This is the Convergent Science Network podcast. Leading researchers in the domain
of neuroscience, brain theory and technology are interviewed by Paul Verschure and Tony Prescott.
All right. It's Paul Verschure with Tony Prescott here for the Convergent Science Network podcast.
And we're here with Daniela Stolzenberg, who was one of the speakers at our school, BCBT school.
And you focus very much on maternal behaviors.
Yes. So why do you think that that's an interesting paradigm to look at?
Well, I started my work in maternal behavior because I was really interested
in how the brain regulates behavior, social behavior.
But I find that maternal behavior is particularly fascinating because there's
such a dramatic change in responsiveness for most mammalian females around the time of birth.
And so I think it's a question that's interesting on several levels.
There's a motivational component, there's a learning component as I spoke about
today, and I think it's a pro-social behavior. So I think you're sort of looking
at multiple different aspects of social behavior.
And obviously it's extremely robust and defining characteristic of mammalian behavior.
But now the framework you took to look at this is what you call epigenetic, right?
So what do you mean with that?
So my interest in epigenetics is really in trying to understand how these initial
experiences with infants lead to long-lasting changes in the behavior of the mother.
And so I see it as sort of an initial experience that can program this memory
and really maintain differences in how neurons are functioning in response to infant stimuli.
And so in that sense, these epigenetic changes that I'm interested in have to
do with sort of an altering of the phenotype of the neuron,
within the circuit that regulates maternal behavior.
So the work is pointing in two directions here. So one is understanding maternal
behavior in mammals and its origins.
And the other is understanding epigenesis in animal development more broadly.
And in your talk, I got the impression
that there are exciting new developments in both of these topics.
Yes. So, I mean, if we go with the epigenesis one, so can you just define for
us more clearly what's the difference between genetics and epigenesis?
Sure. I think about it really on a molecular level.
So when I think about epigenetics, I'm thinking about alterations in gene expression
that are not associated with changes in DNA sequence.
And so for me, the most straightforward difference between genetics and epigenetics is
is this sort of molecular distinction, the distinction between alterations that
are not coded within the DNA sequence,
but that are affecting when and how genes are transcribed versus the actual,
a gene or a gene mutation.
And so, for example, I could give you an example.
Within the context of maternal behavior, one of the things that we know is that
is that dopamine neural systems are important for the display of maternal behavior.
So if we took a genetic standpoint, we could look at mutations in the dopamine
gene, dopamine receptor genes, and see how that influences maternal behavior.
If we took an epigenetic standpoint, we might say that some sort of experience,
might upregulate dopamine receptors for long periods of time or up-regulate
the dopamine gene for long periods of time, but there's no actual mutation or
alteration in the sequence of the gene.
Does that... It does make sense, but I'm still trying to get the difference
between epigenetics and development.
When is an aspect of development epigenetic rather than just developmental? I...
Again, I sort of always go back to this idea that it's a molecular difference.
And so when an aspect of development is epigenetic, it really has to do with
alterations to either the histone proteins or methyl marks that are on the DNA.
So you're taking a very literal, it's got an impact on the DNA inside the cell
in some way, and then it's epigenetic.
Whereas a developmental change which for instance
involved uh i don't know uh alterations to
the strength of some synapses it's not epigenetic well it it could be depending
upon what the mechanism is so i think i'm taking sort of a mechanistic definition
and that's how i think about it um is from this sort of mechanistic approach
um and so uh so you know again when
I think about epigenetics and thinking about these mechanisms that drive changes
in gene expression that can be influenced by the environment.
So, and I talked about that a little bit today in terms of neural activity can drive these changes.
But there's, I think, a certain amount of epigenetic mechanisms that are involved
in sort of the naturally developing organism.
And I think it's sort of convenient that activity dependent changes can modify
these processes that are occurring naturally.
And in the theme of our week, which is evolution and development or evo-devo,
what's interesting about these epigenetic processes is that they can be passed
down through the generations. They can be selected for.
And you think that's the case. In your talk, you made a clear case as to why
that might be true of kind of maternal behavior in mammals.
Yes. But what was it that led you to the suspicion that we could analyze maternal
behavior from an epigenetic approach?
I think in terms of maternal behavior, I think that it really comes down to
thinking about it as a learning and memory question.
And when you think about learning and memory, there's a couple of sort of known
mechanisms that underlie the consolidation of learning.
And so one of these has to do with new protein synthesis, the turning on of genes.
And those processes are sometimes regulated by CREB.
And CREB recruits a CREB-binding protein, which is a histone acetyltransferase.
And so the question of how these new proteins become synthesized,
how these genes get turned on, and whether or not that process lasts for a long
time, and how that plays a role in the consolidation of learning and the maintenance of memories,
those things are being explored from an epigenetic perspective.
Perspective, and I've always been very interested in learning and memory.
And so I spent a lot of time reading about these things, and I had never thought
about epigenetics from that standpoint before, but it's sort of like a cellular memory, really.
And when I thought about it in that context, and I thought about my own research
having to do with the same sort of question, really, which is this experience
that's driving this behavioral change that lasts for a long period of time. It seemed that um.
There might be an epigenetic mechanism underlying it. So the clue,
or one of the clues for why it might be epigenetic rather than some other form
of learning and memory was that, I guess, it happens in mammalian species,
all different mammalian species.
It happens, I guess, quite abruptly at the point where a mother gives birth.
They suddenly begin to express these maternal behaviors. And once the change
has happened, it stays with you.
