's a developing just exactly the same until we perturb it. If we create hypoxic ischemic injury, or if we do a viral inflammation or bacterial inflammation, then we see, you know, the sexes kind of fly apart. And that's called a latent, sometimes called a latent sex difference or divergent sex differences. And that's also seen at the cellular and molecular level. But if I look anywhere in the hypothalamus, it'll find sex differences, hippocampus, definitely, there's some cortical regions. I don't do cortex. I don't have a good answer to your question. Except for to say that more often than not, if you keep digging you'll find this extra now one thing I have people come to me all the time with I've got a six seven so if I was like, oh my god, I'm so excited. What am I going to do? And I'll look at their data on me like Yeah, I think you just have noise you know, I don't really anything that's like less than one fold is just you know it. A lot of times it's Texas or not difference. I don't feel like I'm giving you a really good answer because that don't have a clear answer. But I'm always telling my lab, they'll get all disappointed when something's not different. And like, it's just as exciting when it's not different. And it's just as important. And what we have to do though, is we have to be as sure about no sex differences, we are about a sex difference, you know, it has to be just as well powered, right? The ends have to be just as large and stuff so. So like, I have a whole new line of inquiry going on in my lab right now where we don't find a sex difference in one brain region and one response, but we do find it in another. And we're asking ourselves, wow, how are the two regions doing this so differently? You know, what, why, again, why is it important that they not be different in one brain region? And that they do be different than another brain?

Nick Jikomes 25:45

I see. So it sounds like it's fair to say that when you look for sex differences in the brain, you if you look hard enough, you will often find them. But nonetheless, there are brain regions like the cerebellum, where it appears to be more or less the same in both sexes.

Margaret McCarthy 25:59

Yeah, and there are probably many other regions where it is more or less the same. You know, there's always the reporting bias as well, right? So the reporting bias is towards finding a difference. And people don't tend to when they don't find a difference, they don't report it or doesn't get attention and et cetera. And of course, then I have a bias because I'm always looking for differences. So I might not be the best person to ask that other people might tell you, Oh, yeah, we never see sex differences.

Nick Jikomes 26:22

And the other thing I want to go back to that I think is interesting is so you mentioned a couple of things. You mentioned the hypothalamic pituitary Natl axis. You mentioned this testosterone surge, or testosterone shower, depending on your taste that happens relatively early in development. With those things in mind, can you kind of connect the dots for people between the gonad sex hormones and brain differences that develop like, how does that start to happen? And how do those things all interact with each other to create some differences?

Margaret McCarthy 26:53

Great, great question. So I think you're right, I didn't, I didn't complete that line of thought, which is so that testosterone surge, what it is doing is getting into the brain, there are receptors in the brain for testosterone, they're not everywhere, right, they're only expressed in certain regions that are going to be responsive to testosterone. Testosterone is also a precursor to estrogen or estradiol. And there's a whole nother whole set of cells that that have receptors for estradiol, or for estrogen, that and then those receptors are actually what we call nuclear transcription factors. So they actually directly interact with the DNA, and they cause the transcription, you know, the turning on or turning off often just as important of particular genes. And then they also actually seem to mediate epigenetic changes so that the turning on or turning off of particular genes is established, relatively permanently, right, throughout throughout life. One of the questions so that's, that's so so what I can do in my research program, is I can take a newborn female rat, and I can inject her with testosterone or estrogen. And I can watch watch experimentally, the process of turning her brain into a male brain. And so it's a great tool experimental tool that I have. So I can I can monitor the process of sexual differentiation in real time. And I can measure various endpoints like I can look at the mRNA for various genes, I can look at the morphology of of neurons morphology of astrocytes, I can look at the immune cell profile, etc, at the time, and that's how I get at the mechanism of sex differentiation, that all in my rodents is happening in the first couple of days of life in humans, it's all happening in utero. So even if we could inject a human with testosterone, which we couldn't, we still wouldn't be able to study it because it's happening in utero. So even in primates is, you know, like rhesus macaques and things. It's all happening in utero, which is, it's, we're fortunate that the postnatal rat remains sensitive to this. That's why we call it the sensitive period, to testosterone. Now, if I take a newborn rat, I can do that up to about a week, once she gets to be about 10 days old. If I try to inject her with testosterone, it's too late. She will be a female with a female brain for her whole life. And likewise, for a male if I take away testosterone really early, I don't let him get exposed to testosterone. He'll I can make his brain female like, but if he gets testosterone once he's exposed, that's it. It's sort of so so kind of an analogy I give is it used to be a thing called lazy eye, you know, you had one one eye that didn't quite look straight. And so the way that doctors used to treat that was the two would cover the good eye and you would make the lazy eye correct itself because it had to work harder. But if you didn't do that early in childhood that I would never realize so there was a sensitive period during which it could happen and then it's too late and that's that's the nature of development, right development has things that if they don't have But at the right time, they'll never happen. The really fascinating question to me though, is in the sex differentiation case, is that everything is happening in a pop a little, looks like a little pinky eraser, right? You know, it's, it's not capable of any kind of reproductive function, it can't even walk, right doesn't even have its eyes open. And in that little pinky pup in that little tiny brain, you're coding for its ability as an adult, to either be, you know, mounting and amid the ejaculating to be aggressive, to be highly anxious, you know, to prefer the odor of females, all of these things that are not going to occur for months, right? And so I'm really fascinated as like, how is that stored in the brain? And how is that coded in the brain? Go all that time. And then it's not expressed until after puberty. It's really we don't know anything about about that.

Nick Jikomes 30:55

Interesting. So so when people think about, like sex hormones, testosterone, estrogen, estrogen and things like that, these are literally molecules that are produced in the body, they go through the bloodstream, and they can get into the brain and not only into the brain, but they physically go inside the cells and directly in some cells, at least, directly sort of control which genes are coming on and off and having their effects through that kind of mechanism.

