Welcome to Week 5
of Cellullar Mechanisms of Brain Function.
We've now learned quite a bit
about excitatory glutamatergic synapses in the mammalian brain.
Glutamatergic synapses account for about 80% of the synapses
and they provide an absolutely fundamental role in brain function.
Most neurons are electrically silent and they need some excitatory drive
in order to drive action potential firing
and create processing and signals across the brain.
But if there were only glutamatergic excitation
in the neuronal networks of the brain,
then there would be explosive activity
and all the cells would be continuously active.
And there's, therefore, a need for inhibition
to balance the excitation.
And it's that inhibition that we're going to be considering this week.
Most of the inhibition in the mammalian brain
comes about for the neurotransmitter gamma-Aminobutyric acid,
known as GABA for short.
Most of the neurons in the brain are glutamatergic neurons
when they fire action potentials.
That action potential propagates down the axon
and from presynaptic specializations, glutamate is released,
and that glutamate has an excitatory effect
on the postsynaptic cells.
It tries to increase the amount of action potential firing
of the postsynaptic cell.
For a GABAergic neuron,
the action potential again propagates down the axon,
but at the presynaptic specialization,
the neurotransmitter that's released is called GABA
and that GABA has an inhibitory influence upon the postsynaptic neurons.
It tries to prevent the postsynaptic neuron
from firing action potentials.
And in the same way that there are many types of excitatory neurons
there are also many types of GABAergic neurons.
The synapses that are formed by these GABAergic neurons
can have very high specificity.
They can target specific compartments like distal dendrites,
the axon initial segment, the soma,
and those synapses are created at very specific locations
and presumably have very specific functions.
During the course of the videos of this week,
we'll consider some of the specificity and general features
of GABAergic synaptic transmission.
Let's consider how a GABAergic synapse
is put together.
We have the axon and presynaptic boutons,
that contain synaptic vesicles,
just like before for the glutamatergic vesicles.
But the synaptic vesicles now need to be packaged
with a different neurotransmitter.
Before we're putting glutamate inside these synaptic vesicles
and now we need to put the neurotransmitter GABA
inside these vesicles.
Interestingly, the neurotransmitter GABA
is synthesized in a single enzymatic step from glutamate
so the main excitatory neurotransmitter Glutamate,
also an amino acid and present in the cytosol of all cells,
is treated by a specific enzyme Glutamic Acid Decarboxylase,
or GAD for short, and that removes this CO2 group from Glutamate
and creates the neurotransmitter GABA.
This enzyme GAD is present in the cytosol
even of the presynaptic boutons
and so GABA is produced right at the place where it's needed
and even some of the GAD enzyme seems to be present
directly on the synaptic vesicle membrane.
So, there's a local production of the inhibitory neurotransmitter GABA
right where we need it.
That GABA is then packaged into synaptic vesicles
through a protein, the vesicular GABA transporter
or VGAT that's present on the synaptic vesicle membrane
it utilizes a proton, an electrical gradient,
across the synaptic vesicle membrane and from that energy
it sucks GABA inside the synaptic vesicle
and concentrates it to a concentration
of, probably, around 100 millimolar.
The action potential then can invade the nerve terminal,
it has a glutamatergic synapse that drives calcium influx
and we have the standard exocytotic release machinery that then drives
vesicle fusion and release of the GABA into the synaptic cleft.
Then it diffuses some nanometers and binds to specific receptors
that are present in the postsynaptic specialization.
Similar to the way that we had ligand-gated ion channels
for glutamatergic synapses,
the fast inhibition that occurs at GABAergic synapses
is also mediated directly by ligand-gated ion channels.
So there are GABA binding ion chanels
and the GABA acts directly on that protein
increases the open probability
and for a GABA receptor that causes an outward current.
So, the electrical current flow is outwards,
and that causes negative charge inside the postsynaptic cell,
and that then is hyperpolarizing,
it's a outward postsynaptic current that inhibits the postsynaptic neuron.
That's how inhibition works.
Let's look in a bit more detail to the ionic conductance
that's mediated by GABA binding to its ligand-gated ion channel.
The GABAa receptor, as it's called,
is the site where GABA released directly from the presynaptic specialization,
binds to the protein, increases the open probability
and the GABAa receptor is permeable to anions,
and in particular, chloride is its major permeability.
So GABA opens a chloride conductance
which is formed by the GABAa receptor.