Yes i guess for the rest of your life if you're a mother yes
yeah and and for you that was a if you
like a those were a cluster of markers which suggested epigenetics
rather than some other mechanism yes and i should add to
that um another clue really um was the fact that um hormones are involved in
facilitating this mother-infant bond and facilitating the consolidation of maternal
learning and um it's been established for quite some time that these steroid signaling pathways.
Modify chromatin.
And that's one of the ways that they lead to these sort of long-lasting effects.
And so it seemed as another clue that particularly since I'm interested in this
idea of hormones and experience somehow being interchangeable,
and I've been really interested in whether or not there's a common molecular
mechanism through which hormones or
experience might be impacting behavior,
it seemed as though maybe this would be a good candidate mechanism.
And in support of this idea that it might be involving CREB and CREB-binding protein,
there's also evidence that CREB is increased in the medial preoptic area,
the central site for maternal behavior,
and that that increase in CREB, you see this in virgin
mice that are interacting with infants and you also see it
in postpartum animals that are interacting with infants can
you just unpack what you mean by kreb oh yes sorry
so um so kreb is activated um
through kreb for stands for cyclic amp response element um binding protein and
uh kreb is a transcription factor that's activated um by neural activity and
so um intracellular signaling cascades as a result of neurotransmitter binding can activate CREB.
And when CREB is phosphorylated, it can turn on genes. And...
This CREB-mediated gene transcription we know plays an important role in the
consolidation of memories.
You mutate CREB, you get disruptions in ability to learn and consolidate.
But now, if we talk about maternal behaviors and also pregnancy,
what is the overall hormonal profile that goes along with that?
Because it's not like a single hormone that will impact that,
right? No, it's definitely not a single hormone.
However, that being said, the rise in estradiol, I think, plays a really important role.
And there are other hormones that can act, I think, downstream of that.
So for example, one of the things that this rise in estradiol does is that it
can turn on oxytocin receptor gene expression. And so it sort of plays this
role in priming the circuit.
And so then when we get these surges in oxytocin, the receptors are there and present.
So I sort of always think about things from estradiol's perspective since it's
sort of the first wave and needed for some of these other hormone effects.
But estradiol could also be a switch sitting at the beginning of a long cascade
of responses to that switch.
Yes, yes. Yeah. As you describe it also now.
So then if you don't want to look at, let's say, the intrinsic components contributing
to changes in behavior and the experiential components that are in the environment,
and that would be key in an epigenetic perspective,
how do you see this trade off against each other?
Is it really like very strictly regimented? Like, okay, in this phase,
we're under hormonal control.
Now birth has taken place and now we're going to be under environmental control.
Or are they more interleaved?
I think they're more interweaved, although I'm not entirely sure what you mean
by environmental control.
You mean the experience? Experience, yes. The environment, right? Right.
So I think that when it comes, I mean, I think that the hormones are allowing
for the experiences to have a greater impact.
And I don't think that the hormone, I think the hormones probably play a role
in increasing attractiveness of infant stimuli.
And in the RAP model, which is really where we know the most about the role
of hormones, I think they're probably playing a role in reducing fear and avoidance
tendencies towards the infants.
But it's the experience of interacting with the infants in the context of those
hormonal changes that really increases maternal care and leads to these long-term changes.
And so if you just had those hormones, for example, if you were to,
and this work was done by Alison Fleming, if you were to remove the pups just
as the female was giving birth, and so when she did these experiments,
she actually sort of had females giving birth in this cage where the pups could fall through.
So she wasn't able to interact with the pups at all, but she did deliver them and give birth to them.
You remove those pups immediately, you don't give her an opportunity to interact.
She doesn't show any changes in her maternal responsiveness towards infants
once those hormones have waned.
So clearly those hormones can play a facilitary role, but I think their role
is fairly useless unless infant stimuli are present for her to interact with
under those hormonal changes.
So these rats that delivered the pups and never handled themselves would not
show any kind of, let's say, enhanced maternal response in future interaction
with any other pups? Yes, that's correct.
This is what you're saying. They behave as though they're the virgins.
Mm-hmm. Okay. Yes, yes. So then what's...
What's the key data that you have obtained to make the case for this sort of
epigenetic interpretation of maternal behavior?
Right now, I think my key work is really with using sodium butyrate,
a histone deacetylase inhibitor.
And so this is a drug which inhibits the deacetylation of histone proteins,
allowing for increases in histone acetylation under certain events.
For example, the experience of interacting with infants.
And so when females are interacting with infants in the context of this particular
drug, we see a facilitation of this learning effect,
suggesting that there may be a histone acetylation component that naturally
occurs when females are interacting with infants,
leading to these long-term changes.
So I think, because we're not molecular scientists, this is difficult for us.
But the exciting thing I thought from your talk is that you think you've identified
a cellular mechanism where experience is impacting directly or indirectly on
the DNA and turning on and off the genes that generate maternal behavior.
And so maybe we should just explore that because there are key ideas in your
talk, like the idea of epigenetic marks,
which seem to be very general and quite powerful ways through which developmental
mechanisms and experience can impact on the expression of genes and therefore
could lead to hereditable influences.
So it's a way in which the environment can select for different phenotypes.
So can you explain this idea of epigenetic marks and how that relates to these
processes you're describing in the cell? Sure, definitely.
So when we talk about epigenetic marks, we're really talking about a variety
of post-translational modifications that can occur to the histone proteins that
DNA is super coiled around.
And so these modifications occur on the tails of the histone proteins,
which sort of protrude out.