Margaret McCarthy 31:21

Precisely. Yes. So there was just a beautiful paper published in Nature by a name woman named Jessica Tolkin at Cold Spring Harbor, in which she really lays out the genes that are she uses very, very modern cutting edge techniques in three different brain regions and shows exactly what's getting turned on early, what's getting turned on in adulthood cetera. I see. People don't appreciate how powerful steroids are they, they tend to think of them like you know, other other hormones, you know, say like insulin or something, but they're a completely different class of compounds.

Nick Jikomes 31:55

Interesting. And so these things go in inside of cells, they control their development, with the caveat that it really depends on what phase of development you're in. If, if it's at the right time, so to speak, they can have a huge effect. And they might have little or no effect if it's if it's too late, and some window of opportunity has closed, correct? Yep, that's exactly right. And then the final piece that was interesting there to me was that a lot of the tracks for the sex typical or sex specific behaviors are being laid down, way before the behaviors ever express themselves. So somehow these changes happen and develop, and then they are sort of latent or stored in the brain only to sort of do what they're going to do later on.

Margaret McCarthy 32:33

Yeah, that's exactly right.

Nick Jikomes 32:36

So one of the things that we'll probably end up talking about, because you've studied, it specifically, has to do with juvenile play behavior in rats, which is sexually dimorphic. And we'll talk about that. But before we get to the rat stuff, specifically, can you just talk about juvenile play behavior in mammals generally? How common is it? And what do we think the the general biological function? It serves us?

Margaret McCarthy 32:59

Yeah. So thanks for that question. So first, a little background on on play. It's juvenile play behavior, as you said, and so it only occurs during a restricted period of time. And it's usually an in our rodents, it's like just around real weaning. And until puberty, and once you get past puberty, you know, interest in play goes away. And instead you're interested in mating and being territorial and etc. And it's a mystery still, to this day, what the purpose of play is, somehow we all think, intuitively, it must be important, right? But but we nobody, and it's debated by, you know, psychologist, psychoanalyst, parents, teachers, and neuroscientists. And it used to be a very intense topic of investigation by neuroscientists back in the 1980s, because back then the rat was the favorite animal model for neuroscience. Then the transgenic the power of the transgenic mouse swept in, in the 90s. And, of course, that just, you know, really pushed aside most of the research in rats, because it was so powerful to have this ability to manipulate the genome. And it turns out mice are actually one of the very, very few species that do not engage in this very complex play behavior. So what do I mean by complex putting behavior when you put two rats together, so just a look just like puppies or kittens, they will chase each other, they will, one will pounce on the other and the other one will roll over onto its back to expose it's ventral. And then they'll jump up and they'll reverse and the other one will chase the other one and I'll do the same thing. So they'll stand up and they'll box with each other. And sometimes they just wrestle, you know, they just roll around and wrestle and we know it's rewarding. They like it, you know, if you let them play in a place that they they conditioned to it, they want to come back to that place. There's no aggression. There's never any biting as when it's going well, it's reciprocal. If you get into a situation where one animal is always dominant Eating the other and sort of pinning it, the play extinguishes because the other ones like you're not any fun, you don't let me know kind of thing. And that's, that's also true, you know, so many species that we know of that we all you know, all of our pets and everything. One of the fascinating things I think about our pets is that they start, particularly dogs, they still play post puberty, which is another interesting question. The reason that I got so interested in it is that across all species that play, males will play more intensely, more physically more frequently than females, females do play, they are very physical, they're just not quite as intense and physical as males. And what fascinates me about that, is it when they are at the age that they're playing, there's no steroid hormones around, right? This is like about like a six or seven year old child, right? They don't have any hormones at this time, right? So all so the sex difference is coded by the hormones early in development, just like adult sex behavior and adult aggression. But it's expressed at the time that there's no hormones around. And so that means you're really comparing apples to apples, right? As opposed to an adulthood when males and females have very different hormonal profiles. And so and then, so I also think that the fact that there's this difference between males and females can maybe open a window into what is the purpose of play? You know, do we really need play kind of thing? And that's, that's one of the avenues that my lab is exploring right now.

Nick Jikomes 36:29

One, I mean, one way, I suppose you could look at that. And I don't know if this has been done. has, has anyone simply tried depriving juvenile rats say, of play behavior and see what the downstream consequences are for their reproductive behavior or something like that?

Margaret McCarthy 36:43

What an excellent idea. Yes. That was done. It has been done many times back in the 80s. But mostly, it was done by just completely isolating the animals. So putting them alone in a cage away from all other animals. And turns out that's about one of the meanest things you can do to a juvenile rat. They're very, very social animals. And it's very, very stressful for them to be isolated. So what we've been doing in my lab, and I should all say it was all only done in males, because all of the research back in the early days was only done in male animals. So one of the things that we've done a graduate student and Ashley Moore caught in the laboratory, is she created a cage, a normal rat cage, and what she put a perforated barrier, plexiglass perforated barrier down the middle, so that you can put an animal on each side and they can see each other, they can hear each other, they can smell each other, they can tiny, tiny, tiny little bit, touch each other, but they can't play. And so she houses them like that through the entire juvenile period. And then she gets them through, takes him back out, puts him back into group housing at puberty, and then they go on to an adulthood. She's measured a whole lot of behaviors. And what she has found is that the males, show impaired impaired sexual behavior, they're slower to interact with a female sexually and they interact less frequently, they show a longer latency, they have fewer amounts, fewer ejaculations, etc. Conversely, they seem to be hyper aggressive. So sometimes people say, Oh, play is a rehearsal for aggression. No play is probably a rehearsal for appropriate aggression, not you know, and so, so they're much quicker to attack a stranger etc. They have a very bizarre response to sort of a social preference test. And it would say also, it's like a hyper exaggerated response, which we think is has to do with dominance hierarchies, and not knowing how to interact to establish dominance hierarchies. And then they show a little bit of cognitive impairment. Fascinatingly, the females are completely normal. They show no effect whatsoever of having been deprived to play. And further, if during in the middle of this period of this, you know, not being allowed to play if we just we free them from these barrier cages and allow them to have a playdate. The males are exuberantly playful, right? I mean, they just show a huge exaggeration in their play, and the females are like, they just are again, unaffected. So so it's a really fascinating difference than why why would the play be important to one sex and not the other? And, and again, yeah, it's just we're just really fascinated by why is it the female species? Are they just resilient to the stress they when they when they sit in the cage together, I keep going like this because they sit right next to each other? They know the other animals across the barrier right and they sit right there and somehow that's just enough socialization for the females I don't maybe the chatting away as

