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.
So Paul Verschure with the Convergent Science Network podcast here at BCPD15
together with Tony Prescott. Scott, and we're talking with Stefan Noctor,
who is a great expert in development of the brain.
And so, Stephen, you started out by laying out this whole problem of,
okay, how can we actually control from an evolutionary perspective the size of a brain?
What are the mechanisms underlying that, right? So, why are you concerned with
that specific question?
Why do you think that's the most important one to answer? Well,
I wouldn't say it's the most important one to answer, but it's certainly interesting to me.
And my initial interest, and it still is my main interest, is understanding
the precursor cells that are producing, they're the engine for growth.
So they're producing all the cells, identifying them,
characterizing them, and then figuring out what cell types they produce under
what conditions and defining their functioning in a normal brain,
normal developing brain.
And hoping that that will establish a firm foundation for understanding disease
processes and how they influence development of the brain.
Right. So what are the different preparations that you look at or have looked
at? I use a variety of animal models.
So the bulk of my work has been in rat.
And we do culture models where we will inject different vectors into the prenatal
brain and make cultured slices and observe the cultured slices under a microscope
for four or five days at a time.
And then to make sure that what we're seeing is something that's physiological
and not a culture artifact.
We'll inject vectors in utero in animals and allow them to survive the same
amount of time and then compare the endpoints to make sure we're ending up with
the same thing to make sure that what we see is real and physiologically relevant.
Right. So the standard model, if you want,
of brain development is this whole idea that we have sort of zones where cells
are being generated that then sort of swim out radially from these zones where
we generate these cells.
Is that sort of the standard model we should still think about with any brain
we look at or any vertebrate brain we look at?
It's true mostly for the excitatory cells. So we spoke a little bit about that
earlier today. But the excitatory cells.
For the most part, have a radial trajectory. They migrate out along these radial
fibers that are sort of like the spokes on a bicycle wheel, and they're deployed
in the growing cortical gray matter.
But the cortical interneurons have a different origin, and they have much longer
roots that are migrating.
They're migrating perpendicular to the radial deployment.
And yeah, that's a whole other field. I'm looking primarily at the excitatory cells.
But now the excitatory cells that you talked about actually are showing a very
rapid growth in terms of numbers, not the sheer numbers.
I mean, you mentioned something like five billion cells in seven weeks,
which sounds astonishing.
In the human brain, yeah, in the human brain, yeah. So in what period is that
growth explosion really taking place?
Well, we're really only now starting to get
a handle on human brain development because there
have not been a lot of experiments and uh there's studies
now showing that um cortical excitatory
neurons in human are being
generated uh up until birth and
then the interneurons may be um just a
little bit longer so throughout pregnancy
but starting at maybe six weeks
and throughout the whole time we don't have
a good rate yet so we don't know if it's tailing
off you know do we reach a maximum at 20 weeks and then it starts tailing
off those sorts of data we don't have yet on the
human brain so it's been well mapped out in mouse and in
rat and to you know to some degree and ferret and
and we're starting to get a better handle on on
primates like a rhesus macaque so now
you um so in
this process of of the the construction
if you want of a neocortex um there
are a number of principles at work and what's really fascinating in your talk
is also how you look at this from a more historical perspective to say look
actually people start to have some ideas about that process early on So what
do you see really as sort of the highlights or maybe the forgotten highlights
of that history that we should bear in mind here?
The highlights of the forgotten history. Well, it was known well over 40 years
ago, actually 50 years ago, that there was a generation of new neurons in the postnatal brain.
And there was some hints, some evidence that it might be occurring in the adult brain too.
And that's something that we began to rediscover in the 90s and early 2000s. Mm-hmm.
But now, so in your work, what you have been sort of also pioneering is to bring
different technologies together to really get a handle on the movement of cells
in the ventricular zone and the subventricular zone that builds the cortex,
but also their specialization and differentiation.
So what are the key technologies that you use for that? up?
So my approach has been, I think, relatively simple, and it just requires patience.
So labeling cells with fluorescent tags, which became available in the late
90s, so that you can label fluorescent cells, and then using an appropriate
vector that will label some of them but not too many.
Because if you have a field of similarly labeled cells, you can't follow any single one of them.
So you need a small population of labeled cells.
And then preparing a a culture condition that allows the cells to move.
So it probably took me about a year to get the culture conditions right so that
the cells would proliferate, divide, and then once the new neurons were born
that they would migrate.
Once that was working, then it was just a matter of being patient enough to
sit there and take pictures every hour,
every half hour, depending on what's going on until you couldn't
take it any longer um but to watch
it happen over like five days these are beautiful trajectories that
you you showed in the in the movies that
you put together sort of time lapse and you really see cells sort of moving
inside a brain and for people that normally think about adult brains or think
about neural network models the thought that these that these actually get up
and move from one side to another and then change direction that's quite astonishing.