And the marks can be phosphorylation, acetylation. So the DNA inside the cell
is wrapped up inside of lots of other proteins.
Yes, right. So we say the DNA is not… You've got to start easy with us.
I'm sorry. You've got to start easy with me. Okay. I'm sorry.
Yes, so the DNA, the phrase is the DNA is not naked in the nucleus,
which we sometimes think, you know, that it is.
I mean, I certainly didn't think much about histones before I got involved in epigenetic research.
Research um and so but but the dna
is not naked in the nucleus it is wrapped around these histone
proteins and so the nucleosome which is
the basic repeating unit um consists of
a core octamer of histone protein so there's four proteins uh histone h uh h
h1 is the linker we have h2 h uh h2a h2b um h3 and h4 and so and then we have
two copies of each one of these and so they form this octamer.
And they have, again, as I said, these long protruding tails,
and these tails are where the modifications take place.
And then around this octamer, you have about approximately 147 base pairs of DNA.
And so the modifications can be acetylation, which is an acetyl group added
to a particular residue.
Frequently, it's a lysine residue of the histone protein tail.
And this can occur on histone H3, histone H4, histone H2A, H2B.
And in addition, you could also have methylation marks that occur on these tails,
the histone protein tails, along with phosphorylation marks and ubiquitination.
I mean, there's many different post-translational modifications.
I think we know the most about acetylation, but I think we're we're starting
to learn a bit about methylation.
So just to be clear, so the DNA is wrapped up in this package with all these
histones. In order for the DNA to become active.
It has to be unwrapped in some way. And by attaching molecules onto these histone
tails, then some external mechanism can decide which of the genes is allowed
to express itself within that cell.
Yes, you can sort of think about it. I like to think about it in the promoter
region of a particular gene.
And this promoter region is where the transcription factors are going to bind
in order to turn on gene transcription. In this promoter region,
you can have histone tails, right, sort of nearby.
And these histone tails then can contain different types of marks.
So I would argue the mark, at least the mark I'm the most familiar with,
but I think a mark that is fairly well known is this acetyl group on histone H3 at lysine 9 or 14.
And this mark is associated with actively transcribing genes across the whole genome.
And so excellent genome-wide sequencing experiments have been done to look at this.
And so this mark, and what acetyl marks typically do, is they can actually sort
of loosen the association of the DNA and the histone.
And so it sort of opens up physically the chromatin, allowing for transcription
factors to more easily access gene promoters.
And so it's easier for the gene to be turned on, essentially.
So the idea is that experience with pups is having some effect on the cell,
so that epigenetic marks are being generated, which are binding onto the histone,
and then unlocking some genes which then generate maternal behavior.
And this is exactly what I think, that with several experiences with the infant,
that what we have is essentially an increase in histone acetylation.
I haven't actually shown this yet. This is in the works.
But what I have shown is an increase in the histone acetyltransferase,
binding protein, binding protein, or CBP.
And so, as I said, you know, CBP was my candidate because it sort of made sense
with the whole Krebs story and learning and memory, et cetera.
And so my hypothesis would be that there'd be an increase in the acetylation of H3K914.
This is a mark that's associated with this Krebs-mediated gene transcription,
CBP action, acetylation, and the consolidation of learning. So that would be my hypothesis.
And there's also, you know, there's several other marks that I'm interested in exploring.
And when you think about learning and memory, you always have to think about
the other side of the coin too, which is, I think, inhibiting the expression
of genes that would be reducing consolidation or reducing.
Acting as a consolidation block.
And so that's, I think, definitely a part of what I'd like to- Yeah.
So there's this threshold process that it has to build up and there's another
process going on, which is trying to deconstruct these.
Absolutely. Now, in terms of the threshold, which I think this is sort of a
perfect segue into, again, why I got interested in this.
When you think about the fact that hormones are facilitating this process,
and with hormones, we don't need so many days of experience, right?
We can have very brief periods of experience and we can get these same effects
in terms of maternal learning.
And so what we know about estradiol is that estradiol can, when it binds to
its receptor, translocates to the nucleus and is a transcription factor,
which then turns on genes.
And there's many genes that have estrogen response elements
or respond to estradiol estrogen receptor
complex um but part of that
complex is actually cbp so
cbp is recruited as part of that complex and
so probably there are increases in histone acetylation um via cbp just with
hormone exposure alone no it doesn't make sense because i thought you made a
point earlier that the the histone acetylation would be dependent on the experience,
because it would be the mediator to get the maternal behavior,
and you already made the point earlier that just being pregnant and giving birth is not it, right?
Yes, well, yes, that's absolutely true.
And so, again, I think that the hormones are there, the hormones in this scenario
where we have the surgeon estradiol.
The pup inputs would also need to be present. And so I don't,
I mean, you raise an excellent point.
And I think the answer to this probably lies in understanding all of the genes
that get turned on and what sort of the order of events is.
So for example, estrogen receptor could be turning on a gene,
which then affects, it could be turning on a transcription factor,
which then affects another gene.
Could be turning on, so they could be turning on different genes.
And I don't know the specifics, and I think we actually know relatively little
about the genes that are involved in these two separate processes.
So I would like to get to behavior, but I don't know… There's a few more steps
that I'd like to understand here.
So the estradiol is sort of priming or facilitating an impact that the behavioral
experience is going to have on the cell.
No, we cannot say that because the virgin mice exposed to pups will also show maternal behavior.
So you don't need the estradiol to do anything.
Well, you don't need the estradiol, but it still plays a facilitary role. But it's not required.
But it's not required. Yeah. I think that's what we're saying is that if it's
there, it makes it easier.