Nick Jikomes 39:44

well Yeah, I mean, that was that was another question I had is when we talk about this player behavior in rats were referring typically to this. Easy to measure easy to see like rough and tumble wrestling type behavior. Conceivably, there's there's other ways that interact that are more difficult to discern.

Margaret McCarthy 40:02

Yes, yes, that's an excellent, excellent point. And in fact, the play is so fast and vigorous, we have to videotape it and then slow it down to score it. And so one of the exciting things that's happening in neuroscience right now is computer vision learning. So you can use artificial intelligence to and so we started this process. There's, there's various programs out there, there's quite a few of them that are developing to see if the computer can see something that we're not seeing, right. And but it's the challenge is that these two animals, they, you know, you can teach the computer to track one and we can get the computer to follow them around as they're like chasing each other registers, then they interact, and then they become a free ball. It's really hard for the computer to tell who's who. So we're working on that. But but you raise an excellent point is we do think that there can be things that are going on that we aren't detecting, there can be ultrasonic vocalizations that they're making to communicate with each other that we can't discern. And, again, these are just technical problems, because we can record ultrasonic vocalizations. But we can't tell which one is vocalizing i, because so things of that sort. So but yeah, I think those are exciting questions to pursue in the future.

Nick Jikomes 41:20

Yeah, well, you said a couple of things that were super interesting. So when we think about depriving the rats of play, a, there was this dimorphism there. So the the deprivation of play affected males more than females, it resulted in subsequent deficits in their adult behaviors, such as the reproductive behavior, and their propensity towards aggression, as you mentioned, which is maybe a little bit counterintuitive to people, but I think not so much once you think about it, if you don't let if you don't let the male rats do rough and tumble play with each other, then they become hyper aggressive, they basically don't know how to appropriately interact with other males and other members of the species later on. And you also said that if you deprive them for a little while, then they become like, really, really excited to play even more. So that implies that this is almost homeostatic Lee regulated the same way that other processes in the body are which, which would also say that something very crucial is happening here.

Margaret McCarthy 42:13

Yeah, that's interesting. I'm glad you said that. I hadn't thought about it in terms of being almost directly regulated. But But we, you're absolutely right. In fact, we know sometimes in our experiments, if we want to do things like we want to look at the the neurons that are activated by a play about right, so we'll look at an immediate early genes expression in a particular brain region after a play, but what we'll do is we'll isolate the animals for 24 hours before the play date. And then we know we'll get this huge exaggeration in play. And so you're right, it's like, what are other times what we do is we let them play together every day for like, 10 days in a row, they meet a playmate in the afternoon, that they go, and it's not their cage, mate, it's not their sibling, it's their playmate, and they go to a neutral arena, it's not in their home cage, and they get about 15 minutes to play. And they'll just steadily play the same amount pretty much across the whole 10 days, kind of thing. So unless we isolate him, and then boom. And if we if we introduce a new playmate that doesn't affect the males, but the females, they're like, I don't know who you are, and they don't they don't play with the introduced animal.

Nick Jikomes 43:24

And now we'll will juvenile male rats preferentially play with other males? Or do they play differently with juvenile males versus females?

Margaret McCarthy 43:32

Yeah, that's a great question. If we look in terms of the intensity of play, the highest is male to male, medium is male to female and lowest is female to female. And when you do mix groups, because what we do now is we started out, we would do like six animals together. And we would just watch the all six animals, which was crazy. Yeah. Because you got to label them and all that kind of stuff. And, you know, it's good question whether or not the males initiated more with other males. And that's where like, if we can get really sophisticated computer learning would be a vision learning would be a good question to ask. But definitely, you know, one of the challenges of studying the behavior is that it does require reciprocity. And so if the other animal is not playing the same, that will kind of extinguish it. And so we've been looking at things like rats with genetic mutations for genes that are are identified in autism, and to see if their play behaviors changed, and it is, and it's reduced. And what we have to do is we have to put the to, like the genetically mutated rats to together like, we can't do a mix, Diane, because it won't reveal the effects but so you have to sort of exaggerate it. But anyway, I whether or not there's a preference for a male with a male, I don't think so. But they'll get they'll have more reciprocity from a male and so that just naturally exaggerates the behavior.

Nick Jikomes 44:54

I see. So, so this play behavior is sexually dimorphic, both in terms of how vigorously and Frequently it happens to males versus females. It's also dimorphic in terms of how important or influential it is on their subsequent adult behavior, because if you deprive males, they have deficits in reproduction and aggressive behavior, as you said, you don't see that in females. So the next question is really, okay, this is super interesting. Where does it come from? And I know that a lot of your work has looked at how this type of difference develops, and how the endocannabinoid system in particular ties into that. So can you start to tell us that story of where this dimorphism comes from?