I mean, obviously it has to happen, but to, and you talked about the distance,
that neurons having to travel being the equivalent of several skyscrapers.
And so that also is an amazing thing to know about brains that we don't often think about.
And there's a special offer, the precursor cells that you're talking about.
And are these cells that are really just there,
at the earlier stages of development or do we
lose these i mean are they are they very kind of so it's a great question so
i think the we don't really have a good handle on the lifespan of an individual
precursor so right so are they always giving rise to somebody else that takes
on the job and continues on and then they pass it on i don't think we really have a good handle so um,
Does a single precursor cell generate neurons dedicated for each of the cortical layers?
There's some hints that maybe they do, but we don't really know that well enough.
The experiments that I did were overlapping.
They would last four or five days, but they wouldn't continue for the entire period.
Right. And then at the end, there's still some left. And where do they go?
Okay. And in hippocampus, presumably, the embryonic cells give rise to precursors
that remain in the hippocampus, because we know it's now well known that you
have continued neurogenesis in the hippocampus throughout life.
Well, dentagyrus, you know? So what makes it a precursor cell?
Is it just that it gives rise to other cells? Yeah.
And to me, that's a crucial point because there's one term that people use,
neuroblast, and some people use it to mean a young immature neuron that's migrating.
Other people use it to mean something that can divide.
But yeah, the fact that it can undergo division. Would it not be possible to
take a cell, a neuron, and turn it into a precursor? I mean, you could...
There are some people who believe that can happen under pathological conditions.
Okay, but under normal conditions, there are the precursors,
and then they give rise to these other neurons which don't proliferate further.
Right, yeah. So, yeah, the century-old dogma that once a neuron is a neuron,
it's not going to divide anymore.
And does each neuronal type have its own precursor, or how does that work?
I think we're still working that out, so what different cell types precursors
can give rise to. So, um, yeah.
One thing that is clear from rodent, and I think it's being worked out in different
models, but for example, the two basic types, the excitatory and the inhibitory,
come from different regions of the brain.
So you have in the basal forebrain in a structure called the medial ganglionic eminence.
There's also another one called the caudal ganglionic eminence that gives rise
to interneurons in the rodent.
And yeah, it's disputed. Some people
feel that the dorsal cortex which gives rise
to excitatory cells may also give rise to some of the
interneurons right so that's being that is being argued
so when you're identifying these
neurons and tracking them you're doing that primarily
based on their response to i mean some
markers inside the cell that you can use to
stain them primarily but then you're also able to
isolate some of these cells later and record from them right and
that that that's the proof that that really
is a neuron yeah if they were mature enough then you
you know or i would be able to distinguish an
interneuron from uh from an excitatory cell they have different firing patterns
right when you stimulate them but at the stages i'm looking at they have a very
immature action potential it's just a little blip it's not a full-blown action
potential um so it's really actually quite difficult to distinguish a neuron
from some of the other cell types that are floating around,
Yeah, you can, you know, with the approach I used, you could just say neuron
or not neuron. The glial cells have a very different response.
But that's something that I always believe. You see those inward voltage-gated
sodium channels, and that's believable, whereas if you stop and you fix the
tissue and you try to do some immunostaining,
first you got to hope that your antibodies are going to penetrate and reach
the cell that you're looking for, and that's not always the case.
And then you may have people who might dispute, well, that marker under certain
conditions can be expressed by different cells.
But now if you look at the taxonomy of precursor cells, so that would mean over
time, they also will differentiate initially.
I assume it would start with just a very few precursor cells initially for neurons.
And these would also differentiate.
So what do we know about that process? So, when do you see the first.
The initial precursor cell emerge, and how is that precursor cell then differentiating
other precursor cells that then form the plates which really start to generate our neurons? Mm-hmm.
Well, the terminology that's used in my field is that before cortical neurons
are being generated, people call them neuropathelial cells.
And then once neurons are being generated, the term switches to radial glia.
But there really aren't any good methods for distinguishing one from the other,
other than the fact that you have neurogenesis going on. Yeah.
So if you would compare the precursor cells for the inhibitory neurons that
are located somewhere else, versus those excitatory neurons on their own.
Yeah, those are different though. Okay, so already with two classes at least.
Yeah, yeah. Once neurogenesis starts, there are markers that exist today that you can put on.
So for example, the TBR2 marker that I used and showed in several of the figures,
that is not expressed in the ganglionic eminence where the interneurons are being generated.
And there are some markers that are more specific for interneurons.
But so once the developmental program really starts and I will have to force
you, I force you to give me a number, how many different types of precursor
cells would we have? Yeah, it's a great question.
You give me a number, you won't leave the room. Okay.
That's a great question. And so for all the different cell types,
I would speculate, and I'm just pulling a number out of the air,
but I would put it at less than 10.
And I could be entirely wrong, but easily five or six.
Do precursor cells have sort of stem cell-like properties that,
you know, there's some sort of equipotentiality there?