Well, does it reduce the threshold of exposure? Yes. I don't think you showed that.
No, I did not show that. No, but that's… I thought in all cases it was four
exposures that we were talking about.
So, in my, that's, so, that's what I've shown. But it's.
Very well established in the literature that estradiol produces
very rapid effects compared to experience takes a
bit longer um what i showed with four experiences with postpartum females which
is correct is i wanted to show that if we match the amount of experience that
we would get the same level of responsiveness between a postpartum animal um
and uh and a virgin or ovariectomized experience to females.
Let's say it's a facilitator.
Let's finish on the mechanism.
And so the cells that this is operating on, you found were a specific group
of cells in the hypothalamus. Is that right?
Yes. So my effects are, in terms of turning on genes, have been found in the
medial preoptic area of the hypothalamus, yes.
And why did you look in that bit of the brain, or did you look more widely?
I did look more widely, but I chose
the medial preoptic area because it's considered the central neural site
for maternal care um and so for example
if you lesion the medial pre-optic area you disrupt maternal behavior um
and if uh if you stimulate it uh
with hormones um uh you
can facilitate the onset of maternal behavior um if
you stimulate it with the dopamine agonist
you can facilitate the onset of maternal behavior and that
work has been been done mostly in rat but so are
you saying that your hypothesis
is that some combination of hormone and experience is switching on a population
of hypothalamic cells so that some some genes which are important for maternal
behavior are now on and previously they weren't yes that's correct that is that
is correct but also can you say to follow up on that.
That it exclusively happens there, so that either estradiol release or exposure
to PAPs does not lead to histone acetylase in any other neuron in the brain.
No, and I don't think that that's the case. I think that absolutely there are
other regions within the circuit that we already know are important for maternal
memory in terms of what's… But they would show the same effect.
They would also show histone acetylase in response to these stimuli.
I think that they would. I just haven't found them yet. Yeah.
So, you know, I've always been very interested in the role of the mesolimbic
dopamine system in maternal behavior and maternal motivation.
And so one of the areas that I looked in addition to the pre-optic area was the nucleus accumbens.
Um the genes that i chose as candidates did
not change in the nucleus accumbens um at
any of the time points so the um so i
most of the work that i showed you was with the 24 hours
after their experience looking at gene expression there
but the um the uh uh time course that i did with um uh 30 minutes and five hours
and 24 hours which um which was a which was a full experiment it just It just
wasn't published because there were no other effects at any other time point, right?
And there were no effects that I could find in the nucleus accumbens.
So I certainly think that something is happening in the nucleus accumbens.
I just could not believe that there would be no change there.
I just haven't located the change yet.
So to get to the behavior, so we have some activity in the hypothalamus that
wasn't there before, which is triggering maternal responses to pups.
And the indicator of that which you've been using is whether a female rat will
retrieve a pup that's isolated away from the nest and would die without help.
Exactly. And there's various measures of that, one being the time it takes for
the female rat to retrieve.
I think you had three pups. Yes. But you were also talking about other measures
of maternal behavior, for instance, licking and grooming.
And I think you said that these don't all appear at once. Is that right?
If it's a virgin rat or if it's just happening that these are maybe triggered
at different times? What you usually see is sort of this stereotyped onset,
which flows sequentially.
So when you put the pups into a virgin female's home cage, which is how we start
all of our experiments, and you sort of scatter them out, what the female typically
will do is she'll sniff them immediately.
Immediately um and she'll sort of run from
each you know pup to pup sort of sniffing she might
sort of handle the pup and lick it a little bit um but
usually um she'll engage in sort
of just this sort of sniffing and walking around and investigating the infants
for um several minutes if this is the first day for example and then what you
typically see is um one of two things sometimes you'll see a female come out
and retrieve start to retrieve the pups immediately
sometimes what you'll see is her actually
attempt to build a nest first and then come
out and retrieve the pups to the nest now i've
worked with rats and with mice and i can tell you that when when you see this
onset of behavior in rat it's very stereotypical and it is very much like a
switch it's a it's a very sort of all of a sudden on behavior and they're very very organized.
And they sort of, when they decide that they're going to retrieve the pups,
they come out, they retrieve the pups, they group them back at the nest,
they lick and groom them, and then they sort of rest,
you know, they become more quiescent in a crouched posture as though they were
nursing. Obviously, they're not lactating.
The process in mouse is a little bit less organized.
It's one of the reasons why I decided to look at experience in mouse.
So again, you might see retrieval right away. You might see nest building first.
And often when they retrieve the pups, they don't actually put them into the
nest on the first time. They put them outside of the nest.
And then they sort of obsessively fling bedding around.
And sometimes the pups fall out. It sort of takes them a little while to figure
out exactly how to get the pups into the nest and how to get on top of the pups in the nest.
Which is something you don't see in rat. And so once the female has sort of
grouped the pups, then she'll start to lick the pups.
And then again, she'll begin to quote unquote nurse.
She'll adopt a crouching position over the pups. And then she typically stays
with the pups for long periods of time.
And I should note when females are on, when I've done these experiments with the HDAC inhibitor.
We don't see differences um really
in in these other behaviors in the
home cage so the experience that an a virgin
female getting regular water versus a virgin female
getting the h-deck inhibitor the experiences they have with the pups
appear to be similar so we don't see more licking and grooming for example um
it's the impact of that experience that seems to be different based on their
subsequent behavior on the maze so the h-deck inhibitor inhibits inhibits the
histone as Histone deacetylase.
