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GERALD SCHNEIDER: OK, well we didn't get too far with our
classes on axon growth last time.
We were talking about the growth cone.
And I want next to talk about an experiment on axonal
pathfinding.
How do axons when they're elongating find their way to
their target?
We'll see that the simple way of thinking about it is that
they just follow some kind of chemical pathway all the way
to their target.
And that there's chemical specificity all along the way.
And that was just that was the hypothesis of Roger Sperry.
He called it the chemoaffinity or chemospecificity
hypothesis.
It's been largely verified, but not in the way Sperry
envisioned.
So let me talk a little bit about that.
First of all, the grasshopper leg gave an interesting
example of how axons can find their target.
Here at the top, imagine a grasshopper leg at a very
early stage of development.
So it's sort of a limb bud there.
And imagine that it's transparent and we can see the
peripheral neurons sending our axon out, growling towards the
central ganglion here.
And it's found that they always
follow a certain pathway.
They've shown there in the light pink the envelope of all
the growth of all the axons of a bunch of grasshoppers.
And you can see they always have certain bends.
They always making a sharp bend here.
And then they turn again towards the central ganglion.
So what is guiding it?
Well it turns out there are nonneuronal cells along the
way, sort of like a glial cell.
The hypothesis was soon proposed that they're using
those as guides, that these are guidepost cells.
So how could you test that?
Well very simply.
Before the first axons ever get here, zap them with a
laser beam.
And they could identify these same cells, both the neurons
and these peripheral so-called guidepost cells.
They could identify them from one grasshopper to the next.
And so it's a big advantage to using these simpler animals
where rather than dealing with whole populations of cells,
which you'd have to do with mammals and other larger
animals, you can sometimes find individually identified
themselves.
And in fact, these cells have names.
Well when they zap these cells, here's the control.
And without those cells there, look what happens.
The axons simply start splaying out, forming multiple
branches, they don't seem to know where to go after they
get to the previous cell.
So that it was proposed then that these were guidepost
cells and the pathway is formed.
What would be happening?
Let's say the axon has gotten to this point.
What is it doing to reach to the next point?
How does it to know to go here rather than to turn around and
go back the other, or to go out towards the surface, or go
down to the other edge of the leg?
The idea is that the philipodia can be very long.
They can up to 200 microns.
And these legs are very small early in development.
So they send out these long philipodia and retract them.
And if they can adhere to one of these guidepost cells, they
could move towards it, literally be
pulled towards it.
And then by a metabolic change after they reach there, they
could start the same process again and go on to the next
guidepost cell.
So it would be a sort of connect the dots type of
guidance system.
It's the pioneering axon that uses those cells and then
later growing axons seem to follow the first one.
Well we studied the mammalian optic track, several people in
my laboratory, a few years back.
And we found that what happens in the grasshopper leg is not
what happens in the mammalian optic track.
The initial pioneering axons grow out, now this is after
the optic chiasm.
They grow up in a wide swath, the full width of the optic
track, and they space themselves.
They space out, even though there's not very many of them
when they first grow.
And they grow up the side of the [UNINTELLIGIBLE] brain,
towards the geniculate body.
And then the later growing ones just fit in between.
And they don't form any noticeable fascicles where
axons could be following each other until much later on.
So it's not like the grasshopper.
So we know that there are other
mechanisms of axon guidance.
Let's go over another experiment on axon growth.
This is a very interesting one.
It's often republished and it's described in the Purves
and Lichtman reading.
I give you the page numbers there.
What did he do?
Well Mauthner cells are these giant neurons in the hind
brain of some fish and amphibians.
And you can find them in growing tadpoles.
At the top you a picture of the hind brain there.
And I've outlined with the red dash line there, the piece of
hind brain that Hillard cut out and then he put it back.
And the top row there, he put them back in the same
orientation.
He just kind of it loose, looked at it out,
stuck it back in.
Well you can see, the axons grew out normally.
But in a few times they actually didn't get on the
wrong side of the brain.
Here's one that rather than crossing stayed ipsilarily.
But otherwise, its trajectory was normal.
There could be mechanical effects that were affecting
the tissue destruction that had affected the
guidance of the axon.