Yeah, depending on the time. Time. So as development proceeds,
they become more and more restricted in what they can produce. Right.
So the earlier you get them, they'll have a wider potential for producing different cell types.
So perhaps some of the precursor cells can produce oligodendrocytes and some types of neurons.
But the further along you get, the more restricted they become.
So your interest in the precursor cells
is potentially that they could be reprogrammed to do different things.
So a long way down the line maybe from here, but you started your talk setting
up all these problems with brain diseases which involve development.
And we could think about reprogramming these cells perhaps, how some cells might
be behaving as they shouldn't, and thinking about the programming that's caused that.
It's a challenging problem, because once you have a fully mature brain,
you know, bringing precursor cells into that equation, it's challenging. Right, yeah.
So now we are generating, so okay, let's say we have five, six different kinds
of precursor cells, and we start to generate our neurons, we start to build
this layered cortex. Mm-hmm.
What you started out by showing was something actually extremely weird,
which is that these neurons start to migrate, or the future neurons start to
migrate, and then they bounce about a little bit between the ventricular zone
and the subventricular zone.
And in this bouncing around, sort of differentiation might happen,
or they might divide, and two cells, you get a sister cell going off, and so on.
So what's your interpretation of this whole process of just bouncing around?
How long does this happen? How much movement do we have between ventricular, subventricular?
What's the speed of this movement? How coordinated is it?
So the speed of the movement has been fairly well characterized.
And it's most rapid during G2 phase. So when they're dropping down to the ventricle,
they can travel 70 to 100 microns in about two hours.
So that's the most rapid speed of transit and then they'll undergo division
at the surface of the ventricle and then in G1 they start moving away traversing
that same 70 to 100 microns roughly but that's very slow that can take 8, 10, 12 hours.
And the main idea right now is that it's a passive movement that those cells
are being forced away from the ventricle as others come down via an active process,
But you would expect that it might then stabilize at some point,
not if it's a passive process.
So why does it not stop? Yeah, I think we need to understand the process better.
But so far, molecular motors have not been found that if you knock them out, they don't move up.
So that under whatever conditions the cell bodies continue moving away.
They can stop movement down, but they haven't been able to stop it going up.
So that's the evidence pointing towards it being passive.
But the ones that are moving down and moving down quickly are sort
of actively tunneling their way to the bottom and they're changing their cell
properties as they migrate is that right they um they're entering prophase so
they they have a cell process which is tethered to the ventricular surface and
that nucleus is being pulled down within that process.
Right and they do a number of interesting things so there was this uh this the
first piece of work pointing it out from 1935, the gentleman,
Frederick Sauer, who discovered that,
pointed out that it wasn't a smooth even movement, but that they would move
down towards the ventricle, and then there'd be a little hitch up,
they'd go up a little bit, and then come back down.
And I noticed that in my time-lapse movies, that that's what they do.
They start coming down, then they seem to stop, they bounce back up,
and then they come down and divide.
So they're always tethered, are they? It's kind of like a yo-yo.
They're constantly tethered, yeah. But then there must be also a form of repellent.
The cells that move towards subventricular zone must be repelling those traveling
towards the ventricular zone.
Otherwise, you would believe the system would never stay in this dynamical state.
Do you have any kind of evidence for this of interaction between these cells?
No, but there's got to be something there.
Okay. Yeah, there's definitely got to be something there.
But then if you look at the migration, so if I'm… How do they establish,
you know, for S-phase and G1, when they're climbing up, they stop at a certain
point, and it's almost always the same point.
So they know where that is, and how they know that is an interesting question.
But do you see this in sort of standard morphogenesis terms,
like there is some sort of gradient maybe of RNAs or some other signaling molecule?
Not that I'm aware of, yes.
But that would be roughly the way to think about it.
Okay. But now, if I'm one of these cells, so here I am, I'm at the ventricular
zone, I'm going to move up.
How many trips do I make up and down?
So that would tell you how many cell cycles they've gone through.
In the time-lapse movies that I did, I've seen them go through two or three times.
On the average, it's two or three. In a time-lapse movie, right.
But in vivo, it could be six, seven, perhaps more.
That's what I was saying earlier. We don't know the life cycle.
So as long as that cell remains, that primary precursor cell remains in the
mitotic cell cycle, it's going to continue that bobbing up and down.
And we don't know if each precursor cell will maintain that movement throughout
the generation of all the cortical gray matter, or if you have overlapping subsets.
So there are people who believe that you have one subset that generates the
upper layers and a different subset that generates lower layers, but that's disputed.
So it's a problem that the field is still working out.
But now, in some sense, we often
think about this also in the cartoon you presented about this migration.
We think about it in terms of a bunch of ping pong balls that are sort of a
big aquarium as it is moving about.
But actually, what's going on really, it's not that these balls,
the somas or whatever you want to call them, are moving about.
