In the last lesson we began
to think about how neurons talk to each other
through the process of chemical synaptic transmission.
An action potential is initiated at the axon initial segment.
It travels down the axon, reaches pre-synaptic specializations
where calcium influx occurs,
and that drives neurotransmitter release.
That, in turn, acts upon receptors in the post-synaptic membrane,
driving post-synaptic potentials.
In this lesson we're going to take a closer look at what happens
inside the pre-synaptic specialization.
How can the action potential drive neurotransmitter release
on such a fast timescale.
There are three key events that need to take place
in the pre-synaptic specialization:
first, synaptic vesicles full of neurotransmitter
need to dock and be primed to be release-ready
in immediate apposition to the pre-synaptic plasma membrane.
The vesicle then waits until an action potential invades the terminal.
That, then, drives an increase in calcium concentration
and then in the third step, the increase in calcium drives fusion
of the vesicular membrane with the plasma membrane,
thus driving exocytosis of the neurotransmitter
inside the vesicle.
Let's begin by thinking about how calcium rises occur
in the presynaptic bouton.
The action potential is traveling down the axon,
it reaches swelling where there are many synaptic vesicles present.
In addition to these synaptic vesicles,
there's also a high concentration of voltage-gated calcium channels.
Voltage-gated calcium channels are similar
to voltage-gated sodium and potassium channels,
except they have a high permeability for calcium,
rather than for any other cation.
The voltage-gated calcium channels also have a similar voltage dependence,
so if this is our membrane potential across the plasma membrane and the bouton,
and this is the open probability of a calcium channel,
then at hyper-polarized potentials, the open probability is low,
and then as we get more positive than around -40 mV
the calcium channels begin to open
and they can then let calcium flood into the cell.
Remember, there's about 2 millimolar of calcium in the extracellular space,
and at resting conditions, there's about 100 nanomolar of free calcium ions
that are present inside the cytosome.
So, there's a strong gradient for calcium to enter
when the open probability of the calcium channel
increases at depolarized potentials.
So let's see what happens when an action potential
invades this pre-synaptic specialization?
Here we see the action potential in a nerve terminal
it has a duration of something like 500 microseconds or one millisecond.
The upstroke, as we know, is driven by the activation
of the voltage-gated sodium conductances.
The voltage-gated potassium conductances drive the repolarization
and smaller than those sodium and potassium currents,
also a calcium current is found in the presynaptic terminal.
It's not activated as rapidly
as the extremely fast voltage-gated sodium conductances,
so there's some delay before the calcium current turns on.
It turns out that the peak of the calcium current
is somewhere on the falling phase of the action potential,
so delayed some hundred microseconds or so
from the peak and the onset of the action potential.
This influx of calcium, the calcium current flow
through the voltage-gated calcium channels then increases the calcium concentration
in the immediate vicinity of the synaptic vesicles,
and it's that increase in calcium concentration
that directly drives vesicle fusion.
It turns out that there's an extremely steep dependence
upon calcium concentration and neurotransmitter release rates.
In this graph, which has logarithmic axes in both the X and Y scales,
so it's a log-log plot,
you see a very steep dependence upon the cytosomic calcium concentration
inside the nerve terminal, and the release rate,
the number of vesicles being released per millisecond.
You'll see that there's a range of calcium concentrations here
that goes from one to ten micromolar.
This is a tenfold increase in calcium concentration,
whereas on the Y axis, you'll see that the release rate increases
by a factor of 10,000.
You can plot a line through data points,
and it turns out that there's a nice linear relationship
between calcium concentration and the vesicle release rate,
and it has a slope of around four.
That's the so-called Hill coefficient, and that tells us
that if there's a tenfold increase in calcium, then there's
a ten to the power of four-fold increase in release rate.
Ten times calcium, 10,000-fold increased release rate.
Here's the relationship then.
The release rate goes as a fourth power of the cytosomic calcium concentration
in the nerve terminal.