That's not certain.
The interesting thing is, what happens if when he takes that
piece out, he turns it 180 degrees and then puts it back?
So now if they follow the course determined by where
they came from, [INAUDIBLE] grow in the wrong direction.
And you can see in every case here, the axon when it grows
out grows [UNINTELLIGIBLE]
instead of caudally?.
It appears is to be getting a cue from the original tissue.
But then when it crosses into the tissue that didn't get
turned around, it turns around.
There seems to be a substrate cue of some sort.
So you can say, well then why doesn't it start going just
back and forth back and forth?
It doesn't do that.
By the time it's turned around and grown caudally, its
response to these cues has changed.
And now it continues caudally.
And it ends up farming fairly normal
connections of Mauthner cells.
It just follows an abnormal trajectory.
So at the time the Purves and Lichtman book was written a
few years back, this was 1985 when they published it, but
they could see these four types of mechanisms.
And I put in bold there the ones that have been verified
in other experiments in a number of different species as
valid mechanisms of axon guidance.
Sterotropism means the shape of the surface that it's
growing on.
So for example, if I did an early brain lesion, which I've
done a lot of in the developing hamster when the
optic track was first forming, I could produce tissue
disruptions that would deflect the axons towards the surface
and they would grow abnormally.
That's an example of this theory of tropism.
I've left the same tissue there, I've just changed the
membrane surfaces that these axons are following.
The second one they called galvanotropism because of the
evidence that slight very small electric currents in
tissues can affect the way axons grow.
And there is evidence for that.
The problem is that the currents that actually exist
in developing tissue are extremely tiny.
And if they are having effects, they don't appear to
be mainly on these long trajectories of axons.
They may have local effects.
And there haven't been many studies of that, a few in
these early years.
But then as more and more evidence for chemical cues
developed and the methods improved for studying various
molecular cues, the emphasis changed.
The only chemical guidance was based on
differential adhesion.
We've mentioned how philipodia have cell adhesion molecules
in their membranes, especially at their tips.
And they will adhere to other cell adhesion molecules.
And that can guide the axon.
So more adhesive substrates will effect axons differently
than the less adhesive substrates.
And if there's no adhesion at all, then the axons actually
have trouble growing at all.
They need some adhesion to be able to grow.
And then finally, chemical effects, chemotropism.
And chemotropic mechanisms can be either by membrane
molecules, so it requires contact of the membrane, or
they can be chemicals that diffuse through the
environment.
And this was known too way back in '85 when Purves and
Lichtman summarized developmental knowledge at
that point.
Well since that time, people have clearly distinguished
four types of chemical guidance.
And it's very clear.
This was a picture published in a review paper that
summarized them.
There are long range cues that involve diffusing molecules.
And then the short range cues that require content.
And the results can be of two types in both cases.
They can be attraction and they can be repulsion of
various sorts, like barriers to growth or
inhibition of growth.
And they list some of the molecules there, some of which
we will take up again a little later today or the next time.
So we have attraction and repulsion.
And repulsion can also mean inhibition of growth.
And these can involve effects of either defusing chemicals
or effects of contact.
So to go to the attraction effects first, we have
cell-cell adhesion.
Of course, this requires memory contact.
These are the CAMs, the cell adhesion molecules.
Extracellular or matrix molecules can be adhesive as
well, like the laminins and the cadherins.
We can follow laminin rich tracks that axons follow in
development, for example.
And they are growth factors that increase growth upon
contact or they act by diffusion.
The contact molecules, many of them are called
[UNINTELLIGIBLE], the diffusable ones are like nerve
growth factors.
And other members of that family of growth factors, the
neurotrophins nerve growth factor was the first of
several members of that same family.
The netrins are a molecule we'll talk about a minute.
And there's several different families of growth factors
that have been discovered.
So the other effect, the inhibition of growth, first of
all, we have midline barriers.
For example, in the superior colliculus, axons from one eye
in an animal were almost all the axons are crossed, axons
from one eye will go to one site, axons from the other eye
will go to the other side.
And why don't they cross the midline?
There appears to be a barrier there.
And to test that, in my lab we've actually cut the radial
cells, or glial cells, that are found at the midline.