And so it's sort of, it's allowing, I like to describe it as it's allowing for
more histone acetylation.
It's not directly acetylating histones. It's allowing for any histone acetyl
transferases that are activated to become activated for longer,
to acetylate histones without HDACs coming along and ripping the acetyl groups
off. But then how do we interpret this effect?
Because the histone STLase is correlated with memory consolidation, right?
So if you now have a disinhibitor of that, you would expect,
what would be the effect you expect on consolidation? It should amplify consolidation.
Yes, yes. But actually, that's not what you see when you apply this HDAC.
No, you do see an amplification of consolidation.
So rather than requiring four experiences with pups, these animals only require two.
Okay. Do you see also an invigoration of the behavior? Like the probability
to build a nest is higher?
So the experiences that they have, again, in the home cage are not different.
So the behaviors that we see them engaging in while they're having these experiences
are not different. It's just how they respond on the maze.
They respond faster and they retrieve more pups.
Okay. So now how do you explain the specificity of this effect?
Book so to examine the
specificity of the effect um because the behavioral
effect that we're seeing is really that they're rescuing quote-unquote these
pups from this sort of novel and fear-inducing environment um the first thing
that i looked at was whether or not these animals had a reduction in anxiety
and we don't see any changes in their behavior on an elevated plus maze with the hdac inhibitor.
Uh, we don't see them responding, um, more to, um, objects that aren't pups,
but are the same size and shape of pups. Um, and,
Those are the two main sort of behavioral specificity experiments that we've done.
But it might be possible that what you're looking at is also a nonspecific memory effect, right?
I imagine you would have your animals perform a maze task or any task involving
memory, that also there you would see amplification of performance because you
just boost memory in a nonspecific way.
I think that that's possible, but I don't think that it's necessarily likely, and I'll explain.
And I'm certainly not the only person who's used sodium butyrate or an HDAC
inhibitor to facilitate behavior.
What you typically will see in experiments when people do this is the only effects
that you get with the HDAC inhibitor rely on whatever the animal was learning
when it was being treated with the HDAC inhibitor.
And I think this speaks to the mechanism of how the inhibitor works.
It's allowing for increases in histone acetylation. It's not globally increasing histone acetylation.
And so what that would mean then is that if other areas of the brain weren't
responsive to pup inputs or if other circuits that would consolidate memories
were not being activated,
which I would argue they wouldn't be because these animals are just interacting
with pups, then you wouldn't get activation of HATs.
In those regions. Sure. And so therefore it would basically not matter.
But then if we would take the same mice and do let's say a place preference task.
Right. Yes. Under the same manipulation you would predict. I would predict yes.
That they would learn this also more rapidly.
I would predict. Yeah well I guess I would predict that.
Because I would say that their motivation is amped up. Because that's one of
the things that I think maternal learning does.
So then the histone acetylase itself.
It's not specific to the maternal behavior, but it is affecting the memory component
that feeds into, in this case, maternal behavior, because that's what you're
triggering in the animal.
Sorry, let me clarify my last statement. When you said conditioned place preference,
I was thinking conditioned place preference for pups.
No, no, no. I would mean just like with using rewards or stimuli.
I don't know. I don't think that it would affect that. I do think it would affect
a conditioned place preference for pups, which people do all the time.
So I think it would affect another sort of memory pup-related task.
I don't think that it would necessarily extend to a general learning and memory.
Although I think the issue with that that would make it difficult to ask that
question is that we already know
that experience with infants facilitates learning and memory generally.
So we wouldn't know if it was necessary. I mean, we'd have to do the full experiment
to ensure that it wasn't just… But you have not done these controls to rule this out. No.
Might be a nice thing to try. Yeah,
well, I agree. There's a long list of experiments that I'd like to run.
So the hypothalamus is a conserved structure in vertebrates,
but maternal behavior is a marker of mammals.
And I don't think other vertebrates care for their young.
Certainly they don't give them the same amount of attention that mammals do.
So it would be interesting, I guess, in the future to find out whether this
mechanism that you've described, if that's how it operates,
whether that is something that originates with early mammals,
and whether it's operating throughout different mammal groups as well as in rodents.
I mean, do you have any ideas in that direction, or do you have goals as to
how you're going to answer those kinds of questions?
I think that's a really interesting point, and I
don't have any experiments planned to address that but i think
it would be very interesting i mean the medial pre-optic area is also
involved in other sorts of things in addition to maternal
care right um but uh but certainly that's you know i mean it's involved in sexual
behavior as well but right um but it definitely does play a very central role
in maternal care so i think it would be interesting do you think this this mechanism
you're describing uh is is going to be happening for instance in primates.
Yes, I definitely think that it could be happening in primates.
But then to look again at the hypothalamus, right?
Hypothalamus to actually trigger behavior has to actually drive other structures,
right? It has to talk with brainstem structures.
We talked about central gray.
And associated areas where you really know behaviors are encoded, right?
So the behavior itself doesn't come from your hypothalamus. Hypothalamus is
like setting if you want to switch or a signal like, okay, now we're going to go maternal, right?
Yes. So what's that circuit in which it's embedded that we should consider in this case?
So that's an excellent question. The circuit and really the way that I think
about it is I am not that interested in the stereotyped behaviors.
I'm interested in the medial preoptic area and its interactions with the mesolimbic
dopamine system to get at sort of this general motivation for infants so that
if I put them in any sort of environment, they would be responsive.
And so the medial preoptic area gets inputs from every sensory domain,
and it projects to many regions.