Margaret McCarthy 45:33

Yeah, yeah. So first, you have to start as what is the neural circuitry of play? Right, what in the brain in general, where does play come from, and there is a very strong consensus in the field now of what we call the social behavior network. And now there's also what's called a core aggression network. And these are brain regions that are interconnected that regulate social behaviors include and then also aggressive behavior, they usually start with the olfactory system, either the main or accessory olfactory system, and then you're going to have projections to the amygdala, you're going to have participation of the prefrontal cortex, you're going to have a component of the reward pathway, the VTA nucleus accumbens, and other BNS T and then you sort of kind of go down to the midbrain to the spinal cord, the and the neural circuitry of play near as we can tell, we don't know it as well as we do the others because a lot of that has been really finely mapped now in the mouse. The nose, like as you play is overlapping with the social behavior network is the same network, which is also fascinating, because then like, Why does play Go away? Right, why doesn't extinguish with puberty. But fascinatingly, the sex difference is only in the medial amygdala, you could you could imagine that being a distributed property, right? That would just, it would be redundant to the entire social behavior network. But it's not. It's only in the middle amygdala. And why do we know that? We know that because if you take a newborn female rat pup, and you put testosterone only in her medial amygdala, three weeks later, four weeks later, when she starts to play, she'll play like a male. You don't have to have the testosterone anywhere else in the brain.

Nick Jikomes 47:17

So there's one spot, medial amygdala, in this case, you put testosterone in a female, so it's at levels that resemble what would be naturally in a male. And later on, when she becomes a juvenile, she'll play like a male. Right?

Margaret McCarthy 47:31

So my question was, what is that testosterone doing? Right? What is its, what's its cellular mechanism of action. And we had been doing some other studies on the hippocampus, where we had seen some sex differences in neurogenesis, actually, with males making more new neurons and females in the first couple days of life, and a new postdoc in the lab, named Deseret cribs craft. And I said, Well, you know, the Migdal is in the same sections as the hippocampus. Why don't you just go and look at the, the newborn cells in the amygdala and see if there's any sex difference there? So she did we do this with a BrdU injection, that's a thymidine analog that labels newborn cells, you know, you have to section the brain and look at it. And she went and counted the newborn cells in the middle amygdala. And lo and behold, she found that there were more newborn cells in the female than then the male. And that was exciting, because, you know, usually everything's always more than the male more than male more than me. And so she's like, Oh, that's cool. So there's more newborn cells in the female than the male. And then she's like, well, now what? You know, I was like, yeah, now what? That's a good question. Well, I have it. Sometimes science occurs in funny ways. I had a colleague named Brett Alger, who studied the endocannabinoid system, he was one of the pioneers of this system. And after you know, at this point, I think we've been colleagues for, I don't know, 20 years or something and every single seminar, every thesis defense, every journal club, every thesis proposal, during the question and answer session, he would stand up and say, you know, endocannabinoids, can explain that whatever, whatever was, whatever phenomena was, literally, so I just literally said to her, I don't know. Let's look at endocannabinoids maybe, maybe they're regulating the That's That's literally how it happened. And so she said, okay, and she took a pan agonist for the CB one CB two receptor, which are the two receptors on which endocannabinoids and also THC act, and she injected animals, we just did a pharmacology experiment, and she was able to reverse the sex difference that she was able to bring down the number of newborn cells in the female to the level of the male, so she was able to eliminate the sex difference. And since we knew from the previous work in the 1980s of Bruce McEwen and Michael meany that the middle amygdala was critical to the sex difference and play behavior. She then looked at social play behavior, and she found that it also the treatment with the pharmaceutical drugs that mimics THC had also sex, reverse the play behavior, and that set us off on this journey to under Stan really precisely what's the endocannabinoids for doing

Nick Jikomes 50:03

so. So just to reiterate, so what so So you give testosterone to a female specifically in the medial amygdala, her subsequent play as a juvenile will look like a male. And the same thing happens if you give an activator of CB one, CB, two,

Margaret McCarthy 50:18

correct. And you don't even have to give it into the amygdala, we just give it under the skin. You give it sub cube.

Nick Jikomes 50:24

I see. And does that happen also with THC itself?

Margaret McCarthy 50:27

Yes, it does. We haven't published that work yet. So yes, but we have. And this is Jonathan Vander isn't who's been carrying on this work. And he's preparing the manuscript now in which if we inject newborn pups with THC at exactly the same time that we would do testosterone it, it does, it has the same effect on the newborn cells. And it has the same except effect on the play behavior. And interestingly, if he gives THC to pregnant rats, earlier, so we're looking at because we're trying to model human cannabis use during pregnancy, we give it much earlier, it has the opposite effect in terms of play behavior, it actually suppresses play behavior. So it promotes late behavior in the young animals, that when you give it to the newborns, and it suppresses it during fetal if it's given during fetal life, and we don't know the mechanism of that. But again, it's just the importance of timing development. It's all about timing. Yeah, you

Nick Jikomes 51:15

can get the opposite effect at two different times with the same treatment. So So is there some kind of interaction you guys are hunting down between the sex hormones like testosterone and the Endocannabinoid receptors? Do we know exactly where that link is?