With a little cut we caused the process that goes up to
the surface to degenerate.
And what happens is that the axons will spread across the
midline if you do that.
And if you remove their competition from the eye, they
will spread right across both sides of the optic tectum.
If you don't do that damage at the midline,
they won't do that.
So there's definitely a midline barrier.
And it's now known that midline radial glia actually
secrete cardioglycans and these appear to be responsible
for that barrier.
There are factors in the oligodendrocytes.
Do you remember what oligodendrocytes are?
They're glial cells in the central nervous system.
They act like the Schwann cells in the peripheral
nervous system in that they form myelin.
Their membranes are wrapped around the membrane of axons,
wrapped right around the axon.
Well when the oligodendrocytes appear in development, at a
very early stage in their membranes, there is a molecule
that appears that has been named Nogo by the discoverer
in Switzerland because it inhibits axon growth.
And it's been proposed it's a major factor that prevents
some axon regeneration.
Although, the research that we and others have done has not
supported that.
And then there's various secreted and transmembrane
proteins that also inhibit growth.
And we'll see some of that now.
When we talk about these effects, I want you to be able
to distinguish trophic effects and tropic effects.
One word has the h, one does not.
Tropic means it influences the direction the axon is growing.
That's what tropic meant.
A tropism is something that affects direction.
In animal behavior we talked about thigmotropism, for
example, an animal that tends to follow a surface, like it
follows the wall in the dark, his direction is controlled by
these surfaces.
So tropic means influencing the direction of growth.
Whereas trophic can mean two kinds of things.
Survival promotion, so without trophic molecules, cells, at
least at a certain station in their development, will die.
They need trophic molecules to survive.
But trophic also means promoting growth.
So more growth factor will make them more vigorous in the
growth of their axon and make those axons have more ability
to compete.
And we can give some examples.
But first let me give one caveat here.
The discovery by Mu-ming Poo's group that these guidance
mechanisms aren't fixed.
He published a paper, the first one in a group of
papers, showing that a molecule can change from
having repulsive effects to attraction effects just by
adding cyclic nucleotides.
So for example, I'm sorry the picture is, let me blow it up
there because I'm only interested here in these
pictures at the top.
OK, in A here.
He's using some semaphorin 3 that's diffusing out of a
little pipette that he's put up here.
And here is the axon growing.
And to see what it does?
It starts turning away.
The only thing that's happening there is there's a
diffusion of semaphorin 3 from that pipette shown by where
the arrowhead is there.
The axon is turning away.
So now he's adding cyclic GMP.
It's actually a agonistic cyclic GMP.
It acts like cyclic GMP.
And look what happens.
It's the same experiment, he just adds that molecule.
Now the axon is turning towards.
Now it's going up the gradient instead of
getting away from it.
And he's plotted the quantitative results.
You can see there's some variability, but it's turning
away here and turning towards here.
He found that cyclic GMP or cyclic AMP, either one could
be used and he could get these kinds of effects.
This is pretty important as you'll see in a minute.
Let's first see how that kind of finding on semaphorin 3,
which was originally named collapsin.
Why do you think it was called collapsin initially?
Its first effect discovered was an inhibition of growth.
And when axons were exposed to the molecule, you got that
phenomenon we saw in the video clip the other day, growth
cones would collapse, pull back, and change direction
before they started growing again.
Well it was found that semaphorin was found
especially in the ventral part of the developing spinal cord,
not in the dorsal horn.
That led to the proposal that these axons of the dorsal
root, coming from the dorsal root ganglion cells, when they
grew in, acted differently because they had a different
response to semaphorin 3.
The largest axons, these, that were coming from sensory
fibers innervating muscle, they were the axons that
formed the monosynaptic reflex.
So they went down in the ventral horn to
contact motor neurons.
They were not repelled by the semaphorin 3.
But the smaller axons that terminated in the dorsal horn,
some of them, actually others would terminate here in the
intermediate region, but never going into the ventral horn.
So the idea was that they were the ones responding to
semaphorin 3.
And there's been some verification of that idea.
Let's leave the spinal cord here for a minute and talk
about the axons that terminate in the dorsal horn on
secondary sensory neurons.