This, of course, is a fast action of GABA,
it's a direct action on a ligand-gated ion channel,
so those conductances open with millisecond timing
and mediates fast inhibition in the mammalian nervous system.
Why is a chloride conductance inhibitory?
We know that the chloride concentrations are distributed in an uneven way
across the plasma membrane.
Intracellularly, we have around 5 millimolar of chloride
and extracellularly we have around 120 millimolar of chloride.
So, there's a strong gradient that wants to put chloride into cells
if you open a chloride selective conductance.
We can look at the Nernst equation and drive quantitatively
what the reversal potential for chloride is.
We take our equilibrium potential equation,
we put in our numbers for 5 millimolar intracellular chloride,
120 millimolar extracellular chloride,
we multiply by 61.5 times the log 10,
the base 10 logarithm of these numbers,
and that gives us a chloride reversal potential of -85 millivolts.
That means that, if GABA acts to open the GABA receptor
and it's a chloride channel that tries to bring the membrane potential
of the postsynaptic cell to -85 millivolts.
That's hyperpolarized,
compared to the normal resting membrane potential of neurons
and it's certainly hyperpolarized
compared to the action potential threshold
that sits at around -45 millivolts.
So, chloride conductances are inhibitory
because the reversal potential for chloride is extremely negative
and well below action potential threshold
So, GABA prevents action potential firing of the postsynaptic cell.
The reason that does that is because of the chloride concentrations,
the way they're distributed comparing the inside and the outside of cells.
A key question that determines the inhibitory properties
of the GABA neurotransmitter, is the chloride concentrations.
So, this is of course very interesting to find out how chloride
is distributed and controlled in neurons.
One reason that we have low cytosolic chloride concentrations
is because there are leaky chloride channels
that are present on all cell membranes.
So, there's a permanent chloride conductance
that's open on cell membranes.
And simply by the fact that the resting membrane potential
is sitting at around -70 millivolts,
that will then contribute to distributing chloride
at around the equilibrium potential of -70 millivolts.
So, having negative potentials drives chloride out of cells
and gives us negative reversal potentials.
But the actual reversal potential for GABA
is more negative than the resting membrane potential
of most neurons.
So, we also need an active process that keeps chloride concentration low.
If we now look at the soma of a cell,
sitting on its plasma membrane
are specific transport proteins,
these are 12 transmembrane proteins,
so they have multiple regions across the plasma membrane
and these are transporters,
so they have multiple binding sites and a slow transport rate
but they take chloride out of the cell.
So, that's how chloride concentrations are low.
This is the so called transporter, called KCC2,
that's neuron-specific so it's only expressed in neurons
and it drives low chloride concentrations
in neurons in particular.
The chloride is, in fact, cotransported by potassium
so there's a one to one stoichiometry of potassium and chloride
that transport together through the KCC2 protein
and it's that potassium that drives the extrusion of chloride.
We'll remember that there's a high concentration of potassium inside cells,
a low concentration of potassium outside,
so there's a strong gradient for potassium to leave
and that's what provides the energy to suck the chloride out of neurons
and keep chloride concentrations low inside the cytosol of neurons.
And it's that low chloride concentration inside neurons
that's absolutely critical for making
that hyperpolarized reversal potential of GABA
that makes GABA an inhibitory neurotransmitter.
Interestingly, it turns out that GABA isn't always inhibitory.
In fact, during very early development,
chloride concentrations are radically different
from how they are in the adult brain.
At very early postnatal stages of rodents
that would then correspond to early stages
of development for human babies
that would be still in the gestation period for a human baby,
but rodents are born less developed than humans,
so, in the rodent postnatal day 0,
there are high chloride concentrations inside the cytosol of neurons,
the chloride concentration is thought to be somewhere around
30 millimolar intracellularly,
and a high concentration of chloride intracellularly
where we leave extracellular chloride to be---
it's normal around 120 millimolar,
means that the reversal potential for chloride is quite depolarized
compared to the adult condition of -80.
So, here at around birth for a rodent, the chloride reversal potential
might be sitting at around -40 millivolts.
That means that when GABA is released and activates a GABAa receptor
chloride actually leaves the cell causing depolarization
of the membrane potential.
So, if we have a typical neuron at rest here
at around -65 millivolts
opening of a GABA conductance at early postnatal days
causes depolarization and might contribute
to exciting the postsynaptic neuron.