So increases in calcium irrespective of action potential or membrane potential
drive neurotransmitter release at resting calcium concentrations
of around 100 nanomolar, the release rate is very, very small.
It's nearly negligible.
There is still a small amount of vesicles
that are fusing spontaneously, even at resting levels.
But that release rate increases very dramatically
in the presence of calcium,
so calcium is really the direct signal that drives vesicle fusion,
and it does so in an extremely steep dependence
where small increases in calcium cause huge changes
in neurotransmitter release rates.
In order for the calcium to be able to work,
there must be calcium-sensitive binding proteins
that bind the calcium and translate that
into the exocytosis of the synaptic vesicle.
In addition, for the process of exocytosis to occur on the 100 microsecond timescale,
there must be an extremely efficient machinery
that's able to couple the calcium signal to vesicle membrane fusion events.
Here we see a schematic drawing of a synaptic vesicle
that's in immediate apposition to the presynaptic plasma membrane.
So these two lines here represent
the phospholipid bilayer of the plasma membrane
and the two black lines here around the vesicle
are the phospholipid bilayers of the synaptic vesicle.
The membrane of the synaptic vesicle
is almost identical in terms of its composition
to the membrane phospholipids of the plasma membrane.
The synaptic vesicle with a diameter of around 40 nanometers
is brought into close apposition with the plasma membrane
through the action of three important proteins
that form the so-called <i>SNARE complex</i>.
The SNARE complex is composed of <i>synaptobrevin</i>
which is a so-called vesicular SNARE;
<i>syntaxin</i>, which is a plasma membrane SNARE;
and <i>SNAP-25</i> that's closely associated to syntaxin.
Through binding to each other with very strong strength,
they pull the plasma membrane and the vesicle membrane
into extremely close apposition with each other.
It may, in fact, be that there's already a pre-bound
where the inner leaflet of the plasma membrane
is already in continuum with the outer leaflet
of the plasma membrane of the synaptic vesicle membrane.
So this would then be the release-competent primed vesicle
ready to be released.
It's brought into close contact with the plasma membrane
through the action of the SNARE complex,
and it's waiting for an action potential to come.
The action potential invades,
opens the voltage-gated calcium channel,
calcium floods in, and calcium then binds to the calcium sensor synaptotagmin
and other vesicle-associated protein.
Synaptotagmin has five binding sites for calcium
and when calcium binds to it, it changes its conformation quite dramatically.
It changes the way it interacts with the SNARE proteins
and perhaps also with the phospholipids in the immediate environment.
The action of calcium on synaptotagmin opens the fusion pore,
and so, there's an opening that's created
where the inner leaflet of the synaptic vesicle membrane
becomes continuous with the outer leaflet of the plasma membrane,
and that creates a pore where the contents of the synaptic vesicle
are now in continuum with the extracellular space.
There's a diffusion of the vesicle of the neurotransmitter across the cleft,
and it can then bind to the post-synaptic receptors
driving post-synaptic potentials.
So the core elements in terms of molecular mechanisms of fusion
are the priming and docking of the event
setting up the vesicle through the SNARE protein
so that it's release-competent,
and synaptotagmin then acts as a calcium sensor
that converts the action potential and calcium influx
into the vesicle fusion event itself,
and all of this occurs on the microsecond timescale.
After the neurotransmitter
has been released from the synaptic vesicle,
the synaptic vesicle is empty,
and there's no neurotransmitter left in it.
Before that synaptic vesicle can be useful again
in terms of releasing neurotransmitter,
it must then be refilled.
The refilling process of synaptic vesicles
occurs through an ATP dependent process.
A so-called <i>vacuolar proton pump</i> in ATPase
uses ATP to pump protons, that is, charged hydrogen atoms,
into the vesicle.
It then becomes acidic inside the synaptic vesicle,
and it also becomes positively charged.
And so, just like there's a membrane potential
across the plasma membrane,
there's also a vesicular potential across the synaptic vesicle membrane,
and it's a combination of the acidification
and the electrical potential here
that gives energy for the vesicular neurotransmitter transporters
that then bring neurotransmitter inside the synaptic vesicle.