And then the axon of the secondary sensory cell
projects down across the midline and then ascends the
cord on the opposite side, like this is the
spinothalamic tract.
That's how the spinothalamic tract originates.
Why do you think they grown down to the floor plate?
Now when they're first doing this, you didn't have this
thick area here.
But the floor plate was still there below the ventricle.
This was the work of Tom Jessell and his coworkers at
Columbia University.
He discovered molecules they called the netrin molecules
that diffused from the floor plate region that specifically
affect those axons from the dorsal horn cells, the
secondary sensory cells, that form the spinothalamic tract.
They have tropic effects on those axons.
But if they're attracted to the floor plate, that's what
gets them down there, why do they cross over and go way out
to the lateral edge and then start ascending?
In other words, if they were being attracted to the floor
plate, why don't they get to the floor
plate and stay there?
Since they don't, we have to assume that a metabolic change
of the sort that Mu-ming Poo and his collaborators
discovered must be acting.
At least conceptually we can understand what's happening.
Even though some of the molecular
mechanisms may not be known.
We've discovered the type of mechanism
that would be needed.
So we know that developing axons have to do a lot more
than simply follow a path and then terminate
when they get there.
They're guided all along the way.
We also know though that they change their mode of growth
when they get into their terminal region.
So I want to take a little time now to discuss that.
Because we know that when they're elongating they grow
about 10 times faster than they grow when they enter
their terminal region, something changes.
We also know that when their elongating they not only grow
faster, but they tend to grow in vesicles.
The later ones tend to vesiculate
with the earlier ones.
We also know that when they enter the terminal region and
start growing more slowly, they start branching.
That's how a terminal arbor forms.
They don't branch like that when they're elongating.
So we studied this in the optic tract.
And some of the work was Golgi study, some of it was filling
of axons with various molecules.
The upper two pictures were from the Golgi work.
And we found that through earlier in development we
could always see the growth cone at the tip.
And some of those, sorry, they were a couple of other methods
not Golgi, but they were Golgi like in the result.
Some of it was a molecule called DiI that would diffuse
through the membrane of the cells.
And we found that they were characterized by these
periodic swellings.
And also by lots of little philipodia.
So the philipodia are not only at the growth cone, they're
also along the axon.
And generally when a branch starts to form, it originates
at one of these swellings.
So in other words, something makes the philipodia adhere to
something, adhesive properties are changing.
And this whole axon is capable of growth at that stage and it
can form branches.
And then it starts to arborize.
And when it starts to arborize, it looks like the
second picture here.
It starts forming arbors in a lot of different places, not
everywhere.
But it forms a lot of very rudimentary arbors.
It's not forming big arbors yet.
But this axon here, its final arbor might end
up being right here.
And then it can grow further.
And it will form arbors both beyond that point and proximal
to that point.
And I'm showing that here, the third picture.
It starts to focalize.
Certain arbors start to become augmented
and the others shrivel.
So it loses some of the branches that it's forming.
Its early exuberance is decreased.
It starts increasing is arbors at certain points.
Here's one that's a little further along.
Notice that the one going further here is totally lost.
And it might form abors elsewhere too.
For example, the optic tract that's arborizing in the
tectum of the midbrain could be forming an arbor in part of
the geniculate body.
The very same axon.
Not all of them do that, some of them will terminate only in
the geniculate body or only in the tectum, but quite a few of
them actually terminate in both.
And we have direct experimental evidence for
that, we also have the Golgi evidence.
And then they focalize further.
Initially they distributed throughout from
superficial to deep.
But then they start increasing their arbor only in certain
layers and they lose the extra branches elsewhere.
So that's a laminar focalization.
And that's already happened before the eyes open in the
hamster, and the rat, and the mouse.
But there continues to be changes in the
way that arbor looks.
And just referred to that as maturation.
The appearance of the terminals, especially as scene
in electron microscopy continues to change for a
while as the animal gets older.
And I also point out here that in our studies of brain
damage, it's in this early period that if you make a
lesion there, they can regrow.
If we do the same thing here or here they don't regrow.