But the aspect of the circuit that I've always been most interested in is the
aspect that seems to regulate these changes in maternal responsiveness,
this maternal motivation or this increase in care.
And that aspect of the circuit is mediated by these pre-optic connections to
the ventral tegmental area, which cause the release of dopamine into the nucleus accumbens.
And the nucleus accumbens receives inputs from the basolateral amygdala,
the prefrontal cortex, and those regions also project to the ventral pallidum.
And so there's important interactions between the nucleus accumbens and the ventral pallidum.
I definitely think that that part of the circuit is another place where memories can be consolidated.
What I think is probably happening in the medial preoptic area,
it's probably the connections between, and I have no evidence for this,
this is just my hypothesis,
the connections between the medial preoptic area and the ventral tegmental area
are somehow strengthened so that we're getting a more consistent and robust
release of dopamine in the nucleus When experienced females interact with pups.
And this idea comes from some work also done by Alison Fleming,
where she shows in RAT that as females, pregnant postpartum females,
as they have experience with infants,
that they have more dopamine release via microdialysis into the nucleus accumbens
than do females that have just interacted with pups.
So is this, could we then interpret this circuit?
If you talk about VTA and a nucleus accumbens, we're talking about the processing
of value and effect and valence and so on, not necessarily the information that we want to reinforce.
But there's a mechanism of reinforcing a behavior.
Behavior so would it then look like the hypothalamus
is just signaling to the rest of the brain okay we're
dealing with pups and and then these effect
related areas vta nucleus accumbens say yeah and
it is great and using that signal you reinforce behavioral patterns that allow
it to be more effective in dealing with pups would that be the cascade yes i
agree and there's some projections that are back go feed back to the medial
pre-optic areas so that would that make But then the medial preoptic area could
be, let's say, your specialized detector,
if you want, for maternally relevant states of the world. Like,
oh, there are pups around.
I should do something. Is this roughly how we should think about it?
Yeah, I think that maybe. Yes, yes. Okay. But then….
So, you would see then this preoptic area in hypothalamus as the signaler who
triggers this behavior, right?
Yes. But then that also means there's already on the one hand a predefined circuit
that says, okay, there's a pup there.
There are stereotype behaviors that you can like collecting pups and dragging them around.
And then there are areas that, let's say, are more frontal in the brain that says, okay,
and this is what the world looks like, and this is the specific properties of
the pup I should learn about, and so on, that then I'm reinforcing by talking,
by driving these more effect-related areas.
Yes. That would be the sort of story. Yes.
If we talk about epigenetic and genetic, then the more epigenetic part would
then be sitting more in these more frontal areas, or not?
So where does the genetic begin and end, and where does it become epigenetic?
I think that really when I think about genetics, I'm thinking about these patterns
of genes that might need to be turned on in order to maintain this maternal state.
And so some of these genes, as I mentioned in my talk, estrogen receptor beta,
I see, for example, upregulated for long periods of time.
Um and you know i should mention
that francis champagne's work which shows that early life
experience can modify initial maternal
responses to infants and postpartum females as regulated by an upregulation
of estrogen receptor alpha and so um so i think this sort of long-lasting change
in estrogen receptor beta um would be the epigenetic part but you can't have
that without without the actual gene of estrogen receptor beta.
In terms of the other areas of the circuit, as I said, I haven't found anything
yet, but I certainly think that there are likely changes there.
It's convenient to focus on the medial preoptic area because although it's playing
this sort of central role and interacts with all these different regions,
it's also always necessary for maternal behavior.
So maternal behavior does not become independent of the medial preoptic area
that we can find in our models.
And so it seems like a really likely place that would need to be permanently
changed and maintained in that maternal state for long periods of time.
So I think when we think of the hypothalamus, we think of a motivational center.
And in a sense, what's happening here is that you're turning on a part of the
hypothalamus, which then motivates animals to express maternal behavior.
The behavior themselves are triggered by other cues like there's a pup there, I'll look after it.
But what the hypothalamus is doing is ensuring that motivational circuits and
the reward circuits operate to reinforce that behavior. So it's a very nice
idea and a nice explanation of what people might call the mothering instinct.
But also what I like about this is
it's a nice example of a non-genetic mechanism, which you can see how...
During evolution, this happens and then it happens repeatedly and reliably sufficient
that it can be passed on to later generations without having to have any genetic
change. Yeah, and I think that's an excellent point.
So, I mean, obviously, you know, I think we have certainly moved past,
as a scientific culture, the idea of, you know, any one gene being critical
for something as complex as mothering.
But certainly, if there was sort of an evolution and changing in terms of responsiveness
towards infants, it makes sense that you would have this epigenetic change where
you're sort of using the genes that you already have.
But this change then is altering the way they're regulated in order to ensure
care. But just for clarity, also to clarify what Tony is saying,
because otherwise I might not agree with him.
Isn't it the case that we could argue that basically genetically a certain potential
set of behaviors and potential behaviors are defined?
But now you need very specific environmental triggers to actually unveil that set of behaviors or not.
Yes. This would be the epigenetic switch. switch yeah we
agree i agree don't you agree with that or get something else in
mind but that's not non-genetic it's just
you need an environmental trigger to now also start
to run a specific also genetically predefined program you have to have an environment
which is predictable in such a way that this happens despite there not being
any genetic modification but it happens and therefore you can rely on it happening
in future generations so that you can consider it as a something that can be selected.
Sure. No, I was just trying to be clear about what you meant with non-genetic,
but that's the trigger, the triggering of genetically predefined behavioral programs as well.