It's much more that the processes are sort of feeling their way around.
They attach at different points and then they start to exert a mechanical force
to pull that soma in one direction or the other. So, so.
If we now start to rethink this whole process in terms of neural processes or
cell processes sort of feeling around in that space and making different points of adhesion.
So how do you imagine that? There's like a whole, a huge amount of spider webs
that sort of are being developed in parallel.
So how do I have to imagine that? Why do you think it don't get entangled,
for instance? Well, they start from the very beginning.
The precursor cells have attachments at the ventricular surface and at the peel
surface or the outer dorsal surface of the brain.
And in the beginning, you have a large number of symmetric divisions that expands
that precursor cell pool.
The existing cells maintain their contacts and
the their new daughter cells will establish new ones through
a mechanism that we don't understand fully but the
processes seem to grow in a radial direction and perhaps they use
neighboring processes as guides so they'll establish contacts and then they
maintain those contacts it seems once they have them they they maintain them
for the most part um then the there's signals external signals which i i believe are involved in,
attracting the cells to the ventricle and away from the ventricle.
And one piece of evidence that supports that is once the precursor cells begin
generating neuronal daughter cells.
If you're watching a whole clone of cells, what you'll see is when the mother
cell, when that nucleus starts moving to the ventricle, the daughter cells are
also making some downward movements too.
So they're perhaps responding to that same signaling factor.
So it's kind of interesting if you look
at an individual cell by itself you don't realize that it's
moving in concert with something else but but for you that's
a signaling system and not mechanical so it's not just a
combination of the two yeah combination yeah okay because you
need the microtubules of course you're fortunate to ask you what kind of receptors
these cells would have and what they would be responsive to yeah they um they
start expressing some of the standard neurotransmitter receptors in the rat
for example at e16 which is not too long after neurogenesis has become so um,
but that would not be on time to explain the basic movement no no,
well um that i don't think that's been worked out so um,
Yeah, starting around 15 or 16, you'll have some glutamate receptor expression.
What's dictating that earlier, yeah. But now in your time-lapse movies,
what I found really astonishing is that you had this very rapid emergence of
different kind of processes that were labeled by these specific cells,
but then also deformations of these cells themselves.
So do you see this being meaningful? like
for you also you saw one is really really squeezed down on
the ventricular zone it was deforming itself in some way there
was some process sticking out laterally yeah which seems completely orthogonal
to the direction which you want to move so how do you interpret these
kinds of variations um well when
i first saw that i thought perhaps this was a culture artifact but
and um just taking fixed
sections of animals that were you know
you're just looking at fixed time points we see some of the same things um
but we don't have we don't
really have an idea for what's guiding that right so
but now from the subventricular zone we
have to migrate further up to actually build the cortex yep right so is that
process under the same control as the one we had just been discussing no i probably
not and so that uh you know what's guiding the migration of newborn cortical
neurons, that's fairly well studied.
And yeah, there's a lot of good ideas for what's regulating that,
but I would assume that that's a different signaling system.
But it's also an inside-out construction system. Yeah.
While the ventricular-subventricular one is maybe not an inside-out construction
system because you keep on cycling between the bottom and the top.
Yeah. Whereas the inside-out, the newborn cells Cells are always migrating to
a position just under a structure called the marginal zone.
And there are a number of cells that are expressing important proteins,
including rilin, that are thought to play an instrumental role in the inside-out lamination.
Because in animals that lack rilin, you end up with a cortex that's almost inverted.
So rather than inside-out, it's outside-in. Right.
I was interested that you described this process where the cells actually head
towards the ventricular zone and then turn back. Initially, yeah.
So, I mean, do we have an explanation of what's going on there? Is it orienting itself?
So what I believe is that first process that emits that's being descended down
is a transient or a vestigial axon.
So it emits that process and the nucleus of the cell starts moving in that direction.
And then that process actually stays there. the cell then develops a new leading
process oriented towards the dorsal surface of the brain and it migrates away
and that initial process seems to stay there and it stays there for several days.
The trailing process ends up becoming the axon, but the mature axonal processes,
it'll be sending collaterals in the white matter that will be going tangential to that.
And then by the time the animal's maturing, this vestigial process in the ventricular zone disappears.
But I believe that that's a signaling mechanism, that's a feedback signal.
There's some kind of anchor that it leaves and then it heads upwards.
As it's heading upwards. And I believe that it's actually a feedback conduit
so but now in part of this process also what you what you illustrated is that
in order to migrate now out of of this whole ventricular subventricular zone,
um we have cell division right because we have to build more more cells but
the the orientation of this division again seems to be very systematic like
at the ventricular zone along a vertical plane and the subventricular zone along a horizontal plane.
Does that make any sense to you? Why would it have a difference there?
So the horizontal orientation for the cells in the subventricular zone,
I believe that that's dictated by the radial glial fibers.