It doesn't mean they don't change in the way they're
growing, but it just means they have trouble
regenerating.
Most of them, there's a few of them that will regrow.
Let's say a little bit--
what time is it here?--
say a little bit about formation the map now.
I mentioned the chemoaffinity hypothesis, right?
So let's say a little more about that because quite a bit
has been discovered in more recent years.
These types of molecules have been found to be largely
responsible for map formation.
When I say the responsible for map formation, they're
responsible for making the map precise.
It's not that there aren't other mechanisms.
By the time the axons reach the midbrain tectum for
example, the axons representing the upper visual
field are already growing mediately and the axons
representing the lower visual field coming from the upper
retina are already growing laterally.
But in fact, it's not a perfect order.
And as far as the nasal temporal axis goes, those
axons are just mixed in with each other.
You clearly have to have some other mechanism for
sorting them off.
Now Sperry assumed that they went to where they were
attracted, almost like a lock and key.
But here's what was discovered.
In the retina you have the EPH receptors.
And the ligand and for those receptors are the Ephrins.
And the Ephrin ligands are found in the tectum or
superior colliculus.
So for example, we only need to deal with one of them here.
Let's deal with that picture there.
First of all, here you see the retina.
And this is how we know it will form the adult axons from
the temporal retina will terminate
anteriorally in the tectum.
those from the nasal retina will terminate posteriorly.
We also know that when they initially grew into the tectum
they weren't distributed so precisely.
OK, now if we look at Ephrin-A3 A3, we see that it's
concentrated in the temporal retina, almost none of it in
the nasal retina.
It's distributed in a gradient like way across the retina.
In Ephrin-A2, the ligand is concentrated
in the caudal tectum.
It's not found in the rostrum or the tectum and its levels
of that molecule are in between.
Now it turns out that when you have Ephrin-A3, it's repelled.
The EPHA3 is repelled by Ephrin-A2.
So those axons won't grow into the caudal tectum.
If it doesn't have or it has very little EPH-A3, it can
grow right through the whole tectum.
And here's how that was demonstrated.
What they've done is taken along little tracks here in
tissue culture, they've gotten the axons to grow.
These came from the temporal part of the retina, these came
from the nasal part of the retina.
And they're going down these tracks.
What are they growing on?
They're growing on tectal membranes.
And they mince up the membranes, so they get this
membrane preparation, all little pieces of membrane.
And they get those from purely anterior tectum and then more
and more posterior tectum.
And they lay them down in these tacks.
Whoops, what did I just do?
Sorry about that.
Notice that if they're coming from temporal retina cells
they get stopped as soon as they start encountering
membranes from the posterior tectum.
If they're coming from nasal retina, it doesn't matter
whether they're encountering membranes from the anterior or
from the posterior tectum, they can keep growing.
So that shows that the mechanism of the EPH receptors
interacting with the Ephrins is a repulsive mechanism, it's
a repelling effect.
And that's how these gradients of these molecules are
governing the distribution of the axons.
Later they discovered that there were gradients in other
redirection as well.
So even though the axons are already organized, they from a
better map with the help of these molecules.
That was discovered by these two different groups, one at
Harvard, Flanagan, and one in Germany, the [UNINTELLIGIBLE]
group.
So with all these chemicals it's all pretty rigid, right?
Well, not quite.
Let's take a developing colliculus
here of a baby hamster.
And here comes the optic tract.
Here are the axons coming in.
And right while those axons are growing in, this
is what we'll do.
We'll use a cautery and we'll burn off the
whole caudal tectum.
What does that do?
What happens is we'll us retinal coordinates.
The temporal retina axons will terminate there, which they
normally do.
But the axons from the nasal retina that would normally
have gone back here, terminate there.
So the whole map ends up in crest in
that remaining tectum.
The superior retina is represented here, the inferior
retina here.
That's normal.
And now the whole map is represented
in the smaller space.
That's called map compression.
So what's happened to these molecules?
It shows that it's not a lock and key kind of effect at all,
it's due tot he gradients in molecules.
And that's been verified by effects of expansion.
That's compression here.
You also get map expansion.
How would you test that?
What's the experiment you'd want to do to test to see if
the map can also expand?