So, this is a glutamatergic synapse, there would be
a vesicular glutamate transporter,
VGlut present on on individual synaptic vesicles
that then sucks glutamate in
and concentrates glutamate inside the synaptic vesicle.
When the vesicle is refilled,
it then needs to move it into these sites where it can be released.
It needs to interact with the SNARE complex.
The SNARE complex needs to bring the membranes
into close apposition for each other
and will then be ready for another action potential
to invade, and release neurotransmitter.
There are various ways in which this synaptic vesicle cycle
is thought to work.
One way is signaled here.
This is a so-called <i>Kiss and Run</i> exocytosis
where there's a brief fusion event, a fusion pore opens,
the contents are diffusing out,
and the empty vesicle then closes, moves aside a little bit,
maybe some nanometers away from the plasma membrane,
refuels with neurotransmitter, and then re-docks.
That's called Kiss and Run exocytosis.
And as a possibility, there's so-called <i>Kiss and Stay</i> exocytosis
where after the vesicle is fused, it simply reseals
and remains docked and simply replenishes its neurotransmitter content
while remaining in a docked conformation.
Finally, another possibility is that this fusion pore enlarges
and the membrane gradually
simply becomes part of the plasma membrane
so the vesicle fuses completely and then becomes part
of the plasma membrane from where it can then be endocytosed
in a KLAF independent manner and then enter into the vesicle cycle.
The fastest forms of exocytosis and recycling are the so-called
<i>Kiss and Run</i> and <i>Kiss and Stay</i> types of vesicle recycling
which can occur on the 50 to 100 millisecond timescale.
And so, the whole process here might occur very rapidly
and help sustain fast rates of neurotransmitter release if necessary.
So we've now seen some of the key steps
that are able to sustain neurotransmitter release
at these extremely fast rates of 100 microseconds or so
between the invasion of the action potential into the bouton
and the release of the neurotransmitter.
The key steps are the priming and docking of the vesicle
so that it's ready to react in case of calcium concentration increases.
So, the SNARE complex forms a key feature in an ATP dependent process
that brings the membranes of the synaptic vesicle
and the presynaptic membrane into close apposition with each other.
The vesicle then waits there release-ready until a calcium signal appears
that's driven through the activation of voltage-gated calcium channels
driven by the voltage depolarization of the action potential
as it invades the bouton.
The calcium binds that cell to synaptotagmin,
and it's that change in conformation of the synaptotagmin protein
that allows the vesicle to fuse with the plasma membrane
releasing the neurotransmitter content.
Now, it should be remembered that the exocytosis,
the release of a vesicle, is a stochastic process.
A given docked and release-ready vesicle
may have something like a 10% chance of being released
when an action potential invades the nerve terminal.
This means that synaptic transmission is unreliable,
but it also means that the process can be highly regulated.
We can increase the efficacy of the synaptic transmission by, say,
increasing the amount of calcium influx into the terminal
or we can also decrease the amount of neurotransmitter release.
It's that ability to regulate synaptic transmission
that's thought to be extremely important.
And so, in the next slides we'll take a little look
at how one might regulate neurotransmitter release.
The first, and perhaps most obvious way
in which we can control neurotransmitter release
is by controlling the amount of calcium that enters into the nerve terminal.
You'll remember that we have an extremely steep dependence of calcium
where if calcium goes from one to ten micromolar,
then we go from release rates of 0.1 to 1,000
so a factor of 10,000 increase in calcium
and typically during an action potential invading the nerve terminal,
we think that we're reaching something like 10 micromolar of calcium.
So by increasing or decreasing the amount of calcium we can also
increase or decrease the amount of neurotransmitter being released.
There's an extremely steep dependence
that will go with the fourth power of calcium.
So small changes in calcium can give rise to very big changes in the amount released
from the presynaptic terminal.
So how do we change the amount of calcium that comes into the presynaptic terminal?
Well, there are two obvious ways.
We can change the action potential waveform.
So if the typical action potential has a duration of 500 microseconds,
the calcium influx occurs during the repolarization
and by the time the membrane potential is hyperpolarized,
the calcium influx has come to an end.
So, by increasing the duration of the action potential
we can then increase the amount of calcium influx that occurs,
and thus, we increase the amount of neurotransmitter release.
That can be done by regulating potassium channels.
So potassium channels govern the repolarization phase
of the action potential,
and if we simply reduce the amount of potassium current,
then we'll delay the action potential waveform
and we'll increase calcium influx into the presynaptic terminal,
and hence, increase neurotransmitter release.
That's one way in which neurotransmitter release
is really being regulated physiologically.
Another obvious way is by acting
on the pre-synaptic calcium channels themselves.
Increasing the open probability
or increasing the voltage dependence of these calcium channels
will, of course, increase the amount of calcium
that floods into the pre-synaptic membrane,
and that's another way that's used heavily inside the nervous system
to regulate neurotransmitter release.
Other possibilities in terms of regulating neurotransmitter release
are, of course, how much neurotransmitter is actually inside a synaptic vesicle.
That's controlled in part by the proton pumps
that give rise to the gradients that drive the uptake of the neurotransmitter
into the synaptic vesicle,
and so, you can change the efficacy of the proton pumping
or the efficacy of the neurotransmitter transporters.
Both of those are used to some extent to regulate neurotransmitter content
inside synaptic vesicles.
And although synaptic vesicles
typically have a size of something like 40 nanometers,
there's also some variance.
So there are some bigger vesicles and some smaller vesicles,
so that's another way in which one can control
the amount of neurotransmitter that's being released.
Presumably, a big vesicle would have
more neurotransmitter content than a small vesicle.
Another key way in which one can release different amounts of neurotransmitter
is by controlling the amount of vesicles
that are being released in response to calcium.
So the complex
machinery associated with the docked synaptic vesicle
here and the calcium channel
can then change its composition in terms of what proteins are present,
what isoforms, there are many different isoforms
of these different proteins, so for example,
synaptotagmin has something like 15 different genes that code for it
and they're expressed differentially at different synapses.
Equally, there can be phophorilation events,
so that can change the structure of these different proteins,
and maybe even the physical arrangement,
whether the calcium channels are close to the synaptic vesicles or far away
can make a big difference to whether the calcium is likely to drive
a lot of neurotransmitter release
or whether the probability will rather be lower.
So we can change the release probability and its sensitivity, of course, to calcium
and that's a key way
in which neurotransmitter release is regulated physiologically.
Equally, of course, it's possible to change the number of vesicles
that are present on the pre-synaptic specialization.
So if we only have one vesicle that's there
and it has a 10% chance of being released when an action potential invades,
we can also put in more synaptic vesicles.
We can simply dock more and more synaptic vesicles,
put them close to calcium channels,
and each one of these might then have a 10% chance
of being released in response to an action potential,
and clearly, the more vesicles we have that are in the release-ready state,
the more likely we are to get neurotransmitter release,
and large amounts of it,
when an action potential invades the presynaptic terminal.
So we've now seen the key events responsible
for coupling an action potential to neurotransmitter release.
The depolarization of the bouton causes the activation
of voltage-gated calcium channels
on the timescale of 100 microseconds or so
from action potential invasion,
calcium rises in the immediate vicinity of the synaptic vesicles
that are release-ready and competent.
The calcium binds to synaptotagmin,
five calcium ions bind to a synaptotagmin molecule,
high affinity, and they then drive the fusion of synaptic vesicle membrane
with the pre-synaptic membrane.
The whole process involves some 50 or so proteins,
all of which play an important role in synaptic release.
They can be regulated in a large number of interesting ways
that are physiologically important.
In the next few lessons we'll consider in more detail
how the neurotransmitter release is regulated by the action
of other neurotransmitters acting on the nerve terminal,
and also by their recent history of action potential firing
in that nerve terminal.