I think the dividing cells are affiliated with a radial glial fiber and they
anchor on that and are kind of pulling themselves apart along that.
And the evidence for that is that in some regions of the brain,
the radial glial cells are not oriented radially, but have more of an S-shaped curve.
So the division, wherever it occurs along that S-shaped curve,
it retains that orientation along the fiber.
And then a further piece of evidence supporting that idea is that when the radial
glial cells have translocated away from the ventricle and they're all gone,
then the orientation of division in the submatricular zone becomes completely random.
So while there are radial glial fibers, they seem to have a preference.
And in our experiments, it was perhaps as many as 75% of the divisions would
be horizontally oriented.
And then as that percentage goes down, and by the time the radial glial cells
are gone, it's a complete random process.
It can also be an expression of just a mechanical bias that you want to have in the system.
Like if I'm at a ventricular zone and I'm dividing, I
have to do the packing and covering the space laterally so
if i if i divide across a vertical plane
at least i create a mechanical force that helps me to
optimize the packing of of my cells along this
lateral plane because i'm expanding radially so
if i have a small deviation in my lateral plane i'm at a whole
gap in my cortex that's pretty significant so sort of to optimize the packing
while in the subventricular zone i want to make sure my cells migrate outward
so now i i initiate the division in a way that already implies a mechanical
force that is biasing my movement in the direction where I want to have myself to go.
Would that be a reasonable interpretation or speculation, let's say?
Yeah, it's one of them. So this has been a.
A question that's been around for a long time. I don't know who first raised the question,
but the most common interpretation has been that divisions along what I would
call a vertical plane at the surface of the ventricle, where both daughter cells
remain at the ventricle,
that would be a vertical plane where they're sitting side by side,
that was initially thought thought to be a symmetric division that doesn't produce neurons.
And occasionally you have a division that's perpendicular to that,
in which one daughter cell remains at the ventricle and the other one is sitting on top of it.
And those were initially thought to be neurogenic because the idea was,
well, that top one is free to leave and migrate towards the cortex.
But I don't know who first proposed that idea, but one of my favorite researchers,
this Frederick Sauer guy, He mentioned it in passing in his article saying,
this is from 1935, saying, well, if this was the case, if this horizontal orientation
actually produced neurons,
there's just not enough of them to produce the millions and billions of cells
because it's an infrequent thing.
So we looked at the incidence of these divisions in rat and a number of other
species and it's, you know, maybe 5% or so. It's a very small percentage.
Um, my belief is, so I actually did a study where I tried to correlate that with outcomes.
That's why in the movies you could see I was measuring the angle of the plane
and, and averaging it across a number of different cells. And it didn't seem to correlate at all.
What correlated with cell fate was the time of development at a certain time
in development, regardless of orientation, you would have a specific outcome. Um.
But still, these horizontal divisions at the ventricular zone are very infrequent.
Yeah. Right? It would not be the standard pattern.
Right. So, the other thing we've seen now is that as I'm migrating out,
so now I'm going to climb.
Your comparison for the human case was seven Empire State Buildings,
I think, no? Four, but… Oh, four, sorry. Still, that's a lot.
Right. It's a tall thing. Okay. So, the ventricular zone and the subventricular
zone will get me to which floor?
Oh, that's a good point. they would still be within the first building,
maybe halfway up the first building. Okay, halfway up the, okay.
So, okay, here we are. And now I'm hitting the subventricular zone.
I'm going to be pushed out. I'm going in neuron.
And then you showed actually in substance gradually as I move along this distance,
I slowly start to express neural-like property, like sodium-dependent responses.
So how should we think about this? Yeah, so it's a matriarchal gradient.
So the earliest that I recorded a newborn neuron is maybe eight hours after
it was generated, and there were already detectable sodium currents in those
cells within eight hours after being born.
As they migrate further along, those responses become stronger and stronger.
Still, in many of the experiments that I did, within four days,
it's not a mature neuron.
It it doesn't have a full blown action potential and it can't fire repeatedly yet.
So they still have a number of days until they're much more mature.
So in some sense, if I know what day we are.
Of the animal, what day of development, and also how far I am from a ventricular
zone, you can sort of predict what kind of physiological properties that cell would have.
Yeah. So that's a pretty deterministic system then. Yeah. Okay.
So, now we have a bit of an idea how we sort of generate, right?
How we generate these many billions of cells that might form a cortex ultimately.
But what was really astonishing was your atom.
Because you said, look, to generate is one thing. We just have to sort of constrain.
You have to put boundaries on that as well.
And you pointed out to a very specific class of glia cells that you think play
an important role in also limiting growth.
So what kind of cell type is that exactly? And how did you discover it?
So the cell type you're talking about, they're called microglia and they're
the immune component cell in the brain.
And we discovered those in the laboratory because we were classifying all of the dividing cells.
We wanted to know what were the different types of precursor cells.