Margaret McCarthy 51:29

Right? So so that this is where the story got really, really interesting. We did all those obvious things, right? So we looked for sex differences in the Endocannabinoid receptors and sex differences in the endocannabinoids themselves. And we did find that in fact, males have a higher level of the endogenous and a cannabinoid called to AG. And no cameras are funny signalling system, right? They are a membrane derived signaling molecule that has so that sort of continuously produced all the time. And so there's like a tone, right, you have like a certain tone of endocannabinoids that you you sit at. And so the conclusion was that males have a higher endocannabinoid tone than females. And if we give females testosterone, they make more and when they have a higher tone, it's easier because they're making more or they're degrading it less. A big part of the unexamined system is it's like, you know, think about it, as you know, well, how to think about basically you make it and you degrade it, and you can either make it faster or degrade it faster, and that will shift the amount either way. So so it was definitely the Endocannabinoid tone, not the receptors, that was different. But then it was like, Well, what, what are the cannabinoids actually doing? And we were thinking about the adult and all the things that are known in the adult about how it regulates synaptic physiology, they have presynaptic, inhibition, depolarization and do suppression, etc. And none of that applied to the development of the amygdala, which it makes sense in retrospect, because it's, there's just not developed yet. It doesn't have all that synaptic activity. And so when we're trying to think about cell Genesis, so the big question was, what, who are those newborn cells? Right, those born? And if of course, you think it's gonna be neurons, and certainly in the hippocampus we had shown it was neurons, but again, in retrospect, it makes sense. It wasn't they weren't neurons at all. It was the glial cells known as the astrocytes, right. So these are the sort of always been considered the support cells of the brain. But we now know they're actually very, very active cells. And they're really important to synaptic physiology and etc. And Deseret first identified that and in fact that it did look like it was the astrocytes that there was more in the females and the males Deseret cribs craft. And then Jonathan Van risin really confirmed that but he added in the really interesting and unexpected twist of that he brought in a third cell type called microglia. Now, the problem with microglia is their name. They are not glia. They are not astrocytes in any way. And they're not micro either, because they're 10% of your brain. What they actually are is our modified immune cell. And it's a modified macrophage. And they are born very early in development. And they they get into your brain and they take up residence and they stay there your whole life. And they're scattered throughout. They're peppered, actually we call it, they're tiled through your entire brain, your entire brain is full of microglia. And they're there as your brain's immune system. They're very, they're unique to the brain. They're nowhere else in the body. And what Jonathan found was that rather than there used to be we thought microglia, we call them quiescent. We thought they just sat there waiting for an accident to happen. And then they would maybe, you know, release side to client kinds or prostaglandins and respond to the injury. With the advent of the transgenic mice and the amazing things that you can do with transgender rights. We were able to visualize them in the living brain and found out that they're not quiescent at all. They're actually Moving around all the time, and they're always checking on the other cells around them. But what Jonathan found, and again, just to repeat, Jonathan van risen, is that these these macrophages, these, these immune cells were, in fact engulfing the newborn cells in large phagocytic cups, that living newborn cells, they were actually then killing and consuming them. Basically, by consuming I mean, they, they, they, they degrade them.

Nick Jikomes 55:28

So so these are effectively like immune cells of the brain. And just like a white blood cell or something, and your body might eat bacteria from an infection, they're eating newborn neurons in this case, newborn astrocytes, newborn astrocytes, okay.

Margaret McCarthy 55:44

Yes, yes, yes, that's exactly right. That's exactly right and in an exquisitely tightly controlled manner, which is what's so awesome fasting and that control is coming from the Endocannabinoid tone. Right, so the higher endocannabinoid tone in the male's acts on the microglia to make them basically give them the munchies. It makes them hungrier, and they consume more of the newborn astrocytes. And so the females wind up with more of those astrocytes surviving, and so that as they get to the time of the juvenile play time, the females medial amygdala has more resonant astrocytes than the male does. And you still say, Well, what does that have to do with with play? Well, what Jonathan Van Morrison is showing now is that those astrocytes are actually inhibiting the play neurons, because they're releasing a adenosine, a chemical called adenosine. So the more astrocytes you have, the more inhibition you have in the play neurons. And the males have escaped from that break, basically, killing off their astrocytes when they were young.

Nick Jikomes 56:49

I see. I don't want to go. I don't know if I want to go on this tangent. But But you said adenosine is involved here. That immediately makes me think about caffeine. Oh, wow. Yeah, I don't know. Okay, so there's some. So basically, development is happening in males and females naturally, under normal conditions, you'll have different levels of endocannabinoids, there'll be higher in males, I think, is what I'm hearing. And that will make the microglia hungrier and they'll eat literally eat and digest these things called astrocytes. And that sort of releases the ability to have more play behavior in juvenile males and suppresses it in females.

Margaret McCarthy 57:28

Exactly. Exactly. And interestingly, they only eat the newborn astrocytes, right? They like the babies, which is once astrocytes in mature they don't seem to consumer.

Nick Jikomes 57:38

So a natural question here is, you know, similar to what I asked before about deprivation of play, you said that if you deprive a male of its ability to do rough and tumble play with other juveniles, it has downstream consequences, negative consequences on the adult behavior, both on the aggression side and the reproduction side. What happens to adult behavior? If you say, masculinize, the females or do the reverse does something similar happened downstream?

Margaret McCarthy 58:08

Yeah, that's an excellent question. And we have not done that experiment. Because I think that so if we gave hormones to do that experiment, it like to peripherally say that it would maximize everything, right, because it would invoke another, another mechanism in the hypothalamus and another mechanism in the hippocampus, etc. If we give the endocannabinoids. That's interesting when we have to do so. So when I give a newborn female testosterone, I turned her brain male. And if I take her to adulthood, and I want to see her do male sex behavior, I have to give her testosterone again, right? Because no, even a normal male will not show Sex behavior without testosterone. But you raise an interesting idea. I have not thought about if we gave the endocannabinoids. Would that would could we get them with just adult testosterone to start to show me like sex behavior? That's a That's an interesting idea. I gotta think about that.

Nick Jikomes 59:10

Yeah, I mean, yeah, I mean, one potential prediction would be that yeah, if you're, if you're masculinizing, the player behavior and the juvenile behavior, play behavior is causally related to adult reproductive behavior, you know, would would, at least in the presence of testosterone would have masculinized female start exhibiting male like mating behavior as an adult?

Margaret McCarthy 59:32

Great idea. Great idea. I like that. I think we'll try it.