Well, yes. It's not like strictly non-genetic. I think that would be a bit too
extreme. No, I think that's true. It's not strictly non-genetic.
I think that any time that you talk about epigenetics, you have to talk about genetics.
They go hand in hand. But what I got from what you were saying,
which I don't know if this is the case, but I thought that you were indicating
that there wasn't some new mutation that arose.
Yes, that's what I'm trying to say. And that mutation then was… Right.
And that's, I think, the best… I mean, this is why I like to sort of rely on
these molecular or cellular definitions of epigenetics. it makes it a lot clearer.
So when you talk about a gene mutation, that is strictly genetic.
Whereas when we talk about epigenetic, we're talking about altered expression of particular genes.
Right, but then the next question in some sense is, okay, how deep and primitive,
if you want, is this genetic pre-specification, right, on which we then run
these epigenetic switches?
And there you showed that male mice, at least the lab mice after exposure can
also display maternal behaviors.
So this might seem confusing because you could argue also from an evolutionary
perspective, we don't need to set up these genetically predefined behavioral
programs to show maternal behavior,
yet your mice show it. So how do you explain that?
Well, I would argue that the circuit that's regulating maternal behavior is conserved.
And I would argue that what we're seeing in these laboratory male mice is we're
seeing really maternal behavior.
So they're not like by parental species where we're seeing individual fathering
behaviors that are different than the mothering behaviors.
We're seeing the male mice display mothering behaviors.
And so what I would argue is that everything that they need to do that is there.
But what typically happens is that those processes are inhibited by,
and this is clear in the rat, that there's really two aspects of the maternal neural circuit.
There's an aspect which is in place to actually inhibit maternal care.
And this circuit is what's active in virgin female rats.
And what prevents them from retrieving pups upon initial exposure,
what causes them to avoid pups.
And this has to do with olfactory inputs, activating the anterior hypothalamus.
And having an inhibitory input on the medial preoptic area.
To my knowledge, no one has looked at that aspect of the circuit in males,
but I wouldn't be surprised if those olfactory inputs about pup-related information
were acting on that part of the circuit and inhibiting the medial preoptic area
from responding to infants.
And perhaps for whatever reason, under laboratory conditions,
these animals are inbred.
They're constantly smelling pups because usually we all, everyone's raised in
the same room and it's sort of an artificial scenario. And I think that.
Maybe there's some sort of habituation that has led to a downregulation of that
circuit or sort of led to that circuit not being activated as much as it would have been activated.
And so all you need to do then is present them with PUPs, and they're not avoiding those PUP stimuli.
And so if you present them with the PUPs enough, you can get the other aspect
of the circuit to increase. And so what I'm referring to here really is this
approach avoidance model of the onset of maternal behavior, which has been established in rats.
And the model really states that avoidance tendencies have to be decreased and
approach tendencies have to be increased.
And when those two lines cross, that's when you get maternal care.
Right. Clear. But it also means in terms of behaviorally, these males display
identical behaviors as the female rats.
So including presenting themselves for feeding.
Yes, they do. But I would argue that that's reflexive. You can see that in rats, too.
And so all you really need for that is pups.
If you orient yourself over the pups, the pups will induce you to show those crouching responses.
Right. Okay. All clear. Tony?
Okay. So we had now this exploration through the epigenetic landscape of maternal behavior.
But what's the next step there? Where do you see this go?
So there's many more questions that I'd like to explore within this particular model.
And some of them I've alluded to, and particularly really defining these mechanisms.
I would love to be able to look at all of the histone changes and really characterize
which marks are coming up during these experiences, when they're coming up and
how long they're coming up for.
So one thing I should note is that epigenetic changes, while they can be stable,
they can also be very dynamic.
And so you can have changes in histone acetylation increasing and decreasing over a couple of hours.
You can also have changes in the activity of other what I would call chromatin
modifiers, so things like DNA methyltransferases.
And I didn't talk about DNA methylation, but one of the things that I have found,
which which I did not present today,
is I found changes in the gene expression of DNMT3 with DNMT3A,
which is a de novo DNA methyl transferase, meaning that it adds methyl groups
that were not previously on cytosine residues.
And what exactly is happening with that change in DNMT3A,
I'm not entirely sure, but it would indicate that maybe there's some methylation
that's being added or modified at least in these animals with experience.
So I think there's a lot of mechanism that needs to be sorted out.
And clearly there's some interesting behavioral experiments that I'd like to do as well.
And I see some of these questions being asked in males as well.
But now, so here you are, upcoming light of epigenetics, right? Right.
So, but if you could now define a single law that we should follow to understand
brain and behavior, what would be this Daniel Stolzenberg law?
A single law? Yeah, one.
I'm not sure if I understand what you mean by a single law.
Well, what would you tell your students? Like you have to follow this rule to
be successful so we can understand the brain. What's the one rule?
What's the Daniel Stolzenberg law to understand brain and behavior?
I think that it's worthwhile to investigate a natural behavior when you're interested
in how the brain is regulated. So ecological validity for you would be key. Yes.
Okay. And then, so Tony likes traveling.
So he will come and inspect your lab four years from now. And then he's going
to carry a little note that I wrote.
And the note will say, look, Tony, you really have to check whether this prediction
that Daniela made four years ago was really confirmed.
So what's the one prediction you would like to make today that Tony can come
and test to see if it actually happened four years from now?
In terms of my data? Yeah, better.
I think, well, the one thing that I would really like to do,
as I said, is characterize these changes.
But I don't want to just sort of characterize them randomly.