And these microglia are mitotic, and they represent about 5% of the dividing
cells at the stages of development we were looking for.
That's how we stumbled across them. And we're really excited to see that they
populate and they specifically colonize the proliferative zones in early stages of fetal development.
So they're very well studied and very well characterized in the adult brain.
And in the adult brain, they have an even distribution throughout the brain,
white matter and gray matter. They have this property of tiling.
But in the fetal brain, they specifically colonize the proliferative zones.
And that was very exciting to us to see that.
So we've been studying interactions between the microglial cells and the precursor cells.
And one of the observations that we've made is that,
They seem to like precursor cells. In fact, they like to eat them.
That's one of the mechanisms that we think is helping to put a brake on cell
genesis because through chance or through evolution,
these cells flood into the brain as cell genesis is going on,
and they specifically populate the precursor cell zones and begin consuming
them, and that gets rid of some of the precursor cells.
So we were not the first to make that observation.
After noticing that, I was able to dig up some papers by immunologists who had
mapped out the distribution in fetal human brain, and they saw that there was
this band in the precursor cell zone.
And their idea was that, well, they must go there in order to divide and make more of themselves.
And we followed that up by looking at where the microglia divide,
and they divide everywhere.
So it doesn't appear that they go to the proliferative zones just to divide.
I think they go there to eat, to feed.
Why are they microglia? I mean, are they like other glia cells?
Yeah, so in the mature brain and in a healthy brain, the soma is very small.
Right. And that's where that term comes from. So they have a very small soma
with these fine ramified processes.
Okay. So that's called a ramified or a resting cell.
In the adult brain, if you had some pathological condition, they
change their appearance they become what people
might call activated the soma gets much bigger
and the processes swell and you have fewer processes they have a completely
different characteristic look and for whatever reasons in the fetal brain they
are just super activated you don't see any of these so-called resting cells
they're they're just all jacked up they're they're ready to go so the environment
it's got a two-stage life cycle this these cells so they've.
They have this active phase where they're running around eating bacteria and other cells.
And then they sort of settle down, grow more processes, and become part of the furniture.
That's what we think, but we don't have a good handle on the life cycle.
The lifespan of an individual microglia. So the guys that are present in the
fetal brain, are they still present in the adult? or do they continue dividing
and producing descendants that will populate the adult brain? We don't know that yet.
That's something that we would like to try to figure out, but we don't know yet.
There's something else that's extremely strange, at least from my naive perspective of these neurons.
They're like space invaders, right? They are not intrinsic to the developing
brain, but they have invaded that brain from this embryonic sac.
So what's going on here? It's fascinating. Fascinating.
We need, you know, for our survival, we need a cell to perform this function.
And evolutionarily, it's worked out that they're introduced at this time,
and it doesn't impede development.
And in some ways, it may be helping it. It's definitely shaping the process.
But then, do they also infiltrate other organs, or they really are specific to the brain?
Traditionally, the microglia are classified as those cells which enter the brain,
And then they have related cell types, which would colonize peripheral or the body.
But in some way, they're like white blood cells. They're sort of having this role of cleaning up.
Yeah. So you have macrophages in the body and then the microglia.
And in general, it's thought that they don't mix very much under normal conditions.
But under pathological conditions, I think it's been shown that the macrophages can enter the brain.
But why don't we call them just macrophages or brain-based or brain-specific macrophages?
Wouldn't that be a more appropriate name for them?
I guess it's the inherited terminology. So we're stuck with that.
So these microglia come from the mother. So this might also be potential. From the yolk sac.
From the yolk sac, right? So this might be also an epigenetic mechanism because
this might also be a way for the mother during gestation to sort of modulate
a developmental process.
Is it also how you think about it? I don't think about it that way.
I guess I think of the yolk sac as part of the fetal organ.
But the question of whether maternal cells can enter the fetus is something that's interesting.
So there have been studies showing bidirectional cell transfer between fetus and mother.
But what proportion of cells that is, I think, remains to be determined.
That is a question that we're looking at. at the, are there fetal,
are there maternal immune cells or maternal cells of any sort in the fetuses?
But now, how did you really discover the effect of this microglia?
Because I could imagine that if you see for the first time a microglia engulf
another cell, that you might think, well, okay, that's some error.
This cannot really be the case. Well, it was, yeah, no, it wasn't difficult
to come to it because the first image my student showed me was this dense band
of microglia colonizing the proliferative zone.
So right away, we just did some staining for the precursor cell markers and
the microglia and immediately saw them.
And lucky for us, we were first working this out in primate because it's much
more prominent in primate than it is in rat.
So in rat, we can find the same things happening, but it's going on at a far
greater pace in the primate brain.
So it was just happening all over the place.
But now, is there some sort of ratio then between the propensity of neurons
to divide and to generate large pools of neurons and the prevalence of these microglia?
Is there some sort of magic balance between these two? That's a great question.