Nick Jikomes 59:36

Interesting. So I mean, what else can you say about this general subject? Is that Is there anything that that we've missed in terms of what your lab has studied with respect to the Endocannabinoid regulation of this particular dimorphic behavior? Or did we kind of cover most of the story there?

Margaret McCarthy 59:51

I think we've covered most of it. Yeah, we're now trying to hunt down you know, further details on exactly which cells have the receptors and what who's making the end contaminants and things. But

Nick Jikomes 1:00:01

I mean, another thing worth talking about. So you mentioned that, you know, if you give some of these things, whether it's hormones or endocannabinoids, you know, it different phases of development can have a very different effects can even have the opposite effect depending on on when you give these things. So can you talk a little bit more about about sensitive periods? And like, what, what is determining that? Why is it that giving, you know, a molecule of some kind, whether it's testosterone or an endocannabinoid or THC or whatever, why what's actually allowing it to have two different effects? You give it relatively early versus relatively late?

Margaret McCarthy 1:00:34

Yeah, yeah. Another really fascinating question why? Why does this what makes the sensitive period end, alright, would be a way of putting it and that's a really, we know that we operationally define the onset of the sensitive period as the time when males feel tested start to make testosterone. But why do females eventually become insensitive? Why can't I just give a female you know, 30 days old, and change her brain into male, and it used to be because we thought, well, the the events that are changing are things like cell death, right, and cell proliferation, and the wiring up of synapses and things like that. And those are all considered permanent endpoints, right? You can't under die. And you know, you can only have me reborn, and then we think of synapses. Synaptic wiring is like, you know, plugging up an extension cord into the wall, right, and they're there, I've made my synaptic connection, and there, it will stay forever. But interestingly, we now know all of those things are much more plastic than we thought synapses come and go. And actually, cells continue to proliferate and continue to die throughout the brain. And it's because we used to talk about the blueprint for sex differentiation, the neural architecture of sex differentiation, I don't, I don't use those words very much anymore. Because one of the surprising discoveries that we made along the way it was, as I mentioned before, those steroid hormones, they are transcription factors, but they also appear to be able to modify the genome epigenetically and epigenetic modifications are when it's, you know, every cell in your body has every single gene, right, but it only expresses a subset of it. So that's why a hepatocyte and your liver stays in hepatocyte. And retinal cells stays a retinal cell, right? Because if they started expressed the wrong genes, you have to silence those genes epigenetically. But the brain appears to have a different set of rules. When it comes to epigenetics. It uses it much more plastically to modify gene expression for you know, in response to experiences and things like that. And one of those experiences is whether or not you've been exposed to hormones. So a graduate student in the lab named Bridget Nugent was very interested in the epigenetic modifications of induced by steroids during development. And she just looked at one of the modifications as DNA methylation. You put a methyl group onto of cytosine that cytosine of mine, cytosine, thymine, adenine, and she found that if she looked in the preoptic area, which controls male sex behavior, there was more methylation on average in females than males. So there was more gene suppression, right and right, and it turned out, that's because in the males in response to the testosterone, they actually turned off the enzyme that puts methyl groups on the DNA. So they expressed more cells and the females silenced cells. Well, she can use a drug called zap valerian, which actually also inhibits that enzyme, the same way that testosterone does. This is actually a drug that's used in cancer therapeutics, because a lot of cancers are escaped from epigenetic suppression. And if she gave that drug late, outside the sensitive period, it kind of peeled off the methylation groups on the DNA, and it made the females sensitive again. So so that so we came to the conclusion that the female brain has to constantly maintain a suppression of the male transcriptome in this particular region, because the male transcriptome sort of wants to burst through and so they, she sort of maintains it in a silent state, but if you peel it back, you can get back to the you can get the mail pinner. Fascinatingly, it turns out, that's also something that's going on in the gonads. I know you think you couldn't get more terminal than an ovary versus a test. It's right. But there's a particular gene in the ovary that if it is silenced at any time, if it's turned off, testicular tissue will begin to develop. And you'll have what's called an ova testes have a mix of ovary and testis. So we have to really, you know, really rethink things about how permanent a lot of these changes are really opens up a whole lot about the plasticity that's potentially there. You know, I think about evolutionarily you know, what is the optimal way to go you could be Mother Nature's like always hedging your bets, you know, you never know When I might need to change back between the sexes kind of thing, and so don't make it permanent. Just make it, you know, enduring.

Nick Jikomes 1:05:09

I see interesting. So you know one of the things we've talked about the dimorphism at the level of the juvenile play behavior, and we've talked about it at the level of you know what's going on in terms of what the cells are doing and things like endocannabinoid tone. So at least for the for the males, for juvenile rats, the males have higher endocannabinoid tone. And that is an important piece of why the play behavior differs between the sexes. We also know that cannabinoids and cannabis products in humans often affect men and women differently. And this brings us to the question of how dimorphic the endocannabinoid system itself tends to be in the brain and in the body. Can you speak a little bit about that?

Margaret McCarthy 1:05:51

Well, you know, I don't know that I can. I don't think I have a lot of expertise on it throughout the party. I've really only studied that in the medial amygdala.

Nick Jikomes 1:06:03

So one of the things I want to ask you too, is it sounds like you've explicitly created rap models for studying the effects of cannabinoids, including THC on development, so, you know, rat models of you know, what is in utero THC exposure, you know, what kind of effect is that going to have on the juveniles and subsequent adults that they develop into? What, what have you done there? And what results have come out in terms of what's already been published? And how does that start to make you think about the the human implications? Yeah.