The reason that chloride concentrations are high during early development,
is that the expression of this transport protein, KCC2,
is extremely low at early stages of development,
so the ability to suck chloride out of the cell hasn't developed yet.
So we end up with high chloride concentrations that give rise
to depolarized chloride reversal potentials
and the excitatory action of GABA.
During these first postnatal days, the expression of KCC2
ramps up dramatically
and that then sucks chloride out from the cytosol of the cell,
the chloride concentration goes down,
and now, when we open the GABAa conductance,
chloride wants to enter the cell and hyperpolarize it
and that's where we get the hyperpolarizing action of GABA,
that in the mature state, brings us to around -80 millivolts
as the reversal potential.
It's interesting to note that chloride concentrations
obviously make a big difference to GABA receptor function.
Early in development, GABA seems to have an excitatory role
it's really only at very, very early postnatal stages.
Already in the rodent, one week after birth,
it's clear that GABA has an inhibitory function.
In addition to developmental changes of chloride,
there are also thought to be differences in chloride concentrations
in different compartments of neurons.
So the KCC2 expression turns out to be different
in dendritic and axonal membranes.
So it may be, that chloride concentrations
might also differ in different neuronal compartments,
and that provides additional complexity to thinking about how
GABAergic inhibition might work.
It's now fun to compare what we know
about the glutamatergic and the GABAergic synapses.
Because they have a great deal of instinct similarities
and parallels in the way that they're set up.
At the glutamatergic synapse,
we obviously want to put glutamate inside the synaptic vesicles.
and that glutamate is packaged
by a specific vesicular glutamate transporter
that's present here, that puts glutamate in the vesicles.
In the GABAergic synapse, we need one additional step,
we need an enzyme that makes the GABA
and then we have a different protein, the vesicular GABA transporter
that puts GABA inside the synaptic vesicles.
Action potentials, neurotransmitter release machinery
are similar at glutamatergic and GABAergic synapses
and postsynaptically, of course, we have different receptors.
But the fast excitatory and the fast inhibitory neurotransmission,
both work the ligand-gated ion channels
so the neurotransmitter directly binds to an ion channel,
opens a conductance,
for glutamate it's AMPA and NMDA,
and that has a reversal potential of around 0 millivolts,
so it's excitatory and depolarizing.
For GABA we have the GABAa receptor,
it's a chloride conductance and that tries to bring us to around -80 millivolts
so that's an inhibitory neurotransmitter.
These ligand-gated ion channels are concentrated immediately next
to the presynaptic specialization.
The way that they're concentrated there is by having binding proteins here
that associate tightly with the ligand-gated ion channels
and hold them tightly in place right there in apposition
to the presynaptic terminal.
The ligand-gated receptors are otherwise
present in low concentrations extrasynaptically
where they can diffuse in the plasma membrane,
but when they encounter one of these PSD95 molecules
and other molecules in the postsynaptic density
they get fixed there, held in place,
and that concentrates the ion channels right next to the release site,
where they need to be.
The same situation is true for the GABAa receptors.
They're also present extrasynaptically,
they diffuse around on the plasma membrane
and they get held in place in a position to the presynaptic specialization
also by a scaffolding protein that binds to the receptors
and that's called Gephyrin.
So PSD95 and Gephyrin seem to play equivalent functions
at glutamatergic and GABAergic synapses respectively.
For the glutamatergic synapses
we noted that there was an interesting structural feature
the spine, that tends to be where glutamatergic synapses are made.
So these spines can grow to specific locations
and the fact that they can grow and appear and disappear,
appears to make them very suitable for plastic changes
and might be involved in the rewiring of the brain during learning
and the storage of memories.
GABAergic synapses on the other hand are typically made directly
on the dendritic shaft or even directly on the soma of the cell,
body of the neuron or on the axon initial segment.
So the GABAergic synapses are placed in slightly different locations
to where the glutamatergic synapses are,
although there can be GABAergic synapses present on some spines,
and it may be that that provides a specific regulation
at the level of individual synapses where we might be able to turn
on or off the function of a glutamatergic synapse
by putting a GABAergic synapse right here on the spine of a glutamate synapse.
Spines always have a glutamatergic input and a small fraction of them
also have an inhibitory input,
but most of the GABAergic synapses are directly on dendrites
or on the cell body.
In addition to the fast action of GABA on GABAa receptors,
and that's a ligand-gated ion channel that mediates fast GABAergic receptors,
there's also a metabotropic receptor,
a 7-transmembrane receptor that works
via G-proteins that then activate
a potassium conductance.