We've noticed that there are different proportions in different species.
There's a higher number of microglia in primates, potentially more in humans
than there are in monkeys.
There's more in monkeys than there are in rodents, more in mammals than there
are in reptiles and birds.
So there are differences across species.
So there might potentially be a way you could program these microglia to serve useful functions.
So for example, brain tumors, could you program them to break down cells in
the brain that were cancerous? That would be exciting.
But can you believe microglia can be made into something so specific?
I mean, how specific are they in their sort of... Yeah, I don't think...
In my system, I don't know.
I don't have an answer for how specific. It seems like they'll eat just about
anything because we find them eating young neurons, glial precursor cells,
neuronal precursor cells. They might even be eating each other.
Have you ever observed that? No, but I'm going to go look for it now.
So what stops them sort of running away and eating the whole brain?
So what are the control mechanisms for these? We don't know the control mechanisms,
but we know they exist because it's not indiscriminate.
So there are regions of the developing brain where it does not happen.
So the pineal gland is one example I brought up in the talk today where you
have the same cell types.
You have these vimentin-expressing, PAX6-positive radioglial cell types in the
developing pineal gland, and they're just...
Out of bounds. They're just not to be touched. They aren't touched.
Later on in the adult pineal gland, then this phenomena begins.
So that's one region. And in the adult brain, if you look at the dentate gyrus
where you have continuing neurogenesis, it doesn't occur there.
And in fact, there's some evidence that they might be supporting cell genesis in a way.
So I think the best approach would be to compare those regions,
to compare pineal gland in a developing animal with the cortex where it's happening.
And that may help us find cues that are guiding the process.
What exists in the pineal gland that tells them, you know, hands off, leave us alone. Right.
But then they have some sort of specificity because so far you haven't seen
that friends eat each other.
Right? Also, what you have not seen, they also limit themselves largely,
not fully, but largely to the proliferation zone. Yeah, they're attracted to it.
There's something that attracts them there. So there is some specificity there.
And for all we know, it could be something as simple as ATP.
We know they like ATP, and there's evidence that precursor cells might be putting
out ATP in large quantities, and it might be as simple as that.
But now, if it's, let's say, ATP-dependent, then you could also argue,
well, maybe these guys are just out there to clean up the mess.
And ATP is sort of a signal that something messy is going on because we're leaking
an intercellular element.
So how about we just interpret these microglia as sitting in this proliferation
zone because this is where we do most of the cell division. vision.
We want to assure that we have no cells coming out of that zone that are sort
of anomalous because they can create havoc in the rest of the system.
So I want to have a super conservative system that is as soon as there's even
the tiniest chance that there's something wrong with any cell, I just destroy it.
So would that be a reasonable way to think about this? Yeah. Another
exciting idea to me
is the potential that these cells could be shaping the
proliferative zone so the proliferative zones have defined boundaries
and this could be potentially a
mechanism for shaping that for keeping the boundaries
you know you stray too far you're gone okay and there's some evidence for that
it's that's something that we're following up on you already have some ways
of uh intervening in in the behavior of these cells uh and to show the effect
of maybe reducing the population size.
So one of the effects then is to increase the number of precursors.
Yeah, when we transiently got rid of them, it would increase that.
So what's the next step there in terms of the kinds of things you'd like to
be able to do with the microglia?
We're developing culture models to put precursor cells with microglia in chambers
and be able to experimentally control the conditions to try to work out the
signaling that attracts them to them. That's one thing that we're doing.
We're also trying to repopulate, so you could delete the microglia from a slice
and repopulating them with microglia from different aged animals to see if there's
any different function.
But to repopulate them, you could isolate them from a postnatal brain,
and that process in and of itself changes them.
There's caveats with everything, but those are the sorts of things that we'll
be trying. But are you able to say already that microglia are a critical part
of the neurogenesis process?
If you don't have them, then you have a pathological brain.
I believe so, but there's other groups that don't. So there's knockout models
where presumably you have no microglia, and they claim that the brain is completely normal.
But as we were talking about earlier, there are different types of immune cells,
and so I think it needs to be looked at whether or not other cells are performing similar functions.
Or it may be that there's many mechanisms and microglia is one of the mechanisms,
but if they're not there, something else will serve the job.
Yeah, I just have a hard time believing that if you remove microglia from the
brain that it's going to develop normally.
But now if we would turn this around and say, okay, we're going to give you
all the tools you want to give us a seven layer cortex, right?
Do you think you would have the mechanisms at hand to do that?
Um, probably not. Or just, let's say, a cortex that is twice as thick.
Twice as thick. We would need to extend the length of neurogenesis, so. Sure.
Yeah, we need to figure out what's promoting it. They enter a phase where the
primary precursor cells are in a steady state.
They continue dividing, and each division produces a copy of themselves and
a neuronal daughter cell. and they maintain that.
So they appear in ferrets, they're in that period longer than they are in rats.