Margaret McCarthy 1:06:35

Okay, so there, we did a collaboration with Matt hill at the University of Calgary, in which our laboratory injected pregnant animals with THC and his laboratory set up to do THC inhalation through a vaping mechanism. And then Matt is the world's leading expert in measuring endocannabinoids and also THC in the brain. It's really it's actually really, really hard to do because they're lipophilic molecules. But anyway, so I measured the THC in the placenta, and in the maternal circulation, and in the fetal brain. And they measured also metabolites with THC, some which which are active, and some of which aren't both the the inhalation mechanism and the injection mechanism, because the argument being well, you know, people don't inject themselves with THC, right, they smoke it, or they eat it. Both of those resulted in an enormous accumulation of endocannabinoids in the placenta. And in some cases, in some time, points it collaboration between maternal plasma, THC and metabolites level and fetal brain levels. So, so an enormous amount was getting into the fetal brain. So that that has implications obviously, for cannabis use during human pregnancy. A woman named Yasmin Hurd at Mount Sinai has done a lot of work on humans and their ingestion of cannabis during pregnancy. It's increasing at a very rapid rate because of perceptions of safety. And I think we need to know you know, if it's safe or not, it might be safer than say alcohol or opiates, but we don't really know yet. And partly to is a lot of the literature that we had on it was done back in the 70s and 80s. And the THC content at marijuana at that time was completely different than you know, people are smoking a lot of oregano oil right now, it's different. Yasmin has found some very spiffy has been heard and at Mount Sinai has found some some deleterious effects in humans. And she's tied that to some specific mechanisms. We so far, you know, we don't see anything like I would say, like deleterious per se, we see like natural variation in behavior. But again, I it's, it's just in our rats, but it really does speak to, I think, kind of like, we know you shouldn't drink alcohol during pregnancy. And if the main question is why smoking cannabis during pregnancy, for some women, it's the nausea. And of course, Nausea is who's very serious, you know, it can be really devastating. But But I, I guess if I were talking to a young person today, I would say don't don't think it's safe compared to alcohol, because we just really don't know yet. is like, you know, the old saying used to be, if you smoke your baby smokes. It's the same for for cannabis.

Nick Jikomes 1:09:41

Yeah. And I suppose that even if we don't know a lot of the details yet, it's it sounds like it's safe to say that certainly cannabinoid exposure in utero is going to have some effect on the developmental trajectory of the brain.

Margaret McCarthy 1:09:55

Yeah, I mean, I think you know, we've done a deep dive into one particular brain Region connected to one particular outcome. But this the endocannabinoid system is one of the very first to develop in the brain. And the receptors for the cannabinoids are considered the most abundant G coupled protein receptors in the brain. So they're so ubiquitous and widespread that it's hard to imagine that there isn't some impact, and also that concentration in the placenta, there and then the cannabinoid receptors are expressed all throughout the reproductive axis. So it might even not just be direct effects on the fetal brain, you start messing with the placenta, you're going to really impact fetal brain development, as well. So which has been shown quite a few studies recently, some of them by Tracy Bale, who's shown that placental abnormalities following maternal stress can impact the developing brain so so yeah, I mean, we, you know, here in the States, we've been really hampered in our ability to study THC. It's extremely difficult to get, I have to have a DEA license. That's the Drug Enforcement Administration, it's takes over a year to get the license, and tremendously tight monitoring of it. And, you know, people always say, well, can't you go out on the corner and buy it, which is absolutely true. But I can't do research with what I can buy on the corner. I can buy it legally, you know, through medicalization, etc. But because the reason it's so I can get x ecodan are easier I can get cocaine easier than I can get THC. Yeah, and the reason is, because there is no agreed upon medical use for THC. There's medical use for oxy codon and cocaine. Right. And so that's one of the reasons there's such a push to legalize the medical use of cannabis. So that because we're really hampered in our ability to study it.

Nick Jikomes 1:11:58

You were getting a little bit of feedback from something on the desk, I think. Yeah, that's me missing. Sorry. No worries. Another area I would love just like your general take on that. I mean, I don't know too too much about this. But, you know, my understanding is, you know, given everything that you studied to do with development, and sexual hormones, and all of this stuff. My understanding is that a lot of the stuff that we're eat humans today in the modern world, a lot of the stuff that we're eating that's coming in our diet, a lot of the stuff in our environment contains stuff, whether it's with trace amounts, or even non trace amounts of drugs and things that are influencing all sorts of stuff in our body, including sex hormones and things like this. So what I mean, what can you say about that? Do you have any concern as a sort of neuro endocrinology person about the effect, the modern human environment is actually having on our development and on sex hormones and all this stuff?

Margaret McCarthy 1:12:52

Yeah, yeah. So you're talking about endocrine disrupting compounds? Yeah. Which are found in plastics and in fertilizers and and fungicide fungicides and things like that. And yeah, it's such a poor, it's such a difficult field. It's a, it's, I used to do some endocannabinoid disrupting research. And it, they're the best evidence that they are, in fact, in the environment, or having damages not dropping sperm count in men, that doesn't seem to be the case, it's an increased incidence of hypostasis. In boys, what does that that's, that's when the urethra does not grow all the way to the tip of the penis, and it exits along the shaft. And it's, you know, it's a devastating condition, and it has to do with insufficient, you know, impaired antigen actions very clear, we understand it very well, kind of thing, the other you know, and there's no doubt if you measure the fat tissue, if you measure breast milk, etc, all of these compounds are in our bodies for sure. You know, it's funny, back in my day, it was led right it was led in the gasoline and things like that, and we knew you know, definitely having effects and got that all cleared out. You know, there's not you don't detect lead and kids blood anymore, but now you can detect this phenols and etc. And it's much it's not as clean cut as the effects of of lead were, but there's certainly the fact that we still allow raid to be used, you know, all of the pesticides and things like that are Yeah, this is big. There, there. It's scary. But that being said, humans are probably healthier now, Americans, you know, other than our abysmal maternal health, maternal fetal health statistics, but just not has, does that have to do with environmental plasticizers you know, the nutrition that we have now is so high that that's, you know, I think those are counteracting forces. You know, puberty keeps advancing in human and particularly in the Western world, I think and particularly in girls who estimated about six months a decade that puberty is occurring earlier and earlier. And it's really hard in that case to separate that out from the the nutrients that we have now the increased light, you know, artificial light, etc. Or endocrine disrupting compounds, you know, what is sorting those out is so hard.