I want to conduct a series of sequencing experiments using chromatin immunoprecipitation,
the method that I talked about earlier today, where you're really,
I like it because you're sort of killing two birds with one stone because you're
asking not only about which epigenetic players might be involved,
but you're asking about where in the genome and what genes they might be acting on.
So today I showed CBP being recruited to estrogen receptor beta.
But if I could ask where in the genome is CBP or acetyl H3K914,
where are these marks when a female is consolidating these maternal experiences?
I think that that would be a really important contribution because it would
give us so much information in such a more rich and global way than what I've been able to do so far.
Okay, great. Then, you know, Stilson Burke, thank you very much for this conversation.
Thank you for having me. Sure.
Well that was fun yeah thank you
thank you i think it helped a lot to clarify things
right to to also get away from this idea
like oh we have this this one sort of magical cell in
the thalamus and the hypothalamus and it gets
switched and then it all happens right yeah definitely circuit um
it's that has been very good it's it's
hard to know exactly what what to
present it's funny because i had all these circuit slides in and
i took them all out at the last minute but um
you know it it's uh i i think
that um i've always really thought about
things from a circuit level and it's just it's just it was very depressing to
me that i could find no changes anywhere else in the circuit which is sort of
what's interesting is that maybe this maternal behavior is just recruiting other
behaviors and modifying them
so slightly so i mean we We talked about cannibalism in various species,
and the fact that monodelphus just cannibalizes its young when it doesn't recognize them.
It's quite interesting, because you can imagine that the retrieval mechanism
is not that different from the cannibal mechanism.
I grab it, and I drag it back here, and then rather than- No, it's very different.
No, Tony, there's a really important difference, because what I understood about
mice, pregnant mice, because mice are living in colonies, right?
And they all, the pups in the end are taken care of by all of them in some way.
It's not really very specific, if I have it correctly, right?
So what these mothers do, they kill the pups of other mothers,
so there's more capacity for those other mothers to take care of their offspring.
Okay, so the cannibalism is very specific, specifically focused.
I'm not saying that they're not deliberately killing certain ones,
but I'm just saying that the actual… You're saying like the actual act.
The actual behavior, the sequence of actions that you do. It actually looks…
It's not that different. You just have to grab a bit tighter.
No, wait, I wanted to say what we call cannibalism.
There are actually many different kind of sub-behaviors underneath that we should
not summarize with that one word. because cannibalism might mean it's for food,
it might mean maternal competition, or it might mean an accident.
If we call all of this cannibalism, I think we're confusing ourselves unnecessarily.
I think I'm making a different point, which is that… Look, I got to run.
I'm sorry. To get maternal behavior, you don't have to evolve a whole lot of new action patterns.
What you have to do is you say, well, in this context… I don't buy that at all.
I don't buy that at all, and I will tell you that during our reception.
Okay, we can talk later. See you later. Yeah, thanks.
I might say to that point, though, Tony, that the behavior looks really different when you see it.
Okay, well, I could be wrong with that example. The way that they approach the pups.
But I agree with what you're saying, generally speaking, in terms of like how
many behaviors does, you know.
You've got to think about how you evolve internal behavior.
And I don't think you evolve a complete set of new behaviors because what you
can do is you can say, I've got some existing behaviors.
I'm going to parameterize them so that I retrieve the pup.
Normally, maybe I'm catching something, bringing it back to eat.
Now I'm catching something and bringing it back to nurse.
Take care, yeah. You don't have to evolve a whole new set of strategies for
fetching that object back to the nest because you already know how to do that thing.
I agree with that, and I would add to that point that something that's really
interesting that I've wanted to look at as well that Francis –.
Yeah, I think it was Francis Champagne looked at. She has this one paper about
weaning, and we really know very little about weaning, and particularly in mice.
And what she did was she kept these pups in the cage with the mothers after
day 21, which is typical weaning time, till day 28.
And what she ended up seeing, and she actually sent me the video,
so I saw this in real life, even though I tried to replicate this,
but I think I did some things wrong and I couldn't see it.
But the females actually, as the pups get older and the females are,
I'm assuming, not wanting them to nurse anymore, they actually pin the pups
and they basically start thrusting against them. Right.
And, I mean, it's just really bizarre. But, you know, it's sort of like we've had this conversation.
And I guess it makes sense how many different behaviors, right,
are available to, I think, to a mouse.
Else and i think that this speaks to exactly what you're saying that
you're not going to sort of evolve this new behavior to get the to
sort of actively wean your offspring instead you're
just going to use a behavior that you already have that clearly the pups are
not going to enjoy i mean downstream i think there's an argument to be had about
what how many different mechanisms there are and how you recruit different mechanisms
but um you know sort of the old ethologist idea of fixed action pattern i mean
it's not it's not completely right it's not
completely dead either but then you can imagine you know sort
of this action pattern is triggered by the stimulus i do
it or i take that action pattern i change it a little bit i've
got a new action pattern which does something else yeah so so i mean you what
you want to say is well okay maternal behavior evolves in mammals what what
has to evolve for that to take place and it it may not be that many steps away
from what you can already do uh as a reptile but certain Certain things obviously have to change.
But in animal-like monodulphus domestica, you can see that it's fairly crude.
So that whether it nurses or cannibalizes an infant is based on possibly a single
cue, whether it smells right.
Yes. And yeah, I think absolutely. I think absolutely that's involved.
Anyway, thank you very much. Thank you.
So now you can relax. Oh, you have another tutorial? The CSN podcast was produced
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