Well you could make the tectum bigger, but it's a lot easier
to just ablate part of the retina.
So one of my graduate students made holes in the retina.
Again, with burn lesions in newborn animals, taking out
little pieces of retina.
And if you made a hole in the retina, if you did that in the
adult, you end up with a hole in the map.
Axons are just not found in one part of the tectum.
If you did that in the baby, they filled in.
The remaining map filled in the gap,
so the map was expanding.
And this has been done in goldfish in regeneration
situations in adult goldfish.
When you take off much of one half of the retina, the
remaining will expand across the tectum.
So he was getting things like that
happening in the baby hamster.
You get both compression and expansion.
That we call plasticity of the developing maps.
To explain what was going on, we refer to experiments on
collateral sprouting.
Have we talk about the collateral sprouting at all?
How many of you remember something
about collateral sprouting?
Let's go through collateral sprouting experiments that
were done now not in the adult, which we probably
mentioned before, but during development.
This is a side view of the hamster brain stem, very much
like a rat, or a mouse, or a developing human.
These are optic tract axons.
And this is the normal route for a one bundle of optic
tract axons coming from the optic chiasm up the side of a
diencephalon, terminations in the geniculate bodies, ventral
and dorsal, and in the superior
colliculus or optic tectum.
There are other pathways coming from neurons there, the
very neurons that are receiving these terminations,
that project forward both to the geniculate bodies,
especially to the ventral one, and to that nucleus sitting
right next to the geniculate body, LP, the lateral
posterior nucleus.
So now here's what we'll do.
We'll take our lesion maker and we'll just ablate the
superior colliculus.
And then we'll wait, we'll let the animal grow up and see
what's happened to the optic tract.
What happens is that this normal
projection here is now missing.
But instead, the axons grow into what's left in deeper
layers that normally don't get a retinal projection.
They sprout in there and form some terminations.
It's not of normal size, but it's clearly there.
But this projection is now missing.
The retinal projection will sprout there in both of these
places where the colliculus normally projects.
It's like they're filling in some of the terminal space
that was made available.
So now the retina ends up projecting more densely than
normal in these two places in the thalamus.
And here's a new projection in the midbrain.
They're showing a type of collateral sprouting.
Well the inferior colliculus normally projects there to the
medial geniculate body.
What if we get rid of that projection and we get rid of
the tectum?
Well what happens is that the retinal axons now will sprout
not only in the LP and in the LGB there, they will actually
sprout right into the auditory thalamus.
So now the retina projects into the auditory system.
And now the, in this case, hamster, it happens in the
ferret, the rat also, most of the experiments have been done
with hamster and ferret, you get the retina projecting not
only to the later geniculate body which projects to the
visual cortex, but also projects to the medial
geniculate body which projects to the auditory cortex.
So now you have an extra visual cortex.
In the absence of normal auditory input to the auditory
cortex, now it's getting retinal input.
And there has been some data collected in Mriganka Sur's
lab, behavioral evidence, that in fact that's a functional
projection.
He's done very nice electrophysiology of that
projection.
He's also done some behavioral work.
So what's happening here?
This is theoretical.
If I take two axons coming into a structure like this
superficial tectum and they share the space like this and
we get rid of one of them, we'll get rid of that one.
So this will all degenerate here.
The remaining axon will spread.
It won't form an arbor that's bigger than normal, but it
will spread over a larger area, as if it's being held in
place by the competition.
And there are many experiments like that, that show that
axons compete in some way.
And competition, we think now, can take several forms.
They can be competing for the terminal space, either because
of the growth factors, they get there, and there's good
evidence for that.
Also we looked with the electron microscope, they
actually compete to occupy the synaptic sites.
In many cases, more than one axon can occupy
the synaptic sites.
But we also know about these contact reactions that have
been seen in tissue culture, that axons will space each
other out because they're affected by each other, they
retract from each other.
That's a kind of competition too, this contact inhibition
of extension it's sometimes called.
OK, so next time I'm going to talk about the ability of
axons to compete and how you can change their competitive
growth vigor by chemical means, by activity, and by
lesions, or pruning effects.
We'll go through those at the beginning of the next hour.