Nick Jikomes 1:15:24

So puberty is advancing. You said in human girls at about six months per decade, it's getting earlier and earlier. Interesting. What about what about and boys? Is that a same thing or something similar happening harder

Margaret McCarthy 1:15:37

to measure puberty onset and boys. So but it's the, it seems more so in girls that they can do by breast development. It's like they move down, like, because there's a thing called precocious puberty, right? So when he goes through puberty way too soon, and it's as low as eight years for African American girls. Oh, wow. That's very precocious. But, yeah,

Nick Jikomes 1:16:03

and for some of these endocrine disruptors, can we just be explicit for people? What, what are some examples of these? And can you just sort of list again, very clearly what types of consumer products they tend to be associated with?

Margaret McCarthy 1:16:16

Well, yes, been a while I'm not as expert on this as I once was, but the bisphenol A's it was one of the big concerns and they were at one point and a lot of plastics, including baby baby bottles and everything. And that's why now you look on the bottom bottom of any plastic bottle, right, and it has that little, that little triangle, and there's a number in the middle. And that tells you that I think that whether or not there's this phenol in there, I don't think any drinking bottles have them anymore. Then there are anti fungicides that are used on like fruit trees and things. flus and all I believe, that are antiandrogen like, and there's, you know, they're on on grapes, and apples. And like, particularly like if you're getting grapes from Central America, which most of our grapes come from Chile, and things like that. Those are a concern. Why? There's, you know, the chemicals that we weed killers, raids and things like that, that are just, I mean, they're just, they're just bad. They're not just endocrine disruptors, they're just nervous system, toxins, you know, anything that you're using to kill, kill plants or animals is going to be killing something in your body as well. So

Nick Jikomes 1:17:28

I see. So there's just a lot, there's a lot of nasty stuff in the fertilizers and yard products that we use in the food that we eat. And in the physical products, in some cases, just plastic bottles and things like that.

Margaret McCarthy 1:17:41

Yes, yes. And it's very, very hard. I mean, there's, there's the number of new chemicals that are manufactured is crazy, crazy and hard to keep track of, and they can just change the single molecule and the, you know, again, just like how I said how hard it was to study THC, it's really hard to study these, these compounds, because the tradition is still sort of the classic toxicology, looking for, you know, lethal dose, you know, 50 kind of thing. And instead of looking for the more subtle, subtle reproductive effects and things, and, you know, there's all this evidence and things like amphibians and reptiles and seeing lots of intersex and, you know, why are all the frogs dying and et cetera? You know, there's even things like birth control, started birth control is phenomenally stable. And so it can be detected in the water columns all the time, because just through through the wastewater, because they're those compounds are designed to be stable so that they can pass through the human liver and not be digested.

Nick Jikomes 1:18:46

Oh, I see. So you're saying that some of the molecules that come from the forms of birth control we use just are so stable, they get out into the environment than we were all ingesting them at some level? Yeah. Interesting. And what are some of those compounds? What do they tend to be? Oh, ethylene Ester

Margaret McCarthy 1:19:03

die all nor progesterone, etc, anything, you know, that's in any of the classic birth control?

Nick Jikomes 1:19:11

Do you think this is related? plausibly, at least, I don't know, if we know the answer. Is it related to you know, the decrease in sperm counts that I know that we've been seeing dramatically?

Margaret McCarthy 1:19:19

Yeah, well, that's why I mentioned that I don't think that that those data are sufficiently reliable. The decrease in sperm count and increase in breast cancer, both suffer from the same problem of our methods of detection and quantification are so much better, right? So if you're better at detecting breast cancer, you're gonna detect more breast cancers, right. And if you get better at quantifying sperm counts, you might be much more accurate. So the last and it's been a while since I've looked at the literature on that, but I don't think I don't think that that's held up worldwide. There are certainly certain pockets where there are big effects on sperm counts and stuff and particularly like, you know, men who work a lot with fertilizers and things like that are heavy metals heavy. medals are always a big problem in industry and stuff. But this kind of global idea of has not held up.

Nick Jikomes 1:20:07

I see. Well, we've covered a lot of ground already. A lot of fascinating stuff that you told us about. Is there anything you want to leave people with? Or or summarize for people that we talked about just to do with the general subject of sex differences in the brain and behavior?

Margaret McCarthy 1:20:21

Yeah, I sometimes I get sort of pushback on that we shouldn't study sex differences in the brain because it can be abused, you know, and that's always a risk with any science and the idea that, you know, it will, it will just confirm existing biases about women's biological constraints. But I would say that that's not, that's not a reason not to, because of the discovery potential of studying sex differences. I don't just do it, like I said, for any. But my rats don't. They don't do mathematics. You know, they they don't write poetry. They don't. There's nothing to do with humans, you know, cognitive abilities or anything in the studies. But what they do reveal is fundamental mechanisms by which the brain develops and enormous, beautiful, extraordinary mechanisms by which nature has found to develop the brain. And right now, the textbooks and science textbooks are written based on the male brain, and they've missed a whole lot of varieties of ways that the problems have been solved in brain development. And, and that's to me is the that's the reason I studied is because it's like, I always have I've got a contrast agent, right. I can see things that you couldn't see if you just studied one sex because you wouldn't have you wouldn't have a way of knowing it was different.

Nick Jikomes 1:21:45

All right. Well, Dr. Margaret McCarthy, thank you for your time. It's been my pleasure.

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