And in primates, they're in that period for even longer than they are in ferrets.
So we need to figure out what the signal is that promotes that and what ends it.
Right. But when you're comparing primates, ferrets, and rats,
are you seeing any big differences, for instance, in the primates in the way
that this process is working, which will explain some of the- We're doing those experiments now.
So we have 10 monkeys that we've saved up and we're hopefully going to be getting the answer.
So I think my postdoc is cutting one of the brains today, I hope.
What's the sort of question you're asking there then? What kind of things might
be different in the primate?
So the subventricular zone is a very important structure and neurogenesis is
beginning before the subventricular zone is present.
So what do the neural precursor cells look like before that zone?
Are they subventricular zone
cells that just happen not to have coalesced into a cohesive structure?
Or is there something different going on at that stage of development when deep
layer neurons are being generated versus later on when you have a substantial subventricular zone?
Those are some of the simple questions we're asking, but we're also...
Looking at how the microglia are interacting with the precursor cells in a more
defined way in the primate model.
Any thoughts you might go to Monodelphus, which I know you have in Davis,
to look at the early origins of cortex or even to reptiles?
So we've looked at turtle. We have crocodile that we're just starting to look
at now. So they're present in turtle. all.
I have some tissue from doves and from chickens and they're present there.
So it's the population of precursor cells with microglia or microglial-like
cells is something that's been around for a long time.
So evolutionarily, it's something that occurred fairly early.
But we've got very distinctive differences between the reptile cortex and the mammalian cortex.
So are there clues there as to what's happening with the mammalian cortex or
what has changed what what other things that stand out for you to me determined
really okay so there's there's just lots of open questions yeah yeah it's exciting
it's a really exciting time i think,
but then to follow up on that and i could also challenge you from another angle
and say well look if you look at it as a comparative lead and you would expect
that the mechanisms to lay down a cortex are sort of piggybacking on the way
i lay down earlier structures in the brain the way i I might develop,
let's say, a brainstem or cerebellum or basal ganglia, right? Or spinal cord.
Or spinal cord, right. So how would I have to configure the specific system
you studied that gives rise to cortex to not give me spinal cord?
It's a great question. That's probably at the genetic level, you know. There are...
No, because in some sense, okay, the cell types might be somewhat different, right?
So there might not be the similar kind of layering, but we also will have precursor
cells that will be giving rise to these proton neurons that have to link up together.
You might also have to wash out the detrius from that system in the same way.
So right now, there's not a clear understanding of what these common principles
might be across different structures, is that correct?
There's definitely something different about spinal cord. then there is forebrain.
So we have looked at this phenomena, the microglia, and they don't appear to
be in the spinal cord at the same stages.
So there are key differences.
But now, do you already see some neuropathologies that you think are linked
to pathological activity of this microglia?
So one potential is, and this is something that many, many people are studying,
is that that if a pregnant woman is exposed to a pathogen at a specific stage of pregnancy,
that this can actually influence the function of the fetal immune cells.
And one example that's fairly well known is that if a woman is exposed to influenza
in the first trimester, then there's a greater likelihood that her child will be schizophrenic.
And that's a potential example. Yeah, well, there are also links between autism
and maternal immune responses.
So the mother is exposed to some pathogen. Her body generates an immune response
to fight off the pathogen.
So she develops a full-blown response. Many cytokines are produced,
which are helpful for her, but they actually get into the fetal compartments
and they can get into the fetal brain.
And that changes how these cells function because that's what they're built to do.
They're built to respond to foreign pathogens, to challenges.
And that can change the equation.
So there's hints that in neurodevelopmental disorders, schizophrenia,
perhaps autism, that this will play a role, that it can change the trajectory
and influence the outcome for the worse. Right.
So now, okay, so we made a lot of progress in some sense in our understanding of how a cortex is built.
And also what we hear now, there's still many, many questions to be answered.
And hopefully you will find those answers anytime soon.
But in this whole trajectory that you're on, understanding the developing brain,
what is Stephen's law that we should follow to understand the brain?
What is Stephen's law that we should follow? I think we're still working that out.
We're still, I like to say that what we do in the lab or my approach,
my way of looking at things is that we're defining normal, understanding very
well what's going on during normal development.
Okay. And now Tony likes traveling, so he'll be in Davis soon. Good, yeah.
It's also five years from now he'll be there. and he's going to come to your
lab and visit you and he's going to put out this piece of paper that says,
okay, Stephen, five years ago you made this prediction and you told me that
now you're going to give me the answer.
So what's the main prediction you would like to sort of commit yourself to today
that Tony's going to get the answer to five years from now?
The signaling that controls the
colonization and the interaction between microglia and precursor cells.
I would really hope that we could have that figured out before five years.
All right, Stephen. That's perhaps ambitious, but that's what we're hoping for.
Okay. Stephen Nocturne, thank you very much for this conversation.
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