So this is a slower way of signaling,
so we have to imagine now that we have
our presynaptic specialization that releases GABA
and postsynapticaly, immediately in apposition to this,
we have our chloride channels that bring chloride into the cell
when the GABA binds here, and that's a fast IPSP,
where if we have an action potential we get a rapid inhibition
through this chloride influx into the postsynaptic cell.
The GABA can then diffuse some distance,
and at some distance to the actual synapse,
so at extrasynaptic locations,
there are these GABAb receptors
and these GABAb receptors work via G-proteins
and they activate a potassium conductance,
a so-called G-protein inward rectifying potassium conductance,
a GIRK channel.
That can give rise to much slower postsynaptic potentials
that are delayed by perhaps 50 milliseconds
before they become activated and they can also last
much longer periods of time, several hundred milliseconds,
and the delays impart because the GABA has to diffuse
and also because there are multiple steps before we can activate
this potassium conductance.
So there's a slow inhibitory postsynaptic potential that's mediated by potassium
and there's a fast IPSP that's a GABAa receptor
and that's the chloride conductance.
In addition to this postsynaptic action on potassium conductances
there's also an action of GABAb on calcium conductances
so the GABAb receptors inhibit calcium influx
that has effects postsynaticaly on excitability,
but perhaps more prominently there are also GABAb
present on presynaptic specializations,
on the nerve terminals themselves
there are GABAb receptors again acting through G-proteins
and here they also act upon calcium channels,
but now they play a very important function
because the calcium channels here are responsible directly
for neurotransmitter release,
and GABAb inhibiting the calcium channels
then reduces neurotransmitter release directly
and most forms of presynaptic boutons
have GABAb receptors present on them
where there's a glutamatergic synapse or a GABAergic synapse
or any other type of synapse, typically GABAb receptors are present
and if you activate GABAb, you inhibit neurotransmitter release
and that's another major way in which GABA is inhibitory.
It has a presynaptic inhibitory action where it reduces neurotransmitter release
through inhibiting calcium channels
and in addition to the fast GABAa conductance
there's also a slow GABAb conductance,
this slow potassium-mediated conductance
that gives rise to a slow IPSPs.
Finally it's worth pointing out that although GABA is by far
the most important inhibitory neurotransmitter
in the main brain,
if we go to spinal cord or the brainstem,
then GABA has a less prominent role
and glycine instead takes over as a major inhibitory neurotransmitter.
Glycine works in almost the identical way as GABA does at the GABAa receptor.
So there are glycinergic presynaptic specializations,
glycine is packaged into neurotransmitter vesicles
and in fact it's the same vesicular GABA transporter that also packages glycine
and postsynaptically we have glycine receptors,
that again, are chloride channels that bring chloride
in through the action of glycine acting on direct ligand-gated ion channels.
So the glycine receptor is very similar to the GABAa receptor
and glycine plays an almost identical role in the spinal cord and brainstem
as GABA does in the main brain.
So in this video, we've got an overview of GABAergic inhibition.
GABA is the main inhibitory neurotransmitter of the brain.
It accounts for about 20% of the synapses
and the other 80% of the synapses
are largely excitatory glutamatergic synapses.
GABA exerts its inhibitory action upon two types of receptors:
GABAa receptors that are present in the postsynaptic specialization
they're held in place by gephyrin that concentrates GABAa receptors
and GABA acts on these GABAa receptors to open directly
a ligand-gated ion channel, it's a chloride conductance
and the reversal potential for chloride makes this inhibitory.
Chloride has a reversal potential at around -80 millivolts
so the action of GABA is to try to bring the postsynaptic membrane
to around -80 millivolts.
That's clearly hyperpolarized to action potential threshold
so GABA has a fast inhibitory action through the GABAa receptor.
GABA also acts upon metabotropic receptors,
GABAb receptors,
and there it drives slow inhibitory postsynaptic potentials
through G-Proteins that activate potassium channels,
the GIRK ion channels,
and it presynaptically inhibits neurotransmitter release
by inhibiting calcium channels that directly are involved
in driving neurotransmitter release.
So GABA inhibits at multiple levels in the brain
and forms a very important counteraction
to the excitatory mechanisms of the glutamatergic synaptic transmission.
Over the next videos of this week,
we'll explore GABAergic inhibition